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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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Novel chimeric receptors can enable Jurkat cells to respond following receptor ligation.(a) Populations of the human T cell line Jurkat (as listed) were incubated with anti-c-myc antibody and the expression of the activation marker CD69 determined. The boxed region represents cells with significant upregulation of CD69. Cells from the library-transduced population were subjected to fluorescence activated cell sorting and cells within the region cloned. CD69 upregulation in response to a positive control, phorbol myristate acetate (PMA) and phytohemagglutinin (PHA) is also depicted, together with upregulation in cells expressing a known active CAR, αerbB2CD28ζ. (b) Four clones were screened for their ability to secrete IL-2 in response to CAR ligation by erbB2 antigen, compared to the known positive control αerbB2CD28ζ. (c) IL-2 secretion from clones following incubation with anti-c-myc antibody (3 experiments pooled). (d) Expression of CARs in Jurkat clones was determined by flow cytometry following staining with anti-c-myc antibody. Four clones and the positive control Jurkat-erbB2CD28ζ are listed. (Error bars = SEM). *p<0.05, **p<0.005.
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pone-0063037-g002: Novel chimeric receptors can enable Jurkat cells to respond following receptor ligation.(a) Populations of the human T cell line Jurkat (as listed) were incubated with anti-c-myc antibody and the expression of the activation marker CD69 determined. The boxed region represents cells with significant upregulation of CD69. Cells from the library-transduced population were subjected to fluorescence activated cell sorting and cells within the region cloned. CD69 upregulation in response to a positive control, phorbol myristate acetate (PMA) and phytohemagglutinin (PHA) is also depicted, together with upregulation in cells expressing a known active CAR, αerbB2CD28ζ. (b) Four clones were screened for their ability to secrete IL-2 in response to CAR ligation by erbB2 antigen, compared to the known positive control αerbB2CD28ζ. (c) IL-2 secretion from clones following incubation with anti-c-myc antibody (3 experiments pooled). (d) Expression of CARs in Jurkat clones was determined by flow cytometry following staining with anti-c-myc antibody. Four clones and the positive control Jurkat-erbB2CD28ζ are listed. (Error bars = SEM). *p<0.05, **p<0.005.

Mentions: We anticipated that the CAR library would contain many non-functional CAR, since only relatively rare events would place stimulatory, costimulatory and/or adaptor molecules of suitable activity in the correct order to trigger T cell function. We wished to select highly functional clones that we could compare with the same anti-erbB2 scFv with our best signaling chain (anti-erbB2-CD28-CD3ζ). We could have screened the library directly into primary T cells, but these are varied and heterogenous and comparing receptors among clones from this population would have been difficult. We therefore screened the library of receptors for their ability to activate the human T cell line, Jurkat, in which all cells are genetically similar and so comparisons could be made between different CARs transduced into this line. After transducing the bulk population of Jurkat cells using the retroviral library, screening was performed by incubating the cell line in culture dishes coated with an antibody specific for the c-myc tag present in the extracellular domain of each CAR (Fig. 1a). In this way, we anticipated identifying CAR formats that could respond to molecular crosslinking producing synapses reminiscent of normal T cell signaling processes. Jurkat cells do not secrete all the repertoire of cytokines as naïve T cells, for instance they do not secrete IFNγ, but they do upregulate CD69 expression which has previously been determined to be a marker of activation [16], [17], and they secrete IL-2, so these two screening options were available for selecting functional clones from the Jurkat cells transduced with our library of CARs. Therefore, following incubation with anti-c-myc Ab, Jurkat cells were stained for CD69 expression. Flow cytometric analysis was then performed, and cells with CD69 upregulated above that of parental (non-transduced) Jurkat cells were isolated and cloned into 96-well culture plates to yield a total of 147 clones (Fig. 2a). These clones were then individually re-screened for CD69 upregulation in response to CAR ligation, and 39 were confirmed as responders. The 21 highest CD69-expressors were screened for IL-2 secretion in response to anti-c-myc antibody, and 11 were demonstrated to produce IL-2. The 4 highest IL-2 secretor clones identified in this initial screen were then selected for further analysis and cultured with anti-c-myc antibody or the sarcoma cell line 24JK transfected to express erbB2 or controls, and IL-2 secretion determined. In this screen, some clones produced levels of IL-2 comparable to, or better than our previously determined functionally optimal CAR, αerbB2-CD28ζ (Fig. 2b and 2c). These clones were also determined to express CARs to varying degrees (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)

Novel chimeric receptors can enable Jurkat cells to respond following receptor ligation.(a) Populations of the human T cell line Jurkat (as listed) were incubated with anti-c-myc antibody and the expression of the activation marker CD69 determined. The boxed region represents cells with significant upregulation of CD69. Cells from the library-transduced population were subjected to fluorescence activated cell sorting and cells within the region cloned. CD69 upregulation in response to a positive control, phorbol myristate acetate (PMA) and phytohemagglutinin (PHA) is also depicted, together with upregulation in cells expressing a known active CAR, αerbB2CD28ζ. (b) Four clones were screened for their ability to secrete IL-2 in response to CAR ligation by erbB2 antigen, compared to the known positive control αerbB2CD28ζ. (c) IL-2 secretion from clones following incubation with anti-c-myc antibody (3 experiments pooled). (d) Expression of CARs in Jurkat clones was determined by flow cytometry following staining with anti-c-myc antibody. Four clones and the positive control Jurkat-erbB2CD28ζ are listed. (Error bars = SEM). *p<0.05, **p<0.005.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646939&req=5

pone-0063037-g002: Novel chimeric receptors can enable Jurkat cells to respond following receptor ligation.(a) Populations of the human T cell line Jurkat (as listed) were incubated with anti-c-myc antibody and the expression of the activation marker CD69 determined. The boxed region represents cells with significant upregulation of CD69. Cells from the library-transduced population were subjected to fluorescence activated cell sorting and cells within the region cloned. CD69 upregulation in response to a positive control, phorbol myristate acetate (PMA) and phytohemagglutinin (PHA) is also depicted, together with upregulation in cells expressing a known active CAR, αerbB2CD28ζ. (b) Four clones were screened for their ability to secrete IL-2 in response to CAR ligation by erbB2 antigen, compared to the known positive control αerbB2CD28ζ. (c) IL-2 secretion from clones following incubation with anti-c-myc antibody (3 experiments pooled). (d) Expression of CARs in Jurkat clones was determined by flow cytometry following staining with anti-c-myc antibody. Four clones and the positive control Jurkat-erbB2CD28ζ are listed. (Error bars = SEM). *p<0.05, **p<0.005.
Mentions: We anticipated that the CAR library would contain many non-functional CAR, since only relatively rare events would place stimulatory, costimulatory and/or adaptor molecules of suitable activity in the correct order to trigger T cell function. We wished to select highly functional clones that we could compare with the same anti-erbB2 scFv with our best signaling chain (anti-erbB2-CD28-CD3ζ). We could have screened the library directly into primary T cells, but these are varied and heterogenous and comparing receptors among clones from this population would have been difficult. We therefore screened the library of receptors for their ability to activate the human T cell line, Jurkat, in which all cells are genetically similar and so comparisons could be made between different CARs transduced into this line. After transducing the bulk population of Jurkat cells using the retroviral library, screening was performed by incubating the cell line in culture dishes coated with an antibody specific for the c-myc tag present in the extracellular domain of each CAR (Fig. 1a). In this way, we anticipated identifying CAR formats that could respond to molecular crosslinking producing synapses reminiscent of normal T cell signaling processes. Jurkat cells do not secrete all the repertoire of cytokines as naïve T cells, for instance they do not secrete IFNγ, but they do upregulate CD69 expression which has previously been determined to be a marker of activation [16], [17], and they secrete IL-2, so these two screening options were available for selecting functional clones from the Jurkat cells transduced with our library of CARs. Therefore, following incubation with anti-c-myc Ab, Jurkat cells were stained for CD69 expression. Flow cytometric analysis was then performed, and cells with CD69 upregulated above that of parental (non-transduced) Jurkat cells were isolated and cloned into 96-well culture plates to yield a total of 147 clones (Fig. 2a). These clones were then individually re-screened for CD69 upregulation in response to CAR ligation, and 39 were confirmed as responders. The 21 highest CD69-expressors were screened for IL-2 secretion in response to anti-c-myc antibody, and 11 were demonstrated to produce IL-2. The 4 highest IL-2 secretor clones identified in this initial screen were then selected for further analysis and cultured with anti-c-myc antibody or the sarcoma cell line 24JK transfected to express erbB2 or controls, and IL-2 secretion determined. In this screen, some clones produced levels of IL-2 comparable to, or better than our previously determined functionally optimal CAR, αerbB2-CD28ζ (Fig. 2b and 2c). These clones were also determined to express CARs to varying degrees (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