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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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Porous polysaccharide scaffolds functionalized with appropriate adhesion molecules and stimulatory cues support rapid migration, robust expansion, and sustained release of T cells. (a) Time-lapse video projections of lymphocyte migration through uncoated (left) and collagen mimetic peptide (CMP)-coated (right) macroporous alginate scaffolds tracked for 30 min; each color represents an individual T cell. Scale bar: 50 μm. (b) Comparison of maximum T cell displacements, based on 30 randomly chosen cells from two independent experiments. (c–d) CMP coating promotes the egress of T cells into surrounding tissue: (c) Schematic and corresponding micrographs of the in vitro assay used to quantify cell migration out of the scaffold (purple) into a tissue mimetic (three-dimensional fibrillar collagen gel; pink). Scale bar: 100 μm. (d) Quantification of viable (trypan blue-excluding) T cells enzymatically recovered from scaffolds versus collagen matrices at indicated time points. (e–g) Incorporating stimulatory microspheres into matrices amplifies T cell expansion and release: (e) Microscopy of scaffolds containing embedded particles. Scale bar: 150 μm. The high magnification confocal images in the lower panel show a single lipid-enveloped mesoporous silica microsphere with IL-15/IL15Rα cytokine (Alexa 488-labeled: green) entrapped in the polymer core and stimulatory anti-CD3/CD28/CD137 antibodies (Alexa 647-labeled: blue) tethered to its phospholipid membrane. Scale bar: 15 μm. (f) Absolute counts of viable T cells in scaffolds fabricated with or without stimulatory microparticles (left panel), and of cells transited from these implants into surrounding collagen matrix (right panel). (g) Representative carboxyfluorescein succinimidyl ester (CFSE) assay of T cells that have exited scaffolds during the 7d test period, in which proliferation was assessed by measuring CFSE dilution (consequent to cell division) using flow cytometry. Mean CFSE fluorescence intensities (MFI) for the lymphocyte populations are indicated at the upper left. Differences in apoptosis were quantified by Annexin V labeling. Data are representative of three independent experiments.
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Figure 2: Porous polysaccharide scaffolds functionalized with appropriate adhesion molecules and stimulatory cues support rapid migration, robust expansion, and sustained release of T cells. (a) Time-lapse video projections of lymphocyte migration through uncoated (left) and collagen mimetic peptide (CMP)-coated (right) macroporous alginate scaffolds tracked for 30 min; each color represents an individual T cell. Scale bar: 50 μm. (b) Comparison of maximum T cell displacements, based on 30 randomly chosen cells from two independent experiments. (c–d) CMP coating promotes the egress of T cells into surrounding tissue: (c) Schematic and corresponding micrographs of the in vitro assay used to quantify cell migration out of the scaffold (purple) into a tissue mimetic (three-dimensional fibrillar collagen gel; pink). Scale bar: 100 μm. (d) Quantification of viable (trypan blue-excluding) T cells enzymatically recovered from scaffolds versus collagen matrices at indicated time points. (e–g) Incorporating stimulatory microspheres into matrices amplifies T cell expansion and release: (e) Microscopy of scaffolds containing embedded particles. Scale bar: 150 μm. The high magnification confocal images in the lower panel show a single lipid-enveloped mesoporous silica microsphere with IL-15/IL15Rα cytokine (Alexa 488-labeled: green) entrapped in the polymer core and stimulatory anti-CD3/CD28/CD137 antibodies (Alexa 647-labeled: blue) tethered to its phospholipid membrane. Scale bar: 15 μm. (f) Absolute counts of viable T cells in scaffolds fabricated with or without stimulatory microparticles (left panel), and of cells transited from these implants into surrounding collagen matrix (right panel). (g) Representative carboxyfluorescein succinimidyl ester (CFSE) assay of T cells that have exited scaffolds during the 7d test period, in which proliferation was assessed by measuring CFSE dilution (consequent to cell division) using flow cytometry. Mean CFSE fluorescence intensities (MFI) for the lymphocyte populations are indicated at the upper left. Differences in apoptosis were quantified by Annexin V labeling. Data are representative of three independent experiments.

Mentions: An effective T cell delivery and release platform must support cell egress and provide stimulatory signals to trigger proliferation. We created macroporous scaffolds from polymerized alginate (a moldable, naturally-occurring polysaccharide already FDA-approved because of its biocompatibility and biodegradability10). Lymphocytes normally migrate along collagen fibers, so we integrated GFOGER (a synthetic collagen-mimetic peptide (CMP) that binds to lymphocytes via the α2β1 collagen receptor11) into the scaffolds using carbodiimide chemistry (Supplementary Fig. 1a, b). Time-lapse microscopy established that T cells migrate through these scaffolds with a velocity similar to those in lymphoid organs (averaging 8.9 μm/min12; Fig. 2a; Supplementary Fig. 1c). Thus, in 30 min they travel 119 μm ± 37 μm (Fig. 2b), whereas lymphocytes in unmodified scaffolds only circulate within their void space (mean displacement: 7 μm ± 4.8 μm; Fig. 2a, b; Supplementary Movie 1). CMP contact also increased viability compared with unmodified alginate or plastic (Supplementary Fig. 1d), perhaps reflecting activation of collagen-dependent pro-survival pathways.


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)

Porous polysaccharide scaffolds functionalized with appropriate adhesion molecules and stimulatory cues support rapid migration, robust expansion, and sustained release of T cells. (a) Time-lapse video projections of lymphocyte migration through uncoated (left) and collagen mimetic peptide (CMP)-coated (right) macroporous alginate scaffolds tracked for 30 min; each color represents an individual T cell. Scale bar: 50 μm. (b) Comparison of maximum T cell displacements, based on 30 randomly chosen cells from two independent experiments. (c–d) CMP coating promotes the egress of T cells into surrounding tissue: (c) Schematic and corresponding micrographs of the in vitro assay used to quantify cell migration out of the scaffold (purple) into a tissue mimetic (three-dimensional fibrillar collagen gel; pink). Scale bar: 100 μm. (d) Quantification of viable (trypan blue-excluding) T cells enzymatically recovered from scaffolds versus collagen matrices at indicated time points. (e–g) Incorporating stimulatory microspheres into matrices amplifies T cell expansion and release: (e) Microscopy of scaffolds containing embedded particles. Scale bar: 150 μm. The high magnification confocal images in the lower panel show a single lipid-enveloped mesoporous silica microsphere with IL-15/IL15Rα cytokine (Alexa 488-labeled: green) entrapped in the polymer core and stimulatory anti-CD3/CD28/CD137 antibodies (Alexa 647-labeled: blue) tethered to its phospholipid membrane. Scale bar: 15 μm. (f) Absolute counts of viable T cells in scaffolds fabricated with or without stimulatory microparticles (left panel), and of cells transited from these implants into surrounding collagen matrix (right panel). (g) Representative carboxyfluorescein succinimidyl ester (CFSE) assay of T cells that have exited scaffolds during the 7d test period, in which proliferation was assessed by measuring CFSE dilution (consequent to cell division) using flow cytometry. Mean CFSE fluorescence intensities (MFI) for the lymphocyte populations are indicated at the upper left. Differences in apoptosis were quantified by Annexin V labeling. Data are representative of three independent experiments.
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Related In: Results  -  Collection

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Figure 2: Porous polysaccharide scaffolds functionalized with appropriate adhesion molecules and stimulatory cues support rapid migration, robust expansion, and sustained release of T cells. (a) Time-lapse video projections of lymphocyte migration through uncoated (left) and collagen mimetic peptide (CMP)-coated (right) macroporous alginate scaffolds tracked for 30 min; each color represents an individual T cell. Scale bar: 50 μm. (b) Comparison of maximum T cell displacements, based on 30 randomly chosen cells from two independent experiments. (c–d) CMP coating promotes the egress of T cells into surrounding tissue: (c) Schematic and corresponding micrographs of the in vitro assay used to quantify cell migration out of the scaffold (purple) into a tissue mimetic (three-dimensional fibrillar collagen gel; pink). Scale bar: 100 μm. (d) Quantification of viable (trypan blue-excluding) T cells enzymatically recovered from scaffolds versus collagen matrices at indicated time points. (e–g) Incorporating stimulatory microspheres into matrices amplifies T cell expansion and release: (e) Microscopy of scaffolds containing embedded particles. Scale bar: 150 μm. The high magnification confocal images in the lower panel show a single lipid-enveloped mesoporous silica microsphere with IL-15/IL15Rα cytokine (Alexa 488-labeled: green) entrapped in the polymer core and stimulatory anti-CD3/CD28/CD137 antibodies (Alexa 647-labeled: blue) tethered to its phospholipid membrane. Scale bar: 15 μm. (f) Absolute counts of viable T cells in scaffolds fabricated with or without stimulatory microparticles (left panel), and of cells transited from these implants into surrounding collagen matrix (right panel). (g) Representative carboxyfluorescein succinimidyl ester (CFSE) assay of T cells that have exited scaffolds during the 7d test period, in which proliferation was assessed by measuring CFSE dilution (consequent to cell division) using flow cytometry. Mean CFSE fluorescence intensities (MFI) for the lymphocyte populations are indicated at the upper left. Differences in apoptosis were quantified by Annexin V labeling. Data are representative of three independent experiments.
Mentions: An effective T cell delivery and release platform must support cell egress and provide stimulatory signals to trigger proliferation. We created macroporous scaffolds from polymerized alginate (a moldable, naturally-occurring polysaccharide already FDA-approved because of its biocompatibility and biodegradability10). Lymphocytes normally migrate along collagen fibers, so we integrated GFOGER (a synthetic collagen-mimetic peptide (CMP) that binds to lymphocytes via the α2β1 collagen receptor11) into the scaffolds using carbodiimide chemistry (Supplementary Fig. 1a, b). Time-lapse microscopy established that T cells migrate through these scaffolds with a velocity similar to those in lymphoid organs (averaging 8.9 μm/min12; Fig. 2a; Supplementary Fig. 1c). Thus, in 30 min they travel 119 μm ± 37 μm (Fig. 2b), whereas lymphocytes in unmodified scaffolds only circulate within their void space (mean displacement: 7 μm ± 4.8 μm; Fig. 2a, b; Supplementary Movie 1). CMP contact also increased viability compared with unmodified alginate or plastic (Supplementary Fig. 1d), perhaps reflecting activation of collagen-dependent pro-survival pathways.

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