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Biopolymer implants enhance the efficacy of adoptive T-cell therapy.

Stephan SB, Taber AM, Jileaeva I, Pegues EP, Sentman CL, Stephan MT - Nat. Biotechnol. (2014)

Bottom Line: Using a mouse breast cancer resection model, we show that the implants effectively support tumor-targeting T cells throughout resection beds and associated lymph nodes, and reduce tumor relapse compared to conventional delivery modalities.In a multifocal ovarian cancer model, we demonstrate that polymer-delivered T cells trigger regression, whereas injected tumor-reactive lymphocytes have little curative effect.Scaffold-based T-cell delivery may provide a viable treatment option for inoperable tumors and reduce the rate of metastatic relapse after surgery.

View Article: PubMed Central - PubMed

Affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

ABSTRACT
Although adoptive T-cell therapy holds promise for the treatment of many cancers, its clinical utility has been limited by problems in delivering targeted lymphocytes to tumor sites, and the cells' inefficient expansion in the immunosuppressive tumor microenvironment. Here we describe a bioactive polymer implant capable of delivering, expanding and dispersing tumor-reactive T cells. The approach can be used to treat inoperable or incompletely removed tumors by situating implants near them or at resection sites. Using a mouse breast cancer resection model, we show that the implants effectively support tumor-targeting T cells throughout resection beds and associated lymph nodes, and reduce tumor relapse compared to conventional delivery modalities. In a multifocal ovarian cancer model, we demonstrate that polymer-delivered T cells trigger regression, whereas injected tumor-reactive lymphocytes have little curative effect. Scaffold-based T-cell delivery may provide a viable treatment option for inoperable tumors and reduce the rate of metastatic relapse after surgery.

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Material-deployed T cells robustly expand in tumor tissue, where they reduce residual disease and relapse. (a–d) Biomaterial-supported T cell implants eradicate tumor cells left behind after surgery. Ten days after firefly luciferase-expressing 4T1 breast tumor cells were transplanted into mammary glands of BALB/c mice, tumors were resected such that ~1% of the tissue remained. All mice were treated with cyclophosphamide one day prior to T cell treatment. The first group of mice received a cell-free scaffold immediately following surgery as a control; all others were treated with 7×106 breast tumor-specific T cells. In group 2, the cells were injected intravenously. The next two groups were injected with T cells directly into the tumor bed, with or without pre-activation using anti-CD3/CD28/CD137 antibodies and IL-15/IL-15Rα. The last group received cells delivered via bioactive scaffolds that were implanted directly into the resection cavity. (a) Sequential bioluminescence imaging of the 4T1 breast tumors. (b) Kaplan-Meier survival curves for treated and control mice. Shown are ten mice per treatment group pooled from three independent experiments; ms, median survival. (c) Longitudinal in vivo bioluminescence imaging of breast tumor-specific T cells retrovirally transduced with click beetle red luciferase (CBR-luc). (d) Tumor-specific T cells labeled with CellTracker Orange were embedded in scaffolds that were fluorescently tagged with Hilyte Fluor 647 and implanted into the luciferase-expressing tumor resection cavity. The scaffold and surrounding tissue were excised and sectioned for histological analysis three days later. Scale bar: 100 μm. Data shown are 1 of 2 independent experiments. (e–h) Launching anti-tumor T lymphocytes from bioactive polymer implants controls inoperable cancer. ID8 ovarian carcinoma cells stably expressing VEGF were injected intraperitoneally and allowed to establish for eight weeks. Mice were given cyclophosphamide one day prior to treatment with lymphocytes expressing chimeric NKG2D receptors (NKG2D CARs) using the administration and stimulation protocols described above (except local injections were intraperitoneal instead of intra-resection cavity). (e) Serial in vivo bioluminescence imaging of ID8-VEGF-luc tumors. (f) Survival of animals following T cell therapy depicted as Kaplan-Meier curves. (g) In vivo bioluminescent imaging of T cells expressing CBR-luc. (h) CBR-luc signal intensities after T cell transfer. Every line represents one animal and each dot reflects the whole animal photon count. Pairwise differences in photon counts between treatment groups were analyzed with the Wilcoxon rank-sum test. Shown are data for ten mice per treatment condition.
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Figure 3: Material-deployed T cells robustly expand in tumor tissue, where they reduce residual disease and relapse. (a–d) Biomaterial-supported T cell implants eradicate tumor cells left behind after surgery. Ten days after firefly luciferase-expressing 4T1 breast tumor cells were transplanted into mammary glands of BALB/c mice, tumors were resected such that ~1% of the tissue remained. All mice were treated with cyclophosphamide one day prior to T cell treatment. The first group of mice received a cell-free scaffold immediately following surgery as a control; all others were treated with 7×106 breast tumor-specific T cells. In group 2, the cells were injected intravenously. The next two groups were injected with T cells directly into the tumor bed, with or without pre-activation using anti-CD3/CD28/CD137 antibodies and IL-15/IL-15Rα. The last group received cells delivered via bioactive scaffolds that were implanted directly into the resection cavity. (a) Sequential bioluminescence imaging of the 4T1 breast tumors. (b) Kaplan-Meier survival curves for treated and control mice. Shown are ten mice per treatment group pooled from three independent experiments; ms, median survival. (c) Longitudinal in vivo bioluminescence imaging of breast tumor-specific T cells retrovirally transduced with click beetle red luciferase (CBR-luc). (d) Tumor-specific T cells labeled with CellTracker Orange were embedded in scaffolds that were fluorescently tagged with Hilyte Fluor 647 and implanted into the luciferase-expressing tumor resection cavity. The scaffold and surrounding tissue were excised and sectioned for histological analysis three days later. Scale bar: 100 μm. Data shown are 1 of 2 independent experiments. (e–h) Launching anti-tumor T lymphocytes from bioactive polymer implants controls inoperable cancer. ID8 ovarian carcinoma cells stably expressing VEGF were injected intraperitoneally and allowed to establish for eight weeks. Mice were given cyclophosphamide one day prior to treatment with lymphocytes expressing chimeric NKG2D receptors (NKG2D CARs) using the administration and stimulation protocols described above (except local injections were intraperitoneal instead of intra-resection cavity). (e) Serial in vivo bioluminescence imaging of ID8-VEGF-luc tumors. (f) Survival of animals following T cell therapy depicted as Kaplan-Meier curves. (g) In vivo bioluminescent imaging of T cells expressing CBR-luc. (h) CBR-luc signal intensities after T cell transfer. Every line represents one animal and each dot reflects the whole animal photon count. Pairwise differences in photon counts between treatment groups were analyzed with the Wilcoxon rank-sum test. Shown are data for ten mice per treatment condition.

Mentions: We used the 4T1 mouse breast tumor resection model (which mimics local and systemic recurrences arising post-surgically) to measure the effectiveness of delivering lymphocytes via scaffolds compared with conventional injection. Ten days after introducing luciferase-expressing 4T1 cells into mammary glands, we resected tumors leaving ~1% residual diseased tissue. In the first of six randomly-assigned groups, we implanted scaffolds containing 7×106 tumor-reactive T cells directly into the resection cavity (Fig. 1a, middle panel). Two groups received the same T cell dose injected either intravenously or directly into the resection bed. For the fourth group, we pre-activated T cells with IL-15 superagonist and anti-CD3, anti-CD28, and anti-CD137 antibodies in vitro prior to local injection. Background effects were measured by implanting mice with acellular scaffolds, and controls received no treatment. Conducting bioluminescence imaging to quantify tumor growth, we found that intravenous injection of T cells prevented metastatic relapse no better than controls (median survival: 21 versus 19 days, respectively; P = 0.21, Fig. 3a, b). Injecting cells directly into the resection cavity produced a temporary reduction in residual tumors, yielding a modest (4 day) survival advantage (P = 0.03). Time to relapse was further prolonged by pre-activating the injected cells (median survival: 30 days versus 25 days; P = 0.048), but did not prevent recurrence. By contrast, none of the animals receiving scaffold-delivered T cells experienced tumor relapse (Fig. 3a, b, Supplementary Fig. 7). Luciferase-negative 4T1 tumors produced the same results (Supplementary Fig. 8). As expected, cell-free scaffolds or those containing non-specific lymphocytes did not produce anti-tumor benefits (Supplementary Fig. 9).


Biopolymer implants enhance the efficacy of adoptive T-cell therapy.

Stephan SB, Taber AM, Jileaeva I, Pegues EP, Sentman CL, Stephan MT - Nat. Biotechnol. (2014)

Material-deployed T cells robustly expand in tumor tissue, where they reduce residual disease and relapse. (a–d) Biomaterial-supported T cell implants eradicate tumor cells left behind after surgery. Ten days after firefly luciferase-expressing 4T1 breast tumor cells were transplanted into mammary glands of BALB/c mice, tumors were resected such that ~1% of the tissue remained. All mice were treated with cyclophosphamide one day prior to T cell treatment. The first group of mice received a cell-free scaffold immediately following surgery as a control; all others were treated with 7×106 breast tumor-specific T cells. In group 2, the cells were injected intravenously. The next two groups were injected with T cells directly into the tumor bed, with or without pre-activation using anti-CD3/CD28/CD137 antibodies and IL-15/IL-15Rα. The last group received cells delivered via bioactive scaffolds that were implanted directly into the resection cavity. (a) Sequential bioluminescence imaging of the 4T1 breast tumors. (b) Kaplan-Meier survival curves for treated and control mice. Shown are ten mice per treatment group pooled from three independent experiments; ms, median survival. (c) Longitudinal in vivo bioluminescence imaging of breast tumor-specific T cells retrovirally transduced with click beetle red luciferase (CBR-luc). (d) Tumor-specific T cells labeled with CellTracker Orange were embedded in scaffolds that were fluorescently tagged with Hilyte Fluor 647 and implanted into the luciferase-expressing tumor resection cavity. The scaffold and surrounding tissue were excised and sectioned for histological analysis three days later. Scale bar: 100 μm. Data shown are 1 of 2 independent experiments. (e–h) Launching anti-tumor T lymphocytes from bioactive polymer implants controls inoperable cancer. ID8 ovarian carcinoma cells stably expressing VEGF were injected intraperitoneally and allowed to establish for eight weeks. Mice were given cyclophosphamide one day prior to treatment with lymphocytes expressing chimeric NKG2D receptors (NKG2D CARs) using the administration and stimulation protocols described above (except local injections were intraperitoneal instead of intra-resection cavity). (e) Serial in vivo bioluminescence imaging of ID8-VEGF-luc tumors. (f) Survival of animals following T cell therapy depicted as Kaplan-Meier curves. (g) In vivo bioluminescent imaging of T cells expressing CBR-luc. (h) CBR-luc signal intensities after T cell transfer. Every line represents one animal and each dot reflects the whole animal photon count. Pairwise differences in photon counts between treatment groups were analyzed with the Wilcoxon rank-sum test. Shown are data for ten mice per treatment condition.
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Figure 3: Material-deployed T cells robustly expand in tumor tissue, where they reduce residual disease and relapse. (a–d) Biomaterial-supported T cell implants eradicate tumor cells left behind after surgery. Ten days after firefly luciferase-expressing 4T1 breast tumor cells were transplanted into mammary glands of BALB/c mice, tumors were resected such that ~1% of the tissue remained. All mice were treated with cyclophosphamide one day prior to T cell treatment. The first group of mice received a cell-free scaffold immediately following surgery as a control; all others were treated with 7×106 breast tumor-specific T cells. In group 2, the cells were injected intravenously. The next two groups were injected with T cells directly into the tumor bed, with or without pre-activation using anti-CD3/CD28/CD137 antibodies and IL-15/IL-15Rα. The last group received cells delivered via bioactive scaffolds that were implanted directly into the resection cavity. (a) Sequential bioluminescence imaging of the 4T1 breast tumors. (b) Kaplan-Meier survival curves for treated and control mice. Shown are ten mice per treatment group pooled from three independent experiments; ms, median survival. (c) Longitudinal in vivo bioluminescence imaging of breast tumor-specific T cells retrovirally transduced with click beetle red luciferase (CBR-luc). (d) Tumor-specific T cells labeled with CellTracker Orange were embedded in scaffolds that were fluorescently tagged with Hilyte Fluor 647 and implanted into the luciferase-expressing tumor resection cavity. The scaffold and surrounding tissue were excised and sectioned for histological analysis three days later. Scale bar: 100 μm. Data shown are 1 of 2 independent experiments. (e–h) Launching anti-tumor T lymphocytes from bioactive polymer implants controls inoperable cancer. ID8 ovarian carcinoma cells stably expressing VEGF were injected intraperitoneally and allowed to establish for eight weeks. Mice were given cyclophosphamide one day prior to treatment with lymphocytes expressing chimeric NKG2D receptors (NKG2D CARs) using the administration and stimulation protocols described above (except local injections were intraperitoneal instead of intra-resection cavity). (e) Serial in vivo bioluminescence imaging of ID8-VEGF-luc tumors. (f) Survival of animals following T cell therapy depicted as Kaplan-Meier curves. (g) In vivo bioluminescent imaging of T cells expressing CBR-luc. (h) CBR-luc signal intensities after T cell transfer. Every line represents one animal and each dot reflects the whole animal photon count. Pairwise differences in photon counts between treatment groups were analyzed with the Wilcoxon rank-sum test. Shown are data for ten mice per treatment condition.
Mentions: We used the 4T1 mouse breast tumor resection model (which mimics local and systemic recurrences arising post-surgically) to measure the effectiveness of delivering lymphocytes via scaffolds compared with conventional injection. Ten days after introducing luciferase-expressing 4T1 cells into mammary glands, we resected tumors leaving ~1% residual diseased tissue. In the first of six randomly-assigned groups, we implanted scaffolds containing 7×106 tumor-reactive T cells directly into the resection cavity (Fig. 1a, middle panel). Two groups received the same T cell dose injected either intravenously or directly into the resection bed. For the fourth group, we pre-activated T cells with IL-15 superagonist and anti-CD3, anti-CD28, and anti-CD137 antibodies in vitro prior to local injection. Background effects were measured by implanting mice with acellular scaffolds, and controls received no treatment. Conducting bioluminescence imaging to quantify tumor growth, we found that intravenous injection of T cells prevented metastatic relapse no better than controls (median survival: 21 versus 19 days, respectively; P = 0.21, Fig. 3a, b). Injecting cells directly into the resection cavity produced a temporary reduction in residual tumors, yielding a modest (4 day) survival advantage (P = 0.03). Time to relapse was further prolonged by pre-activating the injected cells (median survival: 30 days versus 25 days; P = 0.048), but did not prevent recurrence. By contrast, none of the animals receiving scaffold-delivered T cells experienced tumor relapse (Fig. 3a, b, Supplementary Fig. 7). Luciferase-negative 4T1 tumors produced the same results (Supplementary Fig. 8). As expected, cell-free scaffolds or those containing non-specific lymphocytes did not produce anti-tumor benefits (Supplementary Fig. 9).

Bottom Line: Using a mouse breast cancer resection model, we show that the implants effectively support tumor-targeting T cells throughout resection beds and associated lymph nodes, and reduce tumor relapse compared to conventional delivery modalities.In a multifocal ovarian cancer model, we demonstrate that polymer-delivered T cells trigger regression, whereas injected tumor-reactive lymphocytes have little curative effect.Scaffold-based T-cell delivery may provide a viable treatment option for inoperable tumors and reduce the rate of metastatic relapse after surgery.

View Article: PubMed Central - PubMed

Affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

ABSTRACT
Although adoptive T-cell therapy holds promise for the treatment of many cancers, its clinical utility has been limited by problems in delivering targeted lymphocytes to tumor sites, and the cells' inefficient expansion in the immunosuppressive tumor microenvironment. Here we describe a bioactive polymer implant capable of delivering, expanding and dispersing tumor-reactive T cells. The approach can be used to treat inoperable or incompletely removed tumors by situating implants near them or at resection sites. Using a mouse breast cancer resection model, we show that the implants effectively support tumor-targeting T cells throughout resection beds and associated lymph nodes, and reduce tumor relapse compared to conventional delivery modalities. In a multifocal ovarian cancer model, we demonstrate that polymer-delivered T cells trigger regression, whereas injected tumor-reactive lymphocytes have little curative effect. Scaffold-based T-cell delivery may provide a viable treatment option for inoperable tumors and reduce the rate of metastatic relapse after surgery.

Show MeSH
Related in: MedlinePlus