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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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CIR-mediated activation of human T cells.(A) Cell-surface expression of CEACAM5 (CEA) on HeLa, HT1080, MDA-MB-231 and MKN45 cells. (B) FACS analysis of CD69 expression by Jurkat, JurkatαCEA-CIR-EGFP and JurkatαNIP-CIR stimulated either with immobilized anti-CD3 mAb or target cells (E∶T = 1∶1; HeLa, HT1080, MDA-MB-231 or MKN45) for 16 hours.
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pone-0007174-g004: CIR-mediated activation of human T cells.(A) Cell-surface expression of CEACAM5 (CEA) on HeLa, HT1080, MDA-MB-231 and MKN45 cells. (B) FACS analysis of CD69 expression by Jurkat, JurkatαCEA-CIR-EGFP and JurkatαNIP-CIR stimulated either with immobilized anti-CD3 mAb or target cells (E∶T = 1∶1; HeLa, HT1080, MDA-MB-231 or MKN45) for 16 hours.

Mentions: To further demonstrated the specificity of CIR-mediated activation, CIR-negative parental Jurkat cells or CIR-positive Jurkat cells (JurkatαCEA-CIR-EGFP or JurkatαNIP-CIR) were co cultured with a panel of human unmodified tumor cell lines (Fig. 4). Only JurkatαCEA-CIR-EGFP cells expressed CD69 after co culturing with CEA-positive MKN45 cells (Fig. 4). Parental Jurkat cells or anti-NIP CIR-expressing Jurkat cells (JurkatαNIP-CIR) could not be activated to express CD69 with any of the tumor cell lines tested (Fig. 4). Furthermore, the viability of JurkatEGFP, JurkatαCEA-CIR-EGFP and JurkatαNIP-CIR cells was not affected when co-cultured for 16 hours in the presence of different types of target cells (data not shown).


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)

CIR-mediated activation of human T cells.(A) Cell-surface expression of CEACAM5 (CEA) on HeLa, HT1080, MDA-MB-231 and MKN45 cells. (B) FACS analysis of CD69 expression by Jurkat, JurkatαCEA-CIR-EGFP and JurkatαNIP-CIR stimulated either with immobilized anti-CD3 mAb or target cells (E∶T = 1∶1; HeLa, HT1080, MDA-MB-231 or MKN45) for 16 hours.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2747005&req=5

pone-0007174-g004: CIR-mediated activation of human T cells.(A) Cell-surface expression of CEACAM5 (CEA) on HeLa, HT1080, MDA-MB-231 and MKN45 cells. (B) FACS analysis of CD69 expression by Jurkat, JurkatαCEA-CIR-EGFP and JurkatαNIP-CIR stimulated either with immobilized anti-CD3 mAb or target cells (E∶T = 1∶1; HeLa, HT1080, MDA-MB-231 or MKN45) for 16 hours.
Mentions: To further demonstrated the specificity of CIR-mediated activation, CIR-negative parental Jurkat cells or CIR-positive Jurkat cells (JurkatαCEA-CIR-EGFP or JurkatαNIP-CIR) were co cultured with a panel of human unmodified tumor cell lines (Fig. 4). Only JurkatαCEA-CIR-EGFP cells expressed CD69 after co culturing with CEA-positive MKN45 cells (Fig. 4). Parental Jurkat cells or anti-NIP CIR-expressing Jurkat cells (JurkatαNIP-CIR) could not be activated to express CD69 with any of the tumor cell lines tested (Fig. 4). Furthermore, the viability of JurkatEGFP, JurkatαCEA-CIR-EGFP and JurkatαNIP-CIR cells was not affected when co-cultured for 16 hours in the presence of different types of target cells (data not shown).

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