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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) and NIP-modified molecules on HeLa, HeLaCEA and HeLa cells labeled with 2.5 µg/mL of the hapten (HeLaNIP). (B) FACS analysis of CD69 expression by JurkatEGFP, JurkatαCEA-CIR-EGFP and JurkatαNIP-CIR stimulated either with immobilized anti-CD3 mAb or target cells (E∶T = 1∶1; HeLa, HeLaCEA or HeLaNIP) for 16 hours.
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pone-0007174-g003: CIR-mediated activation of human T cells.(A) Cell-surface expression of CEACAM5 (CEA) and NIP-modified molecules on HeLa, HeLaCEA and HeLa cells labeled with 2.5 µg/mL of the hapten (HeLaNIP). (B) FACS analysis of CD69 expression by JurkatEGFP, JurkatαCEA-CIR-EGFP and JurkatαNIP-CIR stimulated either with immobilized anti-CD3 mAb or target cells (E∶T = 1∶1; HeLa, HeLaCEA or HeLaNIP) for 16 hours.

Mentions: We then examinated the activity of the FLAG-tagged anti-CEA CIR as a functional receptor molecule. JurkatαCEA-CIR-EGFP cells could be activated to secrete IL-2, upon antigen-mediated ligation of their binding domain by plastic immobilized CEA (iCEA) or membrane-bound CEA (mCEA) (Fig. 2). We next investigated the expression of the very early activation antigen CD69 in JurkatαCEA-CIR-EGFP and JurkatEGFP cells co-cultured overnight in the presence of CEA-negative (HeLa) or CEA-positive (HeLaCEA) cells (Fig. 3A). The results of a representative experiment are shown in Figure 3B. On average CD69+ cells accounted for 40±10% of JurkatαCEA-CIR-EGFP cells stimulated with CEA-positive cells. This result is similar to that observed after stimulation of JurkatαCEA-CIR-EGFP cells with plastic immobilized anti-CD3 mAb (Fig. 3B). In these conditions the peak expression of cell surface CD69 occurred between 12 and 24 h (data not shown). Stimulation of JurkatαCEA-CIR-EGFP and JurkatEGFP cells with plastic immobilized anti-CD3 mAb or stimulation of JurkatαCEA-CIR-EGFP cells with HeLaCEA cells resulted in a marked increase in EGFP reporter expression as compared to unstimulated 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) and NIP-modified molecules on HeLa, HeLaCEA and HeLa cells labeled with 2.5 µg/mL of the hapten (HeLaNIP). (B) FACS analysis of CD69 expression by JurkatEGFP, JurkatαCEA-CIR-EGFP and JurkatαNIP-CIR stimulated either with immobilized anti-CD3 mAb or target cells (E∶T = 1∶1; HeLa, HeLaCEA or HeLaNIP) for 16 hours.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0007174-g003: CIR-mediated activation of human T cells.(A) Cell-surface expression of CEACAM5 (CEA) and NIP-modified molecules on HeLa, HeLaCEA and HeLa cells labeled with 2.5 µg/mL of the hapten (HeLaNIP). (B) FACS analysis of CD69 expression by JurkatEGFP, JurkatαCEA-CIR-EGFP and JurkatαNIP-CIR stimulated either with immobilized anti-CD3 mAb or target cells (E∶T = 1∶1; HeLa, HeLaCEA or HeLaNIP) for 16 hours.
Mentions: We then examinated the activity of the FLAG-tagged anti-CEA CIR as a functional receptor molecule. JurkatαCEA-CIR-EGFP cells could be activated to secrete IL-2, upon antigen-mediated ligation of their binding domain by plastic immobilized CEA (iCEA) or membrane-bound CEA (mCEA) (Fig. 2). We next investigated the expression of the very early activation antigen CD69 in JurkatαCEA-CIR-EGFP and JurkatEGFP cells co-cultured overnight in the presence of CEA-negative (HeLa) or CEA-positive (HeLaCEA) cells (Fig. 3A). The results of a representative experiment are shown in Figure 3B. On average CD69+ cells accounted for 40±10% of JurkatαCEA-CIR-EGFP cells stimulated with CEA-positive cells. This result is similar to that observed after stimulation of JurkatαCEA-CIR-EGFP cells with plastic immobilized anti-CD3 mAb (Fig. 3B). In these conditions the peak expression of cell surface CD69 occurred between 12 and 24 h (data not shown). Stimulation of JurkatαCEA-CIR-EGFP and JurkatEGFP cells with plastic immobilized anti-CD3 mAb or stimulation of JurkatαCEA-CIR-EGFP cells with HeLaCEA cells resulted in a marked increase in EGFP reporter expression as compared to unstimulated 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