Paresh Vishwasrao. M.Sc. Zoology, Molecular Biology, University of Pune, MH, India

Transcription

1 Paresh Vishwasrao 1 Phone #: (201) vishwasrao.paresh@gmail.com Webpage: Education: Ph.D. Translation Immunology and Immunotherapy, Academic Med Center (AMC), Amsterdam. The Netherlands. (Anticipated) Columbia Center for Translational Immunology (CCTI), New York City. USA M.Sc. Zoology, Molecular Biology, University of Pune, MH, India PGDFA Forensic Anthropology University of Pune, MH, India B.Sc. Microbiology, University of Pune, MH, India Research Experience: Research Tech University Medical Center Groningen, (UMCG) Groningen. 07/2010 to 07/2011 The Netherlands. Research Assistant The Helmholtz Zentrum, München. 05/2009 to 03/2010 Germany. Research Student Ludwig Maximillans Universitat (LMU), München. 05/2008 to 03/2009 Germany. Publications: Li. H. W*, Vishwasrao.P*, Holzl. M. A, Chen. S, Choi. G, Zhao, G and Sykes. M. Impact of Mixed Xenogeneic Porcine Hematopoietic Chimerism on Human NK Cell Recognition in a Humanized Mouse Model. Am J Transplant Volume 17, (2017). (doi: /ajt Epub 2016 Aug 10) Zinjarde, S. S., B. V. Kale, P. V. Vishwasrao and A. R. Kumar "Morphogenetic behavior of tropical marine yeast Yarrowia lipolytica in response to hydrophobic substrates." J Microbiol Biotechnol 18(9): (2008). Manuscripts in preparation/submission: Hao Wei Li, Steven Shao, Paresh Vishwasrao, Markus A. Hölzl, Kallie Jiang, Mohsen Khosravi Maharlooei, Guiling Zhao, and Megan Sykes. Impact of mixed xenogeneic porcine hematopoietic chimerism on anti- pig natural humoral responses in a humanized mouse model. (expected- Blood). (Manuscript in Submission)

2 2 Gongbo Li, Nolan Beatty, Paresh Vishwasrao, Bin Yu, Justin Boucher, Paul Park, Yongliang Zhang,and Marco L Davila. Mutation of the 41BB co- stimulatory domain enhances CAR signaling and mouse CD19- targeted CAR T cell function. (expected- Journal of Clinical Investigation). (Manuscript in Submission) Paresh Vishwasrao, Gongbo Li, Justin Boucher, Bin Yu, Yongliang Zhang and Marco L Davila. Immunotherapy Target evaluation for myeloid diseases. (undecided) (in preparation) Professional Summary: Motivated and independent immunologist in training with +6 years of experience in wide range of topics, signal transduction, molecular oncology, immunotherapy. Ability to carry out and interpret data to T cell and NK cell biology. Acquired mouse surgery skills and developed xenogeneic chimeric humanized mouse model to determine human B, NK and T cell tolerance towards pig immune system. Strong scientific background in immune- oncology and in vivo tumor mouse modeling. Contributed to ideas with ongoing projects in different domains related to Medulloblastoma, Cord Blood transplantation, Humanized Mouse Model and Immunotherapy. Validated screening protocols for establishment of specific targets in designing of CARs. Designed 14 color flow panel and perform SPADE analysis. Effectively communicated scientific data to a diverse audience and presented nationally and internationally at various conferences at ICI- 2013, ASH- 2014, FOCIS and ASBMT Currently working on Patient- Derived Xenograft (PDX) mouse model for AML CAR- immunotherapy. Managing lab co- ordination with primary AML patient samples. Mentored students from disciplines of Biology and Biochemistry and rotating graduate students. Research Interests: T- cell immunotherapy has significant potential, as T cells can expand in numbers to eradicate large volume of a disease, traffic to disparate areas of the body to eradicate residual tumor, and endow patients with long- lived tumor immunity. My research is dedicated to developing gene- engineered cell therapies that target cancer cells in pre- clinical animal models for cancer. The goal of this research is to identify optimal cell therapies that can then be evaluated in cancer patients using genetically engineered T cells. Clinical success in most ongoing trials of T reg therapy for autoimmune and GVHD, check point inhibitors such as PD- 1 and CTLA- 4, CAR- T cells for B cell malignancies and Tissue infiltrating lymphocytes (TILs) for solid tumors has turn my attention to bench- to- bed side work. This field is fast evolving and I wish to continue and contribute to improve clinical efficacies. Combing my previous research experience, I would like to continue working in the field of Immuno- oncology. Research skills: SPADE, LSRII, FACS analysis, ique, ELISA- Luminex, PCR, Cloning, RT- PCR, MacVector, Prism and DNA star H. Lee Moffitt Support Grant Prof. Marco Davila Cancer Immunotherapy Work Support Grant Prof. Megan Sykes NIH Grant P01AI and R01 AI Ph.D. Scholarship Dr. C.W Choi Dutch Cancer Society (KWK; UVA ) Research Associate 12/2015 to 04/2017 Department of Clinical Science H. Lee Moffitt Cancer Center, Tampa. Florida.

3 3 Developed two different cell flow panels- first 14 multi- color flow panel for detection of cell surface markers and second 8- multi- color flow panel for intracellular staining. Designed bi- specific CARs for AML myeloid panel with use of MacVector and FlowJo. Performed studies on human primary AML samples, isolation of human T and NK cells from whole blood and co- culture assay. Co- ordinated time- dependent crucial T- cell assays, viral transduction protocols for various CARs. Patient derived Xenograft (PDX) mouse model to identify human AML markers for in vivo modeling. Effectively carried out in vivo modeling mouse model, FACS, SPADE analysis and CTL assay. Isolation of mouse T cells, proliferation and activation with CD3/CD28 beads. Demonstrated flexible work hours with the impeding need of task and meeting the deadlines with different projects on hand. Immunotherapy target evaluation for Myeloid diseases We designed bi- specific CARs targeting antigens CD33/CD123 and/or CD33/TIM3. To compare the co- expression of various tumor markers on AML to identify candidates for multi- antigen targeting, we are evaluating myeloid and T cells from patients with myeloid malignancies to determine their suitability as an adoptive T cell therapy. Currently working on Patient- Derived Xenograft (PDX) mouse model for AML CAR- immunotherapy to validate the anti- tumor efficiency of shared bi- specific CARs. (In Preparation) Mutation of the 41BB co- stimulatory domain enhances CAR signaling and mouse CD19- targeted CAR T cell function. (Gongbo Li*,Nolan Beatty*, Vishwasrao- expected 2017) We demonstrate that replacement of the mouse 41BB intracellular domain with its human counterpart restored CAR signaling and yielded similar survival and persistence to the mouse CD28 CAR. Furthermore, targeted mutations of 5 amino acids on the mouse 41BB domain enhanced CAR signaling, NFKB activation, anti- apoptotic gene expression and cytokine production. Our study has implications for improving CAR design and provides a platform for future studies of CAR T cell biology in animal models. (In Submission) Ph.D. Exchange (Anticipated by the end of 2017) 11/2011 to 11/2015 Academic Med Center. (AMC) Amsterdam, The Netherlands. Columbia University Medical Center (CUMC) Columbia Center for Translational Immunology (CCTI), New York Performed surgeries for transplantation of human thymic tissue under kidney capsule on mouse. Demonstrated isolation of human stem cells from human fetal liver (FLV): injection of these cells in - vivo in NSG lead to a successful pleiotropic population of human immune system. Successfully developed a complex humanized mouse model for tolerance studies and Communicated two research articles in peer- reviewed journals. Experience with human primary cell culture and isolation. Capable of designing complex immune- cell culture experiments addressing a specific hypothesis. Impact of Mixed Xenogeneic Porcine Hematopoietic Chimerism on Human NK Cell Recognition in a Humanized Mouse Model. (Li*, Vishwasrao* et al. 2016) Our results showed that induction of human NK cells in pig/human mixed chimeras did not lead to enhanced loss of pig chimerism. NK cells from most pig/human mixed chimeric mice showed either specifically decreased cytotoxicity to pig cells or global hyporesponsiveness in in vitro cytotoxicity assay, indicating that mixed xenogeneic chimerism resulted in partial or complete tolerance of human NK cells to pig cells. Mixed

4 4 xenogeneic chimerism did not hamper the maturation of human NK cells, but was associated with an alteration in NK cell subset distribution in the bone marrow. Impact of mixed xenogeneic porcine hematopoietic chimerism on anti- pig natural humoral responses in a humanized mouse model. (Li*, Vishwasrao* et al expected at the end of 2017) Our results showed that natural anti- pig cytotoxic antibodies were detectable in the serum of humanized mice. These antibodies were predominantly IgM. The lower levels of anti- pig natural antibodies were not due to the binding of antibodies to pig cells. Mixed pig/human chimerism was not associated with differences in B cell phenotype, but with a lower numbers of human B cells in bone marrow and lower percentages of B cells in several lymphoid tissues. Our data suggest that induction of mixed xenogenic chimerism downregulates human natural humoral responses to pig xenoantigens and support the use of this approach to inducing human B cell tolerance to xenografts in clinical settings. (In Submission) Research Technician III 07/2010 to 07/2011 Dept. of Hematology and Oncology University Medical Center Groningen, (UMCG) Groningen, The Netherlands Role of mutant KRAS and BMI1 in hematopoietic cells The project includes elucidating the molecular mechanisms involved in the regulatory roles of K- RAS and BMI1 in the human Hematopoietic Stem Cells (HSCs) and progenitors as well as in the leukemic compartment in the Acute Myeloid Leukemia (AML). We aimed to dissect the long- term self- renewal (LT- HSC) and maintenance of BMI1 in the cord blood cells mainly CD34+. We reported that the myelo- monocytic lineage with CD11b and CD11c populations arouse during maintenance which was supported by BMI1. Research Assistant 05/2009 to 03/2010 Dept. of Hematology. Helmholtz Zentrum, Munich, Germany Role of a tumor suppressor and transcription factor, CDX2 in the establishment of AML My role as a assistant was to examine the details of signal transduction pathway upstream of CDX2, to determine if an alteration in the pathway leads to the establishment of leukemia in current work model system. Our in vivo experiments, along with CFC assays and FACS analysis of BM cells retro- virally transduced with CDX2 showed ERK pathway and STAT5 was involved in signal transduction. We also performed an assay for in vitro on a phosphorylation inhibitor, U0126 for ERK1/2, utilizing western blot and RT- PCR analysis to establish the link between ERK1/2 regulations with downstream factor CDX2 in a range of AML cell lines. Research Student 05/2008 to 03/2009 Experimental Genetics, Germany Ludwig Maximillans Universitat (LMU). Munich Germany Zebrafish as a model for studying signaling pathway in Medulloblastoma. I worked with Dr. Reinhard Köster on generating expression constructs for establishing a brain tumor model in zebrafish.. I leanred Zebrafish husbandry and crossing zebrafish lines, selectively for conical NOTCH pathway. I learned basic molecular biology methods, cloning, PCR, DNA- purification and in vitro mrna synthesis. Also acquired knowledge in zebrafish embryology and genetics including fish husbandry, zebrafish embryo injection, mrna in situ hybridization and fluorescence stereomicroscope imaging

5 Workshops & Conferences/Poster presentations: 5 CCTI Travel Grant The Federation of Clinical Immunology Societies (FOCIS) SAN DIEGO, USA 2015 Human NK cell Tolerance in Humanized Mouse Model Spinoza Travel Grant International Conference of Immunology (ICI)- MILAN, ITALY 2013 Establishment of a humanized mouse model to investigate the impact of mixed xenogeneic hematopoietic Vishwasrao P, Li G, Yu B, Boucher J and Davila M. Immunotherapy Target Evaluation for Myeloid Diseases. American Society for Blood and Marrow Transplantation (ASBMT). Orlando, FL Vishwasrao. P, Holzl. M, Li. H, Choi.G and Sykes.M. Impact of Porcine- Human Mixed Hematopoietic Chimerism on Human NK Cell Response to Porcine Cells. The American Society of Hematology (ASH). San Diego References: Prof. Davila, Marco L. Director Bone Marrow Transplantation Dept of Clinical Science H. Lee Moffitt Cancer Center Marco.Davila@moffitt.org Asst Prof. HaoWei Li Columbia Center For Translational Immunology (CCTI) Columbia University Medical Center (CUMC), New York, USA hl2591@cumc.columbia.edu Prof. Julien Zuber Faculty of Nephrologist Service de Transplantation Rénale Adulte, Hôpital Necker, France. julien.zuber@aphp.fr Dr. C.W Choi Faculty of Medicine, Dept of Hematology University Medical Center Groningen (UMCG), The Netherlands c.w.choi@umcg.nl Dr. Ruchira S. Ranaweera Dept of Clinical Science H. Lee Moffitt Cancer Center ruchira.ranaweera@moffitt.org

6 Cover Letter 6 As a master s student of molecular biology, I was more confident learning about biochemistry and pathways in cancer biology classes. The interest peaked me further to concentrate focusing on oncology and hematology. For a brief period I taught classes of virology and oncology. Classroom teaching helped me prepare my lectures and broaden my view about oncology and stem cell research. In Germany, at Klinikum Med III,KKG- Leukemia Hämatologikum HelmholtZentrum Munich, my role as a student was to examine the details of signal transduction pathway upstream of CDX2 to determine, if changes in this pathway altered the establishment of leukemia in our model systems. In this capacity, I carried out in vivo experiments with mice, along with CFC assays and FACS analysis of BM cells retro- virally transduced with CDX2. I have also worked in vitro on a phosphorylation inhibitor, U0126 for ERK1/2, utilizing western blot and RT- PCR analysis to establish the link between ERK1/2 regulation with downstream factor CDX2 in a range of AML cell lines. In Groningen, The Netherlands I worked in a project, to elucidate the molecular mechanisms which involve the regulatory roles of KRAS and BMI1 in the human Hematopoietic Stem Cells (HSCs) and progenitors as well as in the leukemic compartment in the Acute Myeloid Leukemia (AML). The project aimed to dissect the long- term self- renewal (LT- HSC) and maintenance of BMI1 in the cord blood cells mainly CD34+. I have experience in techniques such as FACS used to trace the myeloid lineage. Further, the progenitor assessment was done through CFC assays. At Columbia University Medical Center (CUMC) in the department of Columbia Center for Translational Immunology (CCTI) in collaboration with Academic Medical Centrum (AMC), Netherlands, we developed a humanized mouse model. My thesis was focused on xenotransplantation as a potential solution for the shortage of organs for transplantation. The impact of mixed xenogeneic porcine hematopoietic chimerism on human NK cell recognition in a humanized mouse model. In this study we have used NSG pig cytokine mice (PCT- NSG) expressing pig IL3, GMCSF and SCF to generate pig/human mixed chimeric mice. However, potent xenograft rejection responses present a formidable barrier. Mixed xenogeneic hematopoietic chimerism has been shown in rat- to- mouse models to induce tolerance among T, B and NK cells. NK cells play an important role in the rejection of xenogeneic tissues. We have shown previously in an allogeneic mixed chimerism model that specific NK cell tolerance is induced. In a rat- to- mouse transplantation model, in contrast, it was observed that induction of mixed xenogeneic chimerism led to a global hyporesponsiveness of recipient NK cells. In this study, we investigated whether pig/human mixed chimerism could tolerate human NK cells in a humanized mouse model. Our results showed that induction of human NK cells in pig/human mixed chimeras did not lead to enhanced loss of pig chimerism. NK cells from most pig/human mixed chimeric mice showed either specifically decreased cytotoxicity to pig cells or global hyporesponsiveness in in vitro cytotoxicity assay, indicating that mixed xenogeneic chimerism resulted in partial or complete tolerance of human NK cells to pig cells. Mixed

7 7 xenogeneic chimerism did not hamper the maturation of human NK cells, but was associated with an alteration in NK cell subset distribution in the bone marrow. In summary, our results demonstrate that mixed xenogeneic chimerism is able to induce human NK cell tolerance to pig cells and support the use of this approach to inducing xenogeneic tolerance in the clinical setting. My current work at Moffitt Cancer Center, Tampa focuses on designing immunotherapies for myeloid diseases have only modest success and the vast majority of patients will ultimately die. Clearly, novel therapies are warranted and adoptive immunotherapies represent an exciting and promising cancer therapy. The success of antibody mediated therapy targeting immune checkpoint underscores the therapeutic potential of counteracting immune inhibition and T cell exhaustion. Other alternative immunotherapies consist of bispecific T cell engagers (BiTEs), which have had some success for leukemia. A major advance for adoptive T cell therapy is the chimeric antigen receptor (CAR), which is a single chain variable fragment (scfv) fused to the signal domains of a T cell receptor. CAR- T cell therapy has significant potential as a cancer therapy because T cells can expand in numbers to eradicate large volume disease, traffic throughout the body, and provide patients with long- lived tumor immunity. In particular, clinical success with CART19 has generated high complete response rates in patients with B- cell acute lymphoblastic leukemia (B- ALL). Currently, CAR T cells are being developed against CD33, CD123, CD44v6, as well as other antigens. Early pre- clinical work suggests that CAR- T cell directed towards CD33 and CD123 are effective, but unfortunately also kill normal myeloid and/or hematopoietic stem cells. Considering the extensive B cell aplasia reported with CD19- targeted CAR T cells, a similar off- tumor, on- target toxicity for myeloid malignancies could result in bone marrow failure and/or death. My work here is to compare the co- expression of various tumor markers on AML to identify candidates for multi- antigen targeting. CAR- T cells with a combination of either of these markers would prove effective targeting against leukemic stem cells leaving normal stem cells unharmed. We are also evaluating T cells from patients with myeloid malignancies to determine their suitability as an adoptive T cell therapy. My research is dedicated to developing gene- engineered cell therapies that target cancer cells in pre- clinical animal models for cancer. The goal of this research is to identify optimal cell therapies that can then be evaluated in cancer patients using genetically engineered T cells. Clinical success in most ongoing trials of Treg therapy for autoimmune and GVHD, check point inhibitors such as PD- 1 and CTLA- 4, CAR- T cells for B cell malignancies and Tissue infiltrating lymphocytes (TILs) for solid tumors has turn my attention to bench- to- bed side work. This field is fast evolving and I wish to continue and contribute to improve clinical efficacies. Combing my previous research experience, I would like to continue working in the field of Immuno- oncology.