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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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Human T cells bearing the αerbB2DAP10ζCD27 CAR inhibit tumor growth in mice.(a) CAR-expressing human T cells were injected on days 0, 1 and 2 intravenously into NOD-SCID mice injected subcutaneously on day 0 with 24JK-erbB2 sarcomas, and tumor growth monitored (9–10 mice/group). (b,c) Localization of T cells was determined on dissociated tumor and tissues on day 4 of tumor growth using flow cytometry (5 mice/group). (Error bars = SEM). *p<0.05, **p<0.005, NS = not significant.
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pone-0063037-g005: Human T cells bearing the αerbB2DAP10ζCD27 CAR inhibit tumor growth in mice.(a) CAR-expressing human T cells were injected on days 0, 1 and 2 intravenously into NOD-SCID mice injected subcutaneously on day 0 with 24JK-erbB2 sarcomas, and tumor growth monitored (9–10 mice/group). (b,c) Localization of T cells was determined on dissociated tumor and tissues on day 4 of tumor growth using flow cytometry (5 mice/group). (Error bars = SEM). *p<0.05, **p<0.005, NS = not significant.

Mentions: In addition to comparisons of T cell function in vitro, their relative ability to function in vivo is also of interest, which may reveal differences in anti-tumor ability or their capacity to persist or accumulate in tumors. To determine this we injected the erbB2+ cell line 24JK-erbB2 subcutaneously in NOD-SCID mice and transferred CAR-bearing T cells or control T cells intravenously. T cells bearing the DAP10ζCD27 CAR inhibited tumor growth to a greater degree than T cells expressing the CD28ζ CAR (Fig. 5a). The relative persistence of CAR T cells was determined by flow cytometric analysis of spleens, lung, blood and tumor four days after tumor cell injection, with T cells injected intravenously on days 0, 1 and 2. Localization of T cells to tumors was found to be comparable for the two CAR T cell populations (Fig. 5b). Interestingly, the frequency of tumor infiltrating human lymphocytes was very low in all groups of mice, perhaps highlighting the importance of an increased T cell cytolytic capacity when only low numbers of T cells localize to tumors. T cell persistence in the lungs and spleen was observed to be similar for both CAR-bearing populations (Fig. 5c). However, CAR-bearing T cells were present at significantly higher frequencies in the blood compared to control vector transduced T cells, and T cells expressing CD28ζ were present at a higher frequency than T cells expressing DAP10ζCD27. These data demonstrated the ability of a CAR with a newly identified combination of signaling moieties to endow T cells with the ability to inhibit tumor growth in mice. These findings support the crucial role played by cytotoxic lymphocytes in eradicating tumors in numerous animal models of disease.


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)

Human T cells bearing the αerbB2DAP10ζCD27 CAR inhibit tumor growth in mice.(a) CAR-expressing human T cells were injected on days 0, 1 and 2 intravenously into NOD-SCID mice injected subcutaneously on day 0 with 24JK-erbB2 sarcomas, and tumor growth monitored (9–10 mice/group). (b,c) Localization of T cells was determined on dissociated tumor and tissues on day 4 of tumor growth using flow cytometry (5 mice/group). (Error bars = SEM). *p<0.05, **p<0.005, NS = not significant.
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Related In: Results  -  Collection

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

pone-0063037-g005: Human T cells bearing the αerbB2DAP10ζCD27 CAR inhibit tumor growth in mice.(a) CAR-expressing human T cells were injected on days 0, 1 and 2 intravenously into NOD-SCID mice injected subcutaneously on day 0 with 24JK-erbB2 sarcomas, and tumor growth monitored (9–10 mice/group). (b,c) Localization of T cells was determined on dissociated tumor and tissues on day 4 of tumor growth using flow cytometry (5 mice/group). (Error bars = SEM). *p<0.05, **p<0.005, NS = not significant.
Mentions: In addition to comparisons of T cell function in vitro, their relative ability to function in vivo is also of interest, which may reveal differences in anti-tumor ability or their capacity to persist or accumulate in tumors. To determine this we injected the erbB2+ cell line 24JK-erbB2 subcutaneously in NOD-SCID mice and transferred CAR-bearing T cells or control T cells intravenously. T cells bearing the DAP10ζCD27 CAR inhibited tumor growth to a greater degree than T cells expressing the CD28ζ CAR (Fig. 5a). The relative persistence of CAR T cells was determined by flow cytometric analysis of spleens, lung, blood and tumor four days after tumor cell injection, with T cells injected intravenously on days 0, 1 and 2. Localization of T cells to tumors was found to be comparable for the two CAR T cell populations (Fig. 5b). Interestingly, the frequency of tumor infiltrating human lymphocytes was very low in all groups of mice, perhaps highlighting the importance of an increased T cell cytolytic capacity when only low numbers of T cells localize to tumors. T cell persistence in the lungs and spleen was observed to be similar for both CAR-bearing populations (Fig. 5c). However, CAR-bearing T cells were present at significantly higher frequencies in the blood compared to control vector transduced T cells, and T cells expressing CD28ζ were present at a higher frequency than T cells expressing DAP10ζCD27. These data demonstrated the ability of a CAR with a newly identified combination of signaling moieties to endow T cells with the ability to inhibit tumor growth in mice. These findings support the crucial role played by cytotoxic lymphocytes in eradicating tumors in numerous animal models of disease.

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