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Next Generation Sequencing of Immunized Mouse Splenocytes to Develop an anti-TIM3 Chimeric Antigen Receptor for Acute Myeloid Leukemia
Poster Title: Next Generation Sequencing of Immunized Mouse Splenocytes to Develop an anti-TIM3 Chimeric Antigen Receptor for Acute Myeloid Leukemia
Submitted on 08 Feb 2018
Author(s): Lawrence Guan, Justin C Boucher, Paresh Vishwasrao, Inimary T Toby, Bishwas Shrestha, Gongbo Li, Maria L Cabral, Scott Christley, Jared Ostmeyer, William H Rounds, Lindsay G Cowell and Marco L Davila
Affiliations: Department of Blood & Marrow Transplant and Cellular Immunotherapy, Department of Immunology, Department of Clinical Science, H. Lee Moffitt Cancer Center, Tampa, FL, 33612. Division of Biomedical Informatics, Department of Clinical Sciences, University of Texas Southwestern Medical Center, Dallas, TX, 75390. Morsani College of Medicine, University of South Florida Health, Tampa, FL, 33612 . Department of Cell Biology, Microbiology, and Molecular Biology, College of Arts and Sciences, University of South Florida, Tampa, FL, 33612.
This poster was presented at USF Research Day 2018
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Poster Information
Generation of new chimeric antigen receptors (CARs) rely on a time
consuming and/or costly process of phage display screening or
hybridoma production, hybridoma screening, and immunoglobulin
(Ig) sequencing. Here, we describe a novel CAR design strategy by
next generation sequencing (NGS) followed by CAR synthesis and
functional validation against TIM3+ targets. NGS allows for direct
identification of rearranged immunoglobulin genes, mutation and
repertoire analysis, and comparison of multiple immunized mice.
Furthermore, by eliminating the need for hybridoma production and
screening we have developed a rapid, economical system for the
development of novel CARs. After immunization of four separate
mice with CHO-TIM3, NGS demonstrated that nearly 70% of Ig
heavy (IgH) and 61% of Ig kappa (Igκ) sequences were accounted for
by 2 and 4 V-J rearrangements, respectively. This suggested that
these rearrangements were enriched for TIM3-reactivity and we
designed pairs of these IgH and Igκ rearrangements. By this process
we generated 8 anti-TIM3 CARs (4 VHS-based CARs and 4 VH8-
based CARs). After their gene-synthesis and cloning into a genetic
construct we produced retrovirus and transduced Jurkat, as well as
primary T cells. Robust gene-transfer was observed with most
constructs (14-64%). Furthermore, we detected antigen-specific
cytokine production by primary T cell or Jurkat stimulated with
TIM3+ targets. We also examined in vitro killing by primary anti-TIM3 human T cells using a Real-Time cell Analysis cytotoxicity assay (Fig. 5). We are now evaluating the in vivo efficacy of anti-TIM3 CAR T cells in immune deficient animal models implanted with TIM3+ targets. This work demonstrates NGS can be used to identify new sequences that can be developed rapidly and inexpensively into CARs. Using this process we created 8 anti-TIM3 CARs and by in vitro screening, are looking towards one of them (VH8-461) for in vivo validation. We are using this same NGS CAR generation system to develop additional anti-AML CARs which can be used in tandem
with anti-TIM3 CARs to treat AML.
Summary: The progress against B-ALL has not been matched in other types of cancers such as Acute Myeloid Leukemia (AML) Additionally, prognosis is poor with as much as 70% of patients, 65 years or older, dying within 1 year of diagnosis. Our evaluation of TIM3 expression on AML blasts and LSC demonstrated that TIM3 is commonly co-expressed on CD33+ blasts. These results led us to hypothesize anti-TIM3 CAR T cells can target and kill AML.References: 1. Davila, M.L., Riviere, I., Wang, X., Bartido, S., Park, J., Curran, K.,
Chung, S.S., Stefanski, J., Borquez-Ojeda, O., Olszewska, M., Qu, J.,
Wasielewska, T., He, Q., Fink, M., Shinglot, H., Youssif, M., Satter, M.,
Wang, Y., Hosey, J., Quintanilla, H., Halton, E., Bernal, Y., Bouhassira,
D.C., Arcila, M.E., Gonen, M., Roboz, G.J., Maslak, P., Douer, D.,
Frattini, M.G., Giralt, S., Sadelain, M., and Brentjens, R. (2014).
Efficacy and toxicity management of 19-28z CAR T cell therapy in B
cell acute lymphoblastic leukemia. Sci Transl Med 6, 224ra225.
2. Yamamoto JF, Goodman MT. Patterns of leukemia incidence in the
United States by subtype and demographic characteristics,
1997-2002. Cancer Causes Control 2008; 19: 379–390.
3. Ngiow S.F., von Scheidt B., Akiba H., Yagita H., Teng M.W., Smyth
M.J. Anti-TIM-3 antibody promotes T cell IFN-gamma-mediated
antitumor immunity and suppresses established tumors. Cancer
Res. 2011;71:3540–35
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