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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

Copy number of chimeric receptors in Jurkat cell lines evaluated by PCR.The intensity of the PCR products from Jurkat cell lines expressing chimeric receptors was compared to standard copy number of parental Jurkat genomic DNA spiked with 1–16 copies of a CAR by (a) PCR and quantified using (b) Metamorph software. Shown is a representative experiment from 3 experiments performed.
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pone-0063037-g003: Copy number of chimeric receptors in Jurkat cell lines evaluated by PCR.The intensity of the PCR products from Jurkat cell lines expressing chimeric receptors was compared to standard copy number of parental Jurkat genomic DNA spiked with 1–16 copies of a CAR by (a) PCR and quantified using (b) Metamorph software. Shown is a representative experiment from 3 experiments performed.

Mentions: Retroviral biology dictates that a single virus integrates one provirus into the host cell genome, but to gain further insight into the viral copy number in the Jurkat clones, and determine if our sequencing strategy had missed some inserts, we isolated genomic DNA and performed quantitative PCR as described in Materials and Methods. The copy number for Jurkat clone 32, bearing the DAP10ζCD27 CAR, was 1.0, indicating that was the only CAR present (Figure 3). Jurkats with the CD28ζ CAR had a copy number of 1.5, suggesting 1 to 2 viral copies per cell. Clone 21 had 3.8 virus copies (most likely 4), which agreed with the number found by sequencing, indicating we had not missed other integrants. Clones 33 and 43 that had 2 CARs each by sequencing, were found to have 1.6 and 2.4 viral copies by PCR (most likely 2), suggesting the sequenced CARs were the only ones present. Interestingly, we did not observe a correlation between viral copy number and the level of CAR expression by Jurkat clones (Fig. 2d).


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)

Copy number of chimeric receptors in Jurkat cell lines evaluated by PCR.The intensity of the PCR products from Jurkat cell lines expressing chimeric receptors was compared to standard copy number of parental Jurkat genomic DNA spiked with 1–16 copies of a CAR by (a) PCR and quantified using (b) Metamorph software. Shown is a representative experiment from 3 experiments performed.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0063037-g003: Copy number of chimeric receptors in Jurkat cell lines evaluated by PCR.The intensity of the PCR products from Jurkat cell lines expressing chimeric receptors was compared to standard copy number of parental Jurkat genomic DNA spiked with 1–16 copies of a CAR by (a) PCR and quantified using (b) Metamorph software. Shown is a representative experiment from 3 experiments performed.
Mentions: Retroviral biology dictates that a single virus integrates one provirus into the host cell genome, but to gain further insight into the viral copy number in the Jurkat clones, and determine if our sequencing strategy had missed some inserts, we isolated genomic DNA and performed quantitative PCR as described in Materials and Methods. The copy number for Jurkat clone 32, bearing the DAP10ζCD27 CAR, was 1.0, indicating that was the only CAR present (Figure 3). Jurkats with the CD28ζ CAR had a copy number of 1.5, suggesting 1 to 2 viral copies per cell. Clone 21 had 3.8 virus copies (most likely 4), which agreed with the number found by sequencing, indicating we had not missed other integrants. Clones 33 and 43 that had 2 CARs each by sequencing, were found to have 1.6 and 2.4 viral copies by PCR (most likely 2), suggesting the sequenced CARs were the only ones present. Interestingly, we did not observe a correlation between viral copy number and the level of CAR expression by Jurkat clones (Fig. 2d).

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