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Engineering T cell function using chimeric antigen receptors identified using a DNA library approach.

Duong CP, Westwood JA, Yong CS, Murphy A, Devaud C, John LB, Darcy PK, Kershaw MH - PLoS ONE (2013)

Bottom Line: We used a directional in-frame library of randomly assembled signaling domains in a CAR specific for the tumor associated antigen erbB2.Several new and original CARs were identified, one of which had an enhanced ability to lyse cancer cells and inhibit tumor growth in mice.This study highlights novel technology that could be used to screen a variety of molecules for their capacity to induce diverse functions in cells.

View Article: PubMed Central - PubMed

Affiliation: Cancer Immunology Research Program, Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria, Australia.

ABSTRACT
Genetic engineering of cellular function holds much promise for the treatment of a variety of diseases including gene deficiencies and cancer. However, engineering the full complement of cellular functions can be a daunting genetic exercise since many molecular triggers need to be activated to achieve complete function. In the case of T cells, genes encoding chimeric antigen receptors (CARs) covalently linking antibodies to cytoplasmic signaling domains can trigger some, but not all, cellular functions against cancer cells. To date, relatively few CAR formats have been investigated using a candidate molecule approach, in which rationally chosen molecules were trialed one by one. Therefore, to expedite this arduous process we developed an innovative screening method to screen many thousands of CAR formats to identify genes able to enhance the anticancer ability of T cells. We used a directional in-frame library of randomly assembled signaling domains in a CAR specific for the tumor associated antigen erbB2. Several new and original CARs were identified, one of which had an enhanced ability to lyse cancer cells and inhibit tumor growth in mice. This study highlights novel technology that could be used to screen a variety of molecules for their capacity to induce diverse functions in cells.

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Related in: MedlinePlus

The chimeric cDNA library is diverse in size and composition.(a) The genetic construct is depicted encoding extracellular single chain anti-erbB2 linked via a c-myc tag and the hinge region from CD8 to the transmembrane domain of CD28. DNA encoding various signaling molecules was randomly ligated into the SfiI site. (b) Plasmids of 20 representative clones were digested with NotI and XhoI and analyzed by agarose gel electrophoresis. (c) DNA from the 20 clones was sequenced and represented as shown (not to scale). Corresponding clone numbers and weights from panel (b) are indicated. Break indicates unsequenced portions.
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pone-0063037-g001: The chimeric cDNA library is diverse in size and composition.(a) The genetic construct is depicted encoding extracellular single chain anti-erbB2 linked via a c-myc tag and the hinge region from CD8 to the transmembrane domain of CD28. DNA encoding various signaling molecules was randomly ligated into the SfiI site. (b) Plasmids of 20 representative clones were digested with NotI and XhoI and analyzed by agarose gel electrophoresis. (c) DNA from the 20 clones was sequenced and represented as shown (not to scale). Corresponding clone numbers and weights from panel (b) are indicated. Break indicates unsequenced portions.

Mentions: Fourteen signaling molecules (Table 1) were ligated in random number and order into the SfiI site of the pSAMEN-anti-erbB2 plasmid (Fig. 1a). The molecules were chosen as representative of primary stimulators, costimulators and adaptors, and although more signaling molecules are present in T cells, the number was kept at 14 for this initial proof of principle study. This generated a library of tumor targeting receptors, which was used to transform E. coli. We first investigated the diversity of the library using restriction enzyme digests of individual clones from E. coli. Using NotI and XhoI that cleave 5′ to the extracellular portion and 3′ to the cytoplasmic portion of the CAR respectively, a diverse range of sizes of molecules were revealed on agarose gel (Fig. 1b). The sizes of CARs varied from 1.1 kb, the size of the empty vector with no cytoplasmic inserts, to >4 kb, indicating a diverse variety of inserts.


Engineering T cell function using chimeric antigen receptors identified using a DNA library approach.

Duong CP, Westwood JA, Yong CS, Murphy A, Devaud C, John LB, Darcy PK, Kershaw MH - PLoS ONE (2013)

The chimeric cDNA library is diverse in size and composition.(a) The genetic construct is depicted encoding extracellular single chain anti-erbB2 linked via a c-myc tag and the hinge region from CD8 to the transmembrane domain of CD28. DNA encoding various signaling molecules was randomly ligated into the SfiI site. (b) Plasmids of 20 representative clones were digested with NotI and XhoI and analyzed by agarose gel electrophoresis. (c) DNA from the 20 clones was sequenced and represented as shown (not to scale). Corresponding clone numbers and weights from panel (b) are indicated. Break indicates unsequenced portions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646939&req=5

pone-0063037-g001: The chimeric cDNA library is diverse in size and composition.(a) The genetic construct is depicted encoding extracellular single chain anti-erbB2 linked via a c-myc tag and the hinge region from CD8 to the transmembrane domain of CD28. DNA encoding various signaling molecules was randomly ligated into the SfiI site. (b) Plasmids of 20 representative clones were digested with NotI and XhoI and analyzed by agarose gel electrophoresis. (c) DNA from the 20 clones was sequenced and represented as shown (not to scale). Corresponding clone numbers and weights from panel (b) are indicated. Break indicates unsequenced portions.
Mentions: Fourteen signaling molecules (Table 1) were ligated in random number and order into the SfiI site of the pSAMEN-anti-erbB2 plasmid (Fig. 1a). The molecules were chosen as representative of primary stimulators, costimulators and adaptors, and although more signaling molecules are present in T cells, the number was kept at 14 for this initial proof of principle study. This generated a library of tumor targeting receptors, which was used to transform E. coli. We first investigated the diversity of the library using restriction enzyme digests of individual clones from E. coli. Using NotI and XhoI that cleave 5′ to the extracellular portion and 3′ to the cytoplasmic portion of the CAR respectively, a diverse range of sizes of molecules were revealed on agarose gel (Fig. 1b). The sizes of CARs varied from 1.1 kb, the size of the empty vector with no cytoplasmic inserts, to >4 kb, indicating a diverse variety of inserts.

Bottom Line: We used a directional in-frame library of randomly assembled signaling domains in a CAR specific for the tumor associated antigen erbB2.Several new and original CARs were identified, one of which had an enhanced ability to lyse cancer cells and inhibit tumor growth in mice.This study highlights novel technology that could be used to screen a variety of molecules for their capacity to induce diverse functions in cells.

View Article: PubMed Central - PubMed

Affiliation: Cancer Immunology Research Program, Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria, Australia.

ABSTRACT
Genetic engineering of cellular function holds much promise for the treatment of a variety of diseases including gene deficiencies and cancer. However, engineering the full complement of cellular functions can be a daunting genetic exercise since many molecular triggers need to be activated to achieve complete function. In the case of T cells, genes encoding chimeric antigen receptors (CARs) covalently linking antibodies to cytoplasmic signaling domains can trigger some, but not all, cellular functions against cancer cells. To date, relatively few CAR formats have been investigated using a candidate molecule approach, in which rationally chosen molecules were trialed one by one. Therefore, to expedite this arduous process we developed an innovative screening method to screen many thousands of CAR formats to identify genes able to enhance the anticancer ability of T cells. We used a directional in-frame library of randomly assembled signaling domains in a CAR specific for the tumor associated antigen erbB2. Several new and original CARs were identified, one of which had an enhanced ability to lyse cancer cells and inhibit tumor growth in mice. This study highlights novel technology that could be used to screen a variety of molecules for their capacity to induce diverse functions in cells.

Show MeSH
Related in: MedlinePlus