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Lymphocyte display: a novel antibody selection platform based on T cell activation.

Alonso-Camino V, Sánchez-Martín D, Compte M, Sanz L, Alvarez-Vallina L - PLoS ONE (2009)

Bottom Line: Since their onset, display technologies have proven useful for the selection of antibodies against a variety of targets; however, most of the antibodies selected with the currently available platforms need to be further modified for their use in humans, and are restricted to accessible antigens.Furthermore, these platforms are not well suited for in vivo selections.Based on an effective interaction between the CIR and the tumor antigen, we demonstrated that combining CIR-mediated activation with FACS sorting of CD69(+) T cells, it is possible to isolate binders to tumor specific cell surface antigen, with an enrichment factor of at least 10(3)-fold after two rounds, resulting in a homogeneous population of T cells expressing tumor-specific CIRs.

View Article: PubMed Central - PubMed

Affiliation: Molecular Immunology Unit, Hospital Universitario Puerta de Hierro, Madrid, Spain.

ABSTRACT
Since their onset, display technologies have proven useful for the selection of antibodies against a variety of targets; however, most of the antibodies selected with the currently available platforms need to be further modified for their use in humans, and are restricted to accessible antigens. Furthermore, these platforms are not well suited for in vivo selections. We present here a novel cell based antibody display platform, which takes advantage of the functional capabilities of T lymphocytes. The display of antibodies on the surface of T lymphocytes, as a part of a chimeric-immune receptor (CIR) mediating signaling, may ideally link the antigen-antibody interaction to a demonstrable change in T cell phenotype, due to subsequent expression of the early T cell activation marker CD69. In this proof-of-concept, an in vitro selection was carried out using a human T cell line lentiviral-transduced to express a tumor-specific CIR on the surface, against a human tumor cell line expressing the carcinoembryonic antigen. Based on an effective interaction between the CIR and the tumor antigen, we demonstrated that combining CIR-mediated activation with FACS sorting of CD69(+) T cells, it is possible to isolate binders to tumor specific cell surface antigen, with an enrichment factor of at least 10(3)-fold after two rounds, resulting in a homogeneous population of T cells expressing tumor-specific CIRs.

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Schematic representation of lentiviral vector constructs.(A) Control monocistronic vector (pRRL-IRES-EGFP) containing only the enhanced-green fluorescent protein (EGFP) gene and bicistronic vector (pRRL-αCEA-CIR-IRES-EGFP) containing the chimeric immune receptor (CIR) gene and the EGFP sequence. LTR, long terminal repeats; ΔGAG, ATG-deleted group specific antigen; RRE, Rev-responsive cis-acting element; CMV promoter; ECMV IRES, enceohalomyocarditis virus internal ribosomal entry site. (B) FACS analysis of EGFP expression after transduction of Jurkat cells with EGFP encoding lentiviral vectors at different MOIs, ranging from 0.25 to 10. (C) FACS analysis of EGFP and cell surface CIR expression after transduction of Jurkat cells with CIR-EGFP encoding lentiviral vectors at MOI of 1.
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pone-0007174-g001: Schematic representation of lentiviral vector constructs.(A) Control monocistronic vector (pRRL-IRES-EGFP) containing only the enhanced-green fluorescent protein (EGFP) gene and bicistronic vector (pRRL-αCEA-CIR-IRES-EGFP) containing the chimeric immune receptor (CIR) gene and the EGFP sequence. LTR, long terminal repeats; ΔGAG, ATG-deleted group specific antigen; RRE, Rev-responsive cis-acting element; CMV promoter; ECMV IRES, enceohalomyocarditis virus internal ribosomal entry site. (B) FACS analysis of EGFP expression after transduction of Jurkat cells with EGFP encoding lentiviral vectors at different MOIs, ranging from 0.25 to 10. (C) FACS analysis of EGFP and cell surface CIR expression after transduction of Jurkat cells with CIR-EGFP encoding lentiviral vectors at MOI of 1.

Mentions: In order to generate a model for validating a selection platform based on molecular and cellular events associated with T cell activation, we constructed the HIV-1 based lentiviral vector pRRL-αCEA-CIR-IRES-EGFP (Fig. 1A), containing a bicistronic expression cassette encoding a TCRζ-based CIR and EGFP, as reporter gene. The CIR comprises a human immunoglobulin signal peptide, a FLAG epitope, and the anti-carcinoembrionyc antigen (CEA) scFv antibody MFE23 [15], fused to the transmembrane and cytoplasmic regions of the human TCRζ-chain [16].


Lymphocyte display: a novel antibody selection platform based on T cell activation.

Alonso-Camino V, Sánchez-Martín D, Compte M, Sanz L, Alvarez-Vallina L - PLoS ONE (2009)

Schematic representation of lentiviral vector constructs.(A) Control monocistronic vector (pRRL-IRES-EGFP) containing only the enhanced-green fluorescent protein (EGFP) gene and bicistronic vector (pRRL-αCEA-CIR-IRES-EGFP) containing the chimeric immune receptor (CIR) gene and the EGFP sequence. LTR, long terminal repeats; ΔGAG, ATG-deleted group specific antigen; RRE, Rev-responsive cis-acting element; CMV promoter; ECMV IRES, enceohalomyocarditis virus internal ribosomal entry site. (B) FACS analysis of EGFP expression after transduction of Jurkat cells with EGFP encoding lentiviral vectors at different MOIs, ranging from 0.25 to 10. (C) FACS analysis of EGFP and cell surface CIR expression after transduction of Jurkat cells with CIR-EGFP encoding lentiviral vectors at MOI of 1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2747005&req=5

pone-0007174-g001: Schematic representation of lentiviral vector constructs.(A) Control monocistronic vector (pRRL-IRES-EGFP) containing only the enhanced-green fluorescent protein (EGFP) gene and bicistronic vector (pRRL-αCEA-CIR-IRES-EGFP) containing the chimeric immune receptor (CIR) gene and the EGFP sequence. LTR, long terminal repeats; ΔGAG, ATG-deleted group specific antigen; RRE, Rev-responsive cis-acting element; CMV promoter; ECMV IRES, enceohalomyocarditis virus internal ribosomal entry site. (B) FACS analysis of EGFP expression after transduction of Jurkat cells with EGFP encoding lentiviral vectors at different MOIs, ranging from 0.25 to 10. (C) FACS analysis of EGFP and cell surface CIR expression after transduction of Jurkat cells with CIR-EGFP encoding lentiviral vectors at MOI of 1.
Mentions: In order to generate a model for validating a selection platform based on molecular and cellular events associated with T cell activation, we constructed the HIV-1 based lentiviral vector pRRL-αCEA-CIR-IRES-EGFP (Fig. 1A), containing a bicistronic expression cassette encoding a TCRζ-based CIR and EGFP, as reporter gene. The CIR comprises a human immunoglobulin signal peptide, a FLAG epitope, and the anti-carcinoembrionyc antigen (CEA) scFv antibody MFE23 [15], fused to the transmembrane and cytoplasmic regions of the human TCRζ-chain [16].

Bottom Line: Since their onset, display technologies have proven useful for the selection of antibodies against a variety of targets; however, most of the antibodies selected with the currently available platforms need to be further modified for their use in humans, and are restricted to accessible antigens.Furthermore, these platforms are not well suited for in vivo selections.Based on an effective interaction between the CIR and the tumor antigen, we demonstrated that combining CIR-mediated activation with FACS sorting of CD69(+) T cells, it is possible to isolate binders to tumor specific cell surface antigen, with an enrichment factor of at least 10(3)-fold after two rounds, resulting in a homogeneous population of T cells expressing tumor-specific CIRs.

View Article: PubMed Central - PubMed

Affiliation: Molecular Immunology Unit, Hospital Universitario Puerta de Hierro, Madrid, Spain.

ABSTRACT
Since their onset, display technologies have proven useful for the selection of antibodies against a variety of targets; however, most of the antibodies selected with the currently available platforms need to be further modified for their use in humans, and are restricted to accessible antigens. Furthermore, these platforms are not well suited for in vivo selections. We present here a novel cell based antibody display platform, which takes advantage of the functional capabilities of T lymphocytes. The display of antibodies on the surface of T lymphocytes, as a part of a chimeric-immune receptor (CIR) mediating signaling, may ideally link the antigen-antibody interaction to a demonstrable change in T cell phenotype, due to subsequent expression of the early T cell activation marker CD69. In this proof-of-concept, an in vitro selection was carried out using a human T cell line lentiviral-transduced to express a tumor-specific CIR on the surface, against a human tumor cell line expressing the carcinoembryonic antigen. Based on an effective interaction between the CIR and the tumor antigen, we demonstrated that combining CIR-mediated activation with FACS sorting of CD69(+) T cells, it is possible to isolate binders to tumor specific cell surface antigen, with an enrichment factor of at least 10(3)-fold after two rounds, resulting in a homogeneous population of T cells expressing tumor-specific CIRs.

Show MeSH
Related in: MedlinePlus