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A caspase 8-based suicide switch induces apoptosis in nanobody-directed chimeric receptor expressing T cells

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Abstract

In accordance with the two-step hypothesis of T cell activation and the observation that stimulation through the T cell receptor (TCR) alone may lead to anergy, we focused on the introduction of co-stimulatory signaling to this type of receptors to achieve optimal activation. Enhanced mRNA and cell surface receptor expression via the co-stimulatory gene fragment (OX40) was confirmed by RT-PCR and flow cytometry. Inclusion of the OX40 co-stimulatory signaling region in series with the TCR led to enhanced antigen-induced IL-2 production after stimulation by MUC1-expressing cancer cell lines as compared to the chimeric receptor without OX40. Moreover, with the aim of maintaining high efficiency, while providing a means of controlling any possible unwanted proliferation in vivo, a regulation system was used. This controls the dimerization of a membrane-bound caspase 8 protein. Toward that goal, pFKC8 and CAR constructs were co-transfected into Jurkat cells, and the level of apoptosis was measured. 24 h after addition of the dimerizer, a 91% decrease in transfected cells was observed.

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Abbreviations

BSA:

Bovine serum albumin

CAR:

Chimeric antigen receptor

CH2-CH3-hinge:

Sequences coding for CH2-CH3-hinge regions of human IgG3

CH2-CH3-hinge–hinge:

Sequences coding for CH2-CH3-hinge-hinge regions of human IgG3

CD28:

cDNA coding for the transmembrane and intracellular part of CD28 (153–220 aa)

CD3ζ:

cDNA coding for the intracellular domain of CD3ζ (52–164 aa)

ELISA:

Enzyme-linked immunosorbant assay

IgG:

Immunoglobulin G

LB:

Luria-Bertani

MW:

Molecular weight

Nb:

Nanobody

PBS:

Phosphate buffered saline

pFKC8:

pC4-MFv2E plasmid containing myristoylation signal-modified FKBP12-modified FKBP12-caspase 8 cassette

pZCHHN:

CD3ζ-CD28-(CH2-CH3-hinge–hinge)-Nb cassette inserted in pCDNA3.1/Hygro(+) plasmid

pZCHN:

CD3ζ- CD28-(CH2-CH3-hinge)-Nb cassette inserted in pCDNA3.1/Hygro(+) plasmid

pZOCHHN:

CD3ζ-OX40-CD28-(CH2-CH3-hinge–hinge)-Nb cassette inserted in pCDNA3.1/Hygro(+) plasmid

pZOCHN:

CD3ζ-OX40-CD28-(CH2-CH3-hinge)-Nb cassette inserted in pCDNA3.1/Hygro(+) plasmid

scFv:

Single-chain fragment variable

TAA:

Tumour-associated antigen

VHH:

Variable domain from a camel heavy chain antibody

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Acknowledgments

We are grateful to Dr. Oliver Jey Broom (KIPA—Krahbichler Intellectual Property Advisors AB, SE-251 10 Helsingborg, Sweden) for his critical language revision of the manuscript. This work was supported by Faculty of Medical Sciences and the Biotechnology committee of Tarbiat Modares University (TMU-88-8-67), Tehran, Iran.

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Authors and Affiliations

  1. Department of Medical Biotechnology, School of Medical Sciences, Tarbiat Modares University, P.O. Box: 14115-331, Tehran, Iran

    Sepideh Khaleghi, Fatemeh Rahbarizadeh & Mohammad J. Rasaee

  2. School of Allied Medical Sciences, Tehran University of Medical Sciences, Tehran, Iran

    Davoud Ahmadvand

  3. CNRS UMR6235, Faculté de Médecine, Université de Nice, 28 avenue de Valombrose, 06107, Nice Cedex 2, France

    Philippe Pognonec

Authors
  1. Sepideh Khaleghi
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  2. Fatemeh Rahbarizadeh
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  3. Davoud Ahmadvand
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  4. Mohammad J. Rasaee
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  5. Philippe Pognonec
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Correspondence to Fatemeh Rahbarizadeh.

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Khaleghi, S., Rahbarizadeh, F., Ahmadvand, D. et al. A caspase 8-based suicide switch induces apoptosis in nanobody-directed chimeric receptor expressing T cells. Int J Hematol 95, 434–444 (2012). https://doi.org/10.1007/s12185-012-1037-6

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  • DOI: https://doi.org/10.1007/s12185-012-1037-6

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