skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

Abstract

Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8{sup +} CAR-T cells had antigen-specific cytotoxic activity.more » • CD4{sup +} CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.« less

Authors:
; ; ; ; ;
Publication Date:
OSTI Identifier:
22596340
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 473; Journal Issue: 1; Other Information: Copyright (c) 2016 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ACCURACY; ANTIGENS; CATTLE; DISEASE VECTORS; ENZYMES; FLUORESCENCE; FUNCTIONAL ANALYSIS; GENES; IMMUNOTHERAPY; INTERFERON; LYMPHOCYTES; MICE; MONOCLONAL ANTIBODIES; NEOPLASMS; POLYMERASE CHAIN REACTION; PUROMYCIN; RECEPTORS; RIBOSOMES; STIMULATION

Citation Formats

Kusabuka, Hotaka, Fujiwara, Kento, Tokunaga, Yusuke, Hirobe, Sachiko, Nakagawa, Shinsaku, E-mail: nakagawa@phs.osaka-u.ac.jp, and Okada, Naoki, E-mail: okada@phs.osaka-u.ac.jp. Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy. United States: N. p., 2016. Web. doi:10.1016/J.BBRC.2016.03.054.
Kusabuka, Hotaka, Fujiwara, Kento, Tokunaga, Yusuke, Hirobe, Sachiko, Nakagawa, Shinsaku, E-mail: nakagawa@phs.osaka-u.ac.jp, & Okada, Naoki, E-mail: okada@phs.osaka-u.ac.jp. Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy. United States. doi:10.1016/J.BBRC.2016.03.054.
Kusabuka, Hotaka, Fujiwara, Kento, Tokunaga, Yusuke, Hirobe, Sachiko, Nakagawa, Shinsaku, E-mail: nakagawa@phs.osaka-u.ac.jp, and Okada, Naoki, E-mail: okada@phs.osaka-u.ac.jp. 2016. "Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy". United States. doi:10.1016/J.BBRC.2016.03.054.
@article{osti_22596340,
title = {Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy},
author = {Kusabuka, Hotaka and Fujiwara, Kento and Tokunaga, Yusuke and Hirobe, Sachiko and Nakagawa, Shinsaku, E-mail: nakagawa@phs.osaka-u.ac.jp and Okada, Naoki, E-mail: okada@phs.osaka-u.ac.jp},
abstractNote = {Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8{sup +} CAR-T cells had antigen-specific cytotoxic activity. • CD4{sup +} CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.},
doi = {10.1016/J.BBRC.2016.03.054},
journal = {Biochemical and Biophysical Research Communications},
number = 1,
volume = 473,
place = {United States},
year = 2016,
month = 4
}
  • Replication-defective amphotropic retrovirus vectors containing either the human ..beta..-globin gene with introns or an intronless ..beta..-globin minigene were constructed and used to study ..beta..-globin expression following gene transfer into hematopoietic cells. The ..beta..-globin genes were marked by introducing a 6-base-pair insertion into the region corresponding to the 5' untranslated region of the ..beta..-globin mRNA to allow detection of RNA encoded by the new gene in human cells expressing normal human ..beta..-globin RNA. Introduction of a virus containing the ..beta..-globin gene with introns into murine erythroleukemia cells resulted in inducible expression of human ..beta..-globin RNA and protein, while the viruses containingmore » the minigene were inactive. The introduced human ..beta..-globin gene was 6 to 110% as active as the endogenous mouse ..beta../sup maj/-globin genes in six randomly chosen cell clones. Introduction of the viruses into human BFU-E cells, followed by analysis of marked and unmarked globin RNAs in differentiated erythroid colonies, revealed that the introduced ..beta..-globin gene was about 5% as active as the endogenous genes in these normal human erythroid cells and that again the minigene was inactive. These data are discussed in terms of the potential treatment of genetic disease by gene therapy.« less
  • The locus activation region (LAR) of the human {beta}-globin-like gene cluster is characterized by a group of four DNase I hypersensitive sites, which arise specifically in erythroid tissues and are required for a normal pattern of {beta}-globin-like expression. The hypersensitive sites are found at positions 6.1, 10.9, 14.7, and 18 kilobase pairs (kbp) 5{prime} of the {epsilon}-globin gene. Recently functional assays of the LAR that tested determinants for all four hypersensitive sites showed that expression of the human {beta}-globin gene was increased to normal or near-normal levels in both transgenic mice and erythroid cells. The authors constructed retroviral vectors withmore » a human {beta}-globin gene and the determinant for a single hypersensitive site and measured {beta}-globin gene expression after retroviral infection of murine erythroleukemia cells. In the context of gene-transfer experiments ultimately aimed at gene therapy, the results show that LAR determinants lead to an increased level of human {beta}-globin RNA expression after retroviral transfer into erythroid cells. But inclusion of LAR determinants in retroviral vectors also entails the potential risk of activating the expression of nonglobin genes in erythroid cells.« less
  • Antigen activation of murine T lymphocytes leads to phosphorylation of three subunits of the murine T cell antigen receptor. Two kinases are activated in this process: protein kinase C which leads to phosphorylation of the ..gamma.. and, to a lesser extent, the epsilon subunits on serine residues and a tyrosine kinase which phosphorylates the p21 subunit. The authors sought to determine whether treatment of these cells with NaF could simulate any of these antigen-induced events. Indeed NaF treatment resulted in breakdown of polyphosphoinositides and production of phosphoinositols. This treatment also resulted in a rise in cytosolic free Ca/sup 2 +/.more » EGTA failed to block this rise suggesting that NaF liberated intracellular stores of Ca/sup 2 +/. Finally NaF treatment resulted in phosphorylation of the ..gamma.. and epsilon chains of the T cell receptor indistinguishable from the effects of phorbol esters. The NaF effect was potentiated by addition of AlCl/sub 3/ consistent with the view that the active moiety is AlF/sub 4//sup -/. The AlF/sub 4//sup -/-induced phosphorylations were abolished in cells in which protein kinase C was depleted by prior treatment with phorbol myristate acetate. All of these observations are compatible with the interpretation that the AlF/sub 4//sup -/ phosphorylation is mediated by protein kinase C. Antigen and anti-receptor antibody-induced receptor serine phosphorylation and phophatidylinositol turnover are blocked by raising intracellular levels of cyclic adenosin monophosphate. In contrast, AlF/sub 4//sup -/-induced effects were in sensitive to cyclic adenosine monmonophosphate« less
  • DNA rearrangements that occurred in the vicinity of T-cell antigen receptor ..beta..-chain gene clusters residing on chromosome 7 were examined in human T-cell acute lymphoblastic leukemia cells. In one patient, it was observed that, for the T-cell receptor ..beta..-chain genes, a D/sub ..beta.. 1/-J/sub ..beta..2.3/ (where D is diversity and J is joining) junction was found on one chromosome, while the other chromosome kept the germ-line configuration. If this D/sub ..beta../-J/sub ..beta../ junction was formed by the customary deletion mechanism, the C/sub ..beta..1/ gene (where C is constant) located between the D/sub ..beta..1/ and J/sub BETA2.3/ loci should have disappearedmore » from this chromosome. The C/sub ..beta..1/ gene indeed was absent from the rearranged chromosome 7, but it was found on chromosome 6 as an inserted segment. The implications of the observations are discussed.« less
  • Radioimmunotherapy (RIT) with α-emitting radionuclides is an attractive approach for the treatment of minimal residual disease (MRD) because the short path lengths and high energies of α-particles produce optimal cytotoxicity at small target sites while minimizing damage to surrounding normal tissues. Pretargeted RIT (PRIT) using antibody-streptavidin (Ab-SA) constructs and radiolabeled biotin allows rapid, specific localization of radioactivity at tumor sites, making it an optimal method to target α-emitters with short half-lives, such as bismuth-213 (213Bi). Athymic mice bearing Ramos lymphoma xenografts received anti-CD20 1F5(scFv)4SA fusion protein (FP), followed by a dendrimeric clearing agent and [213Bi]DOTA-biotin. After 90 min, tumor uptakemore » for 1F5(scFv)4SA was 16.5 ± 7.0 % injected dose per gram (ID/g) compared with 2.3 ± 0.9 % ID/g for the control FP. Mice treated with anti-CD20 PRIT and 600 µCi [213Bi]DOTA-biotin exhibited marked tumor growth delays compared to controls (mean tumor volume 0.01 ± 0.02 vs. 203.38 ± 83.03 mm3 after 19 days, respectively). The median survival for the 1F5(scFv)4SA group was 90 days compared to 23 days for the control FP (p<0.0001). Treatment was well tolerated, with no treatment-related mortalities. This study demonstrates the favorable biodistribution profile and excellent therapeutic efficacy attainable with 213Bi-labeled anti-CD20 PRIT.« less