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, 20 (1), 51-61

Chimeric Receptor mRNA Transfection as a Tool to Generate Antineoplastic Lymphocytes

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Chimeric Receptor mRNA Transfection as a Tool to Generate Antineoplastic Lymphocytes

Peter M Rabinovich et al. Hum Gene Ther.

Abstract

mRNA transfection is a useful approach for temporal cell reprogramming with minimal risk of transgene-mediated mutagenesis. We applied this to redirect lymphocyte cytotoxicity toward malignant cells. Using the chimeric immune receptor (CIR) constructs anti-CD19 CIR and 8H9 CIR, we achieved uniform expression of CIRs on virtually the entire population of lymphocytes. We reprogrammed CD3+ CD8+, CD3+ CD4+, and natural killer (NK ) cells toward autologous and allogeneic targets such as B cells, Daudi lymphoma, primary melanoma, breast ductal carcinoma, breast adenocarcinoma, and rhabdomyosarcoma. The reprogramming procedure is fast. Although most of the experiments were performed on lymphocytes obtained after 7-day activation, only 1-day activation of T cells with anti-CD3, anti-CD28 antibodies, and interleukin-2 is sufficient to develop both lymphocyte cytotoxicity and competence for mRNA transfer. The entire procedure, which includes lymphocyte activation and reprogramming, can be completed in 2 days. The efficiency of mRNA-modified human T cells was tested in a murine xenograft model. Human CD3+CD8+ lymphocytes expressing anti-CD19 CIR mRNA inhibited Daudi lymphoma growth in NOD=SCID mice. These results demonstrate that a mixed population of cytotoxic lymphocytes, including T cells together with NK cells, can be quickly and simultaneously reprogrammed by mRNA against autologous malignancies. With relatively minor modifications the described method of lymphocyte reprogramming can be scaled up for cancer therapy.

Figures

FIG. 1.
FIG. 1.
Cytotoxicity of CTLs transfected with anti-CD19 CIR mRNA against CD19+ tumor cells. (A) Structure of anti-CD19 CIR, which contains a signal peptide, VL and VH domains of single-chain anti-CD19 antibody, a transmembrane (TM) domain, and signal transduction domains originated from 4-1BB and CD3 ζ proteins. (B) Cytotoxicity of mRNA-transfected CTLs against target cells loaded with 51Cr. The vertical axis represents normalized chromium release, and the horizontal axis indicates the effector-to-target (E:T) ratio. CTLs, after the standard 7 days of activation, were electroporated with CIR mRNA or without mRNA (mock ). K562 cells were used as a CD19-negative control. All other target cells expressed CD19 protein.
FIG. 2.
FIG. 2.
CIR mRNA activity under various conditions. CTLs were obtained after short (1 day) or standard (7 days) ex vivo activation with anti-CD3/CD28 beads and IL-2, and were electroporated with CIR mRNA or without mRNA (mock). Surface CIR expression was visualized with a goat anti-mouse (Fab)2 polyclonal antibody conjugated with biotin and streptavidin PerCP (x axes). (A) Expression of anti-CD19 CIR in CTLs activated for 1 day (panel 1) or for 7 days (panel 2) (Elion et al., 2007); expression of anti-CD19 CIR δ(4–1BB) in CTLs activated for 7 days (panel 3). (B) Cytotoxicity of CTLs under the conditions indicated in (A). Targets, CD19+ autologous B cells, were loaded with 51Cr and analyzed at the indicated E:T ratios.
FIG. 3.
FIG. 3.
Cytotoxicity of various types of lymphocytes against autologous targets. CD8+ T cells, CD4+ T cells, a 1:1 mix of both (CD8+ and CD4+ cells), and NK cells were transfected with anti-CD19 CIR mRNA or without mRNA (mock). Targets, autologous CD19+ B cells, were loaded with 51Cr and analyzed for cytotoxicity at the indicated E:T ratios.
FIG. 4.
FIG. 4.
Cytotoxicity of anti-CD19CIR+ CTLs against tumor cells transfected with CD19 mRNA. (A) Expression of CD19 protein in two target cell lines, K562 and A2058, 18 hr after their electroporation with CD19 mRNA (CD19+) or without mRNA (CD19). Surface receptor expression was visualized with PE-conjugated anti-CD19 monoclonal antibody. (B) Cytotoxicity of CTLs transfected with anti-CD19 CIR mRNA against targets characterized in (A). Targets were loaded with 51Cr and analyzed for cytotoxicity at the indicated E:T ratios.
FIG. 5.
FIG. 5.
Cytotoxicity of 8H9 CIR+ CTLs against some solid tumors. (A) Structure of 8H9 CIR, which contains signal peptide (Spratt), VH and VL domains of single-chain 8H9 antibody, a transmembrane domain (TM), and signal transduction domains originated from CD28 and CD3ζ proteins. (B) Cytotoxicity of CTLs isolated from a melanoma patient against autologous melanoma and other tumors. CTLs obtained after the standard 7 days of ex vivo activation were electroporated with 8H9 CIR mRNA or without mRNA (mock) and tested against various tumor cells. We have verified that 8H9 antigen, gp58, was expressed on all solid tumor targets used, but not on K562 cells (negative control). Targets were loaded with 51Cr and analyzed for cytotoxicity at the indicated E:T ratios.
FIG. 6.
FIG. 6.
In vivo activity of anti-CD19 CIR+ CTLs. Twenty-four NOD/SCID mice injected with 3 × 106 luciferase (ffLuc)-expressing Daudi cells, and developed exponentially growing tumors by day 3 after injection were imaged and divided into three groups: treated with RPMI medium (1), mock transfected (2), or anti-CD19 CIR-transfected (3) CTLs. Groups received injections of 5 × 106 CTLs on days 3, 6, and 9. The mice were imaged on days 3, 6, 9, 12, and 15. (A) Pseudocolor image representing light intensity and anatomic localization of the ffLuc Daudi cells in representative mice. (B) Longitudinal monitoring of the bioluminescent signals of ffLuc+ Daudi cells injected into 3 groups of 24 NOD/SCID mice. Data are presented as geometric means of total photon flux normalized for exposure time, surface area, and initial signal for each group of mice; error bars represent the geometric SD.

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