- Email: [email protected]
720. Triple-Negative Breast Cancer Cells and Tumor Endothelium Are Killed by Targeting Tumor Endothelial Marker 8 (TEM8)
Cancer – Immunotherapy, Cancer Vaccines II 718. AAV-Mediated Local Anti-EGFR Antibody Gene Expression in CNS Delays Tumor Growth and Increases Survival in a Human Glioblastoma Xenograft Model
Maria J. Chiuchiolo,1 Martin J. Hicks,1 Yie Xie,1 Fan Fan,1 Jonathan B. Rosenberg,1 Jonathan P. Dyke,1 Kosuke Funato,2 David F. Havlicek,1 Douglas J. Ballon,1 Viviane Tabar,2 Dolan Sondhi,1 Stephen M. Kaminsky,1 Ronald G. Crystal.1 1 Weill Cornell Medical College, New York, NY; 2Memorial SloanKettering Cancer Center, New York, NY. Epidermal growth factor receptor (EGFR) is an attractive target for the treatment of glioblastoma (GBM), where deregulated expression or activity of EGFR has been associated to tumor development, progression and spread, and decreased survival. Systemic administration of an anti-EGFR antibody (CetuximabTM) reduces cellular proliferation in a variety of cancer models, but for GBM, efficacy is limited by the blood-brain barrier, where only ∼ 0.1% of circulating antibodies reach the brain. To circumvent these limitations, we have developed a strategy to deliver the cDNA sequence for the anti-EGFR antibody directly to the CNS via adeno-associated virus (AAV) gene transfer vectors, resulting in local, persistent expression in the CNS. Efficacy of this therapeutic approach was evaluated by assessment of survival of NOD/SCID immunodeficient mice after CNS administration of human U87MG glioblastoma cells modified to express wild type EGFR to the striatum, either at the same time or 8 days prior to the administration of an AAVrh.10based vector coding for the anti-EGFR monoclonal antibody Cetuximab (AAVrh.10CetMab). Localized expression of the antibody was confirmed by Western analysis and ELISA. Simultaneous administration of AAVrh.10CetMab and GBM xenograft to the CNS was associated with survival of the treated animals compared to controls that received PBS (AAVrh.10CetMab treated 50±5 days vs PBS-treated, 31±2 days, p<0.0001). Administration of the vector 8 days after tumor cells delivery, reduced tumor growth by 3-fold at 4 wk as measured by CNS MRI (p<0.05), and increased survival of the antibody treated mice (AAVrh.10CetMab-treated 36±4 days vs PBS-treated, 30±3 days, p<0.02). Likewise, CNS administration of the AAVrh.10CetMab vector 3 wk after mice received patientderived early passage GBM primary cells to the CNS increased survival in the antibody-treated animals with respect to the controls (AAVrh.10CetMab treated 98±3 days vs PBS-treated, 88±3 days, p<0.03). The data demonstrates that local delivery of an anti-EGFR antibody by an AAVrh.10-derived vector reduced GBM tumor growth and increased survival in mice with a cell line expressing EGFR and patient-derived xenografts. AAV-mediated antibody gene transfer directly to the CNS may provide an effective treatment of glioblastoma, with therapeutic levels of antibody expression in the tumor milieu.
719. Combination of ICOS and 4-1BB in a Third Generation CAR Exhibits Enhanced T Cell Persistence and Increased Antitumor Effect
Sonia Guedan,1 Prachi Patel,1 Shannon E. McGettigan,1 Avery D. Posey,1 Omkar Kawalekar,1 John Scholler,1 Brian Keith,1 Carl H. June.1 1 Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA. T cell persistence is likely to promote long-term antitumor effects after adoptive T cell transfer; however, T cells expressing chimeric antigen receptors (CARs) have not persisted well in patients with solid tumors in trials reported to date. Here, we hypothesized that CD4+ and CD8+ T cells may need distinct costimulation signals to persist. To test this hypothesis, we compared the in vivo antitumor effects Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
and persistence of combined CD4+ and CD8+ T cells redirected with CARs containing different costimulatory domains (CD28, 4-1BB or ICOS). Using multiple mouse tumor models, we demonstrate that the ICOS intracellular domain enhanced the in vivo persistence of CAR-expressing CD4+ T cells. Persistence of CD8+ T cells was highly dependent on the intracellular domain used to redirect CD4+ T cells; specifically, CD4+ T cells expressing an ICOS-based CAR significantly increased the persistence of CD8+ T cells expressing either CD28- or 4-1BB-based CARs. We further demonstrate that the combination of ICOS and 4-1BB in a third generation CAR (ICOSBBz) is more effective at eliminating large pancreatic tumors than either 4-1BB or ICOS alone. T cells redirected with ICOSBBz showed a less differentiated phenotype with superior proliferation and in vivo persistence. These results indicate that ICOSBBz-based CARs are promising therapeutics for clinical testing.
720. Triple-Negative Breast Cancer Cells and Tumor Endothelium Are Killed by Targeting Tumor Endothelial Marker 8 (TEM8)
Tiara Byrd,1,2 Kristen Fousek,1,2 Antonella Pignata,2 Christopher Szot,3 Kevin Bielamowicz,2 Amanda Wakefield,2 Steven Seaman,3 Bradley Fletcher,4 Meenakshi Hegde,2 Brad St Croix,3 Nabil Ahmed.1,2 1 Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, TX; 2Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX; 3National Cancer Institute, National Institutes of Health, Frederick, MD; 4Medicine, University of Florida, Gainesville, FL. Background: Triple-negative breast cancer (TNBC) refers to a subset of breast cancers that are HER2, estrogen-receptor and progesterone-receptor negative. Associated with an aggressive phenotype and high incidence of recurrence, and devoid of the aforementioned receptors, there are currently no targeted therapies for TNBC. Tumor Endothelial Marker 8 (TEM8) is one of nine gene products up-regulated in the tumor vs. normal endothelium and is overexpressed in TNBC. Purpose: To investigate whether TEM8 could serve as a viable target for TNBC therapy. Methods/Results: To validate the target antigen we conducted double immunofluorescence staining of a cohort of 6 TNBC specimens for TEM8 and the pan-endothelial cell marker (CD31). In all 6 cases TEM8 was overexpressed, when compared to normal adjacent breast tissue. TEM8 staining was not restricted to the CD31 positive cells, but also detected in tumor parenchymal cells. The immortalized TNBC cell lines (MDA-MB-231, MDA-MB-436, MDA-MB-468 and Hs578T) expressed endogenous levels of TEM8 protein as revealed by western blot. To target TNBC using T cells we designed in silico a novel TEM8-specific chimeric antigen receptor (CAR) molecule. This CAR consisted of a TEM8 recognition extracellular domain single-chain variable fragment (scFv) derived from the monoclonal antibody, L2, a connecting transmembrane chain, followed by CD28, 41BB and CD3-zeta chain intracellular signaling domains. The TEM8 CAR-encoding DNA construct was synthesized and then sequence verified. Retroviral transduction was used to integrate the TEM8 CAR transgene in HEK 293T, then on primary T cells. Over 90% of primary human T cells expressed the TEM8 specific CAR, as indicated by flow cytometry. TEM8 specific T cells recognized and killed TEM8 positive cells in standard 4 hour 51Cr release cytotoxicity assays, effectively targeting both TNBC and tumor endothelium cell lines. Further, TEM8 CAR T cells secreted immunostimulatory cytokines in coculture. In contrast, there was minimal reactivity against TEM8 negative targets or by non-transduced T cells from the same blood donor. Adoptively transferred TEM8 specific CAR T cells resulted in
S287
Cancer – Immunotherapy, Cancer Vaccines II a significant decline in the tumor growth of vascularized orthotopic breast cancer xenografts in a murine model, compared to nontransduced T cells (p=0.03). Conclusion: We conclude that TEM8 specific CAR T cells could serve as a tumor and vascular targeted therapy for TNBC.
721.
Safety of Multiple Doses of CAR T Cells
Kristen Fousek,1,2 Rayne Rouce,2 Nabil Ahmed,1,2 Stephen Gottschalk,1,2 Barbara Savoldo,2 Gianpietro Dotti,1,2 Carlos Ramos,2 Chrystal Louis,2 Cliona Rooney,1,2 Malcolm Brenner,1,2 Helen Heslop.1,2 1 Translational Biology and Molecular Medicine Program, Baylor College of Medicine, Houston, TX; 2Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children’s Hospital, Houston Methodist Hospital, Houston, TX. Chimeric antigen receptor (CAR) T cells have demonstrated promising results for cancers in pre-clinical models and early phase trials. However, some groups have observed serious adverse events (SAEs) and toxicities attributable to the administration of CAR T cells, the most clinically significant being cytokine release syndrome (CRS). In addition, a recent case report described a patient who developed an anaphylaxis reaction to CAR T cells, ultimately attributed to receipt of multiple doses of cells. As there are a number of ongoing clinical trials at our institution utilizing CAR T cells to treat various hematologic and solid malignancies and several patients have received multiple doses, we performed a retrospective review to assess whether early or late infusion toxicities were observed with subsequent infusions. We identified 45 of over 200 patients who received more than one dose of CAR T cells between January 2009 and December 2014. We assessed patient characteristics including type of malignancy, disease status at the time of subsequent infusions, cell product (autologous vs. allogeneic), presence or absence of prior lymphodepletion, number of infusions, dosing schedule, and the relationship to development of CRS. Each characteristic was examined for any correlation to developing a SAE. Our results indicate that at our institution, repeated CAR T cell infusions are well tolerated, with the majority of grade 3-4 adverse events being hematologic and electrolyte abnormalities. Furthermore, the majority of SAEs reported after repeated CAR T cell infusions were attributed as unrelated to the infusion itself. However, further evaluation of a larger cohort is necessary to determine whether an association between the timing of repeated infusions and CRS and other SAEs exists. These findings and our continued evaluation of patients receiving multiple infusions will help us to ensure the safety of administering multiple doses of CAR T cells in the future.
722. Overcoming EBV Tumor Specific T-Cell Anergy in Rapidly-Generated EBVST-Cells for Adoptive Transfer Therapy
Sandhya Sharma,1 Serena Perna,1 Natalia Lapteva,1 Minhtran Ngo,1 Vicky Torrano,1 Rayne Rouce,1 Carlos Ramos,1 Catherine Bollard,1,2 Helen Heslop,1 Cliona Rooney.1 1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston Methodist Hospital, Texas Children’s Hospital, Baylor College of Medicine, Houston, TX; 2Center for Cancer and Immunology Research, Children’s National Health System, Washington, DC. Up to 30% of Hodgkin and non-Hodgkin lymphomas carry the Epstein-Barr virus (EBV) genome and express the viral latency proteins EBNA1, LMP1, LMP2 and BARF1;a type 2 latency pattern of EBV gene expression. We previously reported that EBV-specific T-cells (EBVSTs) directed to LMP1 and LMP2 expanded from the blood of lymphoma patients produced complete tumor responses in over 50% of patients. To shorten and simplify the EBVST production S288
time, we removed viral-vector components from our manufacturing process, replacing these components with dendritic cells pulsed with peptide libraries (pepmixes) spanning type 2 latency antigens. Responder T-cells are then expanded by restimulation with pepmixpulsed, autologous, activated T-cells and HLA-negative K562 costimulatory cells in the presence of IL-4 and IL-7. Despite enhanced antigen specificity from EBVSTs generated from healthy donors, we were unable to consistently generate patient-derived EBVSTs with significant activity against the Type 2 antigens. We hypothesized that patient T-cells were anergized by their immunosuppressive tumors. IL-15 has been shown to rescue tolerant or anergized CD8+ T-cells and we found that substitution of IL-15 (5ng/mL) for IL-4 (in combination with IL-7 (10ng/mL)) improved CD4+ and CD8+ T-cells’ specificity for the EBV Type 2 latency antigens by up to 10-fold. By increasing the concentration of IL-15, we achieved significantly higher fold expansion (3 fold mean increase in absolute EBVST number) and further enhanced specificity for type 2 latency EBV antigens (high vs. low IL-15 concentration: EBNA1: 216±273 vs. 29±48, LMP1:145±253 vs. 44±63, LMP2: 636±548 vs. 106±74 and BARF1: 80±100 vs. 29±22; SFC/105 cells; n=5). There was no increase in the absolute numbers of NK-cells despite high doses of IL-15. Enhanced antigen-specificity correlated with increased cytotoxic effect, with an increase in mean % specific lysis of EBV pepmix-pulsed autologous activated T-cell targets from 5 to 66% at a 20:1 E:T ratio in the low vs. high dose IL-15 conditions (n=5). We have infused EBVSTs manufactured using all three conditions into 17 patients with multiply-relapsed, EBV-positive lymphoma as adjuvant therapy after stem cell transplantation or chemotherapy in 8 patients and as treatment for disease in 9 patients. Of patients in remission at the time of infusion, two with IL-4/7-grown EBVSTs and 6 with IL-15/7 grown EBVSTs remain in remission. Of patients with disease at the time of infusion, one receiving IL-4/7-grown EBVSTs had stable disease and 3 had progressive disease, while of 5 patients with IL-15/7-grown EBVSTs, one had stable disease, one had a partial response, one had a complete response and two are too early to assess. We will continue to modify our manufacturing process in an attempt to further increase the specificity and function of EBVSTs from patients with relapsed or refractory lymphoma.
723. Oncolytic Adenovirus Breaks Tumor Tolerance To Adoptive T-Cell Therapy By Recruiting Immune Cells To and Promoting Their Activity in the Tumor
Siri Tähtinen,1 Susanna Grönberg-Vähä-Koskela,1 Dave Lumen,3 Maiju Merisalo-Soikkeli,1 Mikko Siurala,1,2 Anu J. Airaksinen,3 Markus Vähä-Koskela,1 Akseli Hemminki.1,2 1 Cancer Gene Therapy Group, Department of Pathology and Transplantation Laboratory, Haartman Institute, University of Helsinki, Helsinki, Finland; 2TILT Biotherapeutics Ltd, Helsinki, Finland; 3Laboratory of Radiochemistry, Department of Chemistry, University of Helsinki, Helsinki, Finland. Background: Despite the fast growing rate in the development of novel adoptive T-cell therapies (ACT), the clinical benefit in established solid tumors has remained modest. Roadblocks to effective immune attack by infiltrating tumor-specific T-cells include several immune evasion mechanisms, which contribute to tumor tolerance. Oncolytic virotherapy on the other hand is the use of cancer cell specific, conditionally replicative viruses in the treatment of cancer. Oncolytic virotherapy has been suggested to enhance epitope spreading by direct oncolysis of tumor cells, leading to enhanced antigen presentation by dendritic cells and increased immunogenicity of the established tumor. Here, we studied the combination of these two established forms of immunotherapy in a highly resistant and poorly immunogenic B16.OVA mouse melanoma model. Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
Maria J. Chiuchiolo,1 Martin J. Hicks,1 Yie Xie,1 Fan Fan,1 Jonathan B. Rosenberg,1 Jonathan P. Dyke,1 Kosuke Funato,2 David F. Havlicek,1 Douglas J. Ballon,1 Viviane Tabar,2 Dolan Sondhi,1 Stephen M. Kaminsky,1 Ronald G. Crystal.1 1 Weill Cornell Medical College, New York, NY; 2Memorial SloanKettering Cancer Center, New York, NY. Epidermal growth factor receptor (EGFR) is an attractive target for the treatment of glioblastoma (GBM), where deregulated expression or activity of EGFR has been associated to tumor development, progression and spread, and decreased survival. Systemic administration of an anti-EGFR antibody (CetuximabTM) reduces cellular proliferation in a variety of cancer models, but for GBM, efficacy is limited by the blood-brain barrier, where only ∼ 0.1% of circulating antibodies reach the brain. To circumvent these limitations, we have developed a strategy to deliver the cDNA sequence for the anti-EGFR antibody directly to the CNS via adeno-associated virus (AAV) gene transfer vectors, resulting in local, persistent expression in the CNS. Efficacy of this therapeutic approach was evaluated by assessment of survival of NOD/SCID immunodeficient mice after CNS administration of human U87MG glioblastoma cells modified to express wild type EGFR to the striatum, either at the same time or 8 days prior to the administration of an AAVrh.10based vector coding for the anti-EGFR monoclonal antibody Cetuximab (AAVrh.10CetMab). Localized expression of the antibody was confirmed by Western analysis and ELISA. Simultaneous administration of AAVrh.10CetMab and GBM xenograft to the CNS was associated with survival of the treated animals compared to controls that received PBS (AAVrh.10CetMab treated 50±5 days vs PBS-treated, 31±2 days, p<0.0001). Administration of the vector 8 days after tumor cells delivery, reduced tumor growth by 3-fold at 4 wk as measured by CNS MRI (p<0.05), and increased survival of the antibody treated mice (AAVrh.10CetMab-treated 36±4 days vs PBS-treated, 30±3 days, p<0.02). Likewise, CNS administration of the AAVrh.10CetMab vector 3 wk after mice received patientderived early passage GBM primary cells to the CNS increased survival in the antibody-treated animals with respect to the controls (AAVrh.10CetMab treated 98±3 days vs PBS-treated, 88±3 days, p<0.03). The data demonstrates that local delivery of an anti-EGFR antibody by an AAVrh.10-derived vector reduced GBM tumor growth and increased survival in mice with a cell line expressing EGFR and patient-derived xenografts. AAV-mediated antibody gene transfer directly to the CNS may provide an effective treatment of glioblastoma, with therapeutic levels of antibody expression in the tumor milieu.
719. Combination of ICOS and 4-1BB in a Third Generation CAR Exhibits Enhanced T Cell Persistence and Increased Antitumor Effect
Sonia Guedan,1 Prachi Patel,1 Shannon E. McGettigan,1 Avery D. Posey,1 Omkar Kawalekar,1 John Scholler,1 Brian Keith,1 Carl H. June.1 1 Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA. T cell persistence is likely to promote long-term antitumor effects after adoptive T cell transfer; however, T cells expressing chimeric antigen receptors (CARs) have not persisted well in patients with solid tumors in trials reported to date. Here, we hypothesized that CD4+ and CD8+ T cells may need distinct costimulation signals to persist. To test this hypothesis, we compared the in vivo antitumor effects Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
and persistence of combined CD4+ and CD8+ T cells redirected with CARs containing different costimulatory domains (CD28, 4-1BB or ICOS). Using multiple mouse tumor models, we demonstrate that the ICOS intracellular domain enhanced the in vivo persistence of CAR-expressing CD4+ T cells. Persistence of CD8+ T cells was highly dependent on the intracellular domain used to redirect CD4+ T cells; specifically, CD4+ T cells expressing an ICOS-based CAR significantly increased the persistence of CD8+ T cells expressing either CD28- or 4-1BB-based CARs. We further demonstrate that the combination of ICOS and 4-1BB in a third generation CAR (ICOSBBz) is more effective at eliminating large pancreatic tumors than either 4-1BB or ICOS alone. T cells redirected with ICOSBBz showed a less differentiated phenotype with superior proliferation and in vivo persistence. These results indicate that ICOSBBz-based CARs are promising therapeutics for clinical testing.
720. Triple-Negative Breast Cancer Cells and Tumor Endothelium Are Killed by Targeting Tumor Endothelial Marker 8 (TEM8)
Tiara Byrd,1,2 Kristen Fousek,1,2 Antonella Pignata,2 Christopher Szot,3 Kevin Bielamowicz,2 Amanda Wakefield,2 Steven Seaman,3 Bradley Fletcher,4 Meenakshi Hegde,2 Brad St Croix,3 Nabil Ahmed.1,2 1 Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, TX; 2Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX; 3National Cancer Institute, National Institutes of Health, Frederick, MD; 4Medicine, University of Florida, Gainesville, FL. Background: Triple-negative breast cancer (TNBC) refers to a subset of breast cancers that are HER2, estrogen-receptor and progesterone-receptor negative. Associated with an aggressive phenotype and high incidence of recurrence, and devoid of the aforementioned receptors, there are currently no targeted therapies for TNBC. Tumor Endothelial Marker 8 (TEM8) is one of nine gene products up-regulated in the tumor vs. normal endothelium and is overexpressed in TNBC. Purpose: To investigate whether TEM8 could serve as a viable target for TNBC therapy. Methods/Results: To validate the target antigen we conducted double immunofluorescence staining of a cohort of 6 TNBC specimens for TEM8 and the pan-endothelial cell marker (CD31). In all 6 cases TEM8 was overexpressed, when compared to normal adjacent breast tissue. TEM8 staining was not restricted to the CD31 positive cells, but also detected in tumor parenchymal cells. The immortalized TNBC cell lines (MDA-MB-231, MDA-MB-436, MDA-MB-468 and Hs578T) expressed endogenous levels of TEM8 protein as revealed by western blot. To target TNBC using T cells we designed in silico a novel TEM8-specific chimeric antigen receptor (CAR) molecule. This CAR consisted of a TEM8 recognition extracellular domain single-chain variable fragment (scFv) derived from the monoclonal antibody, L2, a connecting transmembrane chain, followed by CD28, 41BB and CD3-zeta chain intracellular signaling domains. The TEM8 CAR-encoding DNA construct was synthesized and then sequence verified. Retroviral transduction was used to integrate the TEM8 CAR transgene in HEK 293T, then on primary T cells. Over 90% of primary human T cells expressed the TEM8 specific CAR, as indicated by flow cytometry. TEM8 specific T cells recognized and killed TEM8 positive cells in standard 4 hour 51Cr release cytotoxicity assays, effectively targeting both TNBC and tumor endothelium cell lines. Further, TEM8 CAR T cells secreted immunostimulatory cytokines in coculture. In contrast, there was minimal reactivity against TEM8 negative targets or by non-transduced T cells from the same blood donor. Adoptively transferred TEM8 specific CAR T cells resulted in
S287
Cancer – Immunotherapy, Cancer Vaccines II a significant decline in the tumor growth of vascularized orthotopic breast cancer xenografts in a murine model, compared to nontransduced T cells (p=0.03). Conclusion: We conclude that TEM8 specific CAR T cells could serve as a tumor and vascular targeted therapy for TNBC.
721.
Safety of Multiple Doses of CAR T Cells
Kristen Fousek,1,2 Rayne Rouce,2 Nabil Ahmed,1,2 Stephen Gottschalk,1,2 Barbara Savoldo,2 Gianpietro Dotti,1,2 Carlos Ramos,2 Chrystal Louis,2 Cliona Rooney,1,2 Malcolm Brenner,1,2 Helen Heslop.1,2 1 Translational Biology and Molecular Medicine Program, Baylor College of Medicine, Houston, TX; 2Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children’s Hospital, Houston Methodist Hospital, Houston, TX. Chimeric antigen receptor (CAR) T cells have demonstrated promising results for cancers in pre-clinical models and early phase trials. However, some groups have observed serious adverse events (SAEs) and toxicities attributable to the administration of CAR T cells, the most clinically significant being cytokine release syndrome (CRS). In addition, a recent case report described a patient who developed an anaphylaxis reaction to CAR T cells, ultimately attributed to receipt of multiple doses of cells. As there are a number of ongoing clinical trials at our institution utilizing CAR T cells to treat various hematologic and solid malignancies and several patients have received multiple doses, we performed a retrospective review to assess whether early or late infusion toxicities were observed with subsequent infusions. We identified 45 of over 200 patients who received more than one dose of CAR T cells between January 2009 and December 2014. We assessed patient characteristics including type of malignancy, disease status at the time of subsequent infusions, cell product (autologous vs. allogeneic), presence or absence of prior lymphodepletion, number of infusions, dosing schedule, and the relationship to development of CRS. Each characteristic was examined for any correlation to developing a SAE. Our results indicate that at our institution, repeated CAR T cell infusions are well tolerated, with the majority of grade 3-4 adverse events being hematologic and electrolyte abnormalities. Furthermore, the majority of SAEs reported after repeated CAR T cell infusions were attributed as unrelated to the infusion itself. However, further evaluation of a larger cohort is necessary to determine whether an association between the timing of repeated infusions and CRS and other SAEs exists. These findings and our continued evaluation of patients receiving multiple infusions will help us to ensure the safety of administering multiple doses of CAR T cells in the future.
722. Overcoming EBV Tumor Specific T-Cell Anergy in Rapidly-Generated EBVST-Cells for Adoptive Transfer Therapy
Sandhya Sharma,1 Serena Perna,1 Natalia Lapteva,1 Minhtran Ngo,1 Vicky Torrano,1 Rayne Rouce,1 Carlos Ramos,1 Catherine Bollard,1,2 Helen Heslop,1 Cliona Rooney.1 1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston Methodist Hospital, Texas Children’s Hospital, Baylor College of Medicine, Houston, TX; 2Center for Cancer and Immunology Research, Children’s National Health System, Washington, DC. Up to 30% of Hodgkin and non-Hodgkin lymphomas carry the Epstein-Barr virus (EBV) genome and express the viral latency proteins EBNA1, LMP1, LMP2 and BARF1;a type 2 latency pattern of EBV gene expression. We previously reported that EBV-specific T-cells (EBVSTs) directed to LMP1 and LMP2 expanded from the blood of lymphoma patients produced complete tumor responses in over 50% of patients. To shorten and simplify the EBVST production S288
time, we removed viral-vector components from our manufacturing process, replacing these components with dendritic cells pulsed with peptide libraries (pepmixes) spanning type 2 latency antigens. Responder T-cells are then expanded by restimulation with pepmixpulsed, autologous, activated T-cells and HLA-negative K562 costimulatory cells in the presence of IL-4 and IL-7. Despite enhanced antigen specificity from EBVSTs generated from healthy donors, we were unable to consistently generate patient-derived EBVSTs with significant activity against the Type 2 antigens. We hypothesized that patient T-cells were anergized by their immunosuppressive tumors. IL-15 has been shown to rescue tolerant or anergized CD8+ T-cells and we found that substitution of IL-15 (5ng/mL) for IL-4 (in combination with IL-7 (10ng/mL)) improved CD4+ and CD8+ T-cells’ specificity for the EBV Type 2 latency antigens by up to 10-fold. By increasing the concentration of IL-15, we achieved significantly higher fold expansion (3 fold mean increase in absolute EBVST number) and further enhanced specificity for type 2 latency EBV antigens (high vs. low IL-15 concentration: EBNA1: 216±273 vs. 29±48, LMP1:145±253 vs. 44±63, LMP2: 636±548 vs. 106±74 and BARF1: 80±100 vs. 29±22; SFC/105 cells; n=5). There was no increase in the absolute numbers of NK-cells despite high doses of IL-15. Enhanced antigen-specificity correlated with increased cytotoxic effect, with an increase in mean % specific lysis of EBV pepmix-pulsed autologous activated T-cell targets from 5 to 66% at a 20:1 E:T ratio in the low vs. high dose IL-15 conditions (n=5). We have infused EBVSTs manufactured using all three conditions into 17 patients with multiply-relapsed, EBV-positive lymphoma as adjuvant therapy after stem cell transplantation or chemotherapy in 8 patients and as treatment for disease in 9 patients. Of patients in remission at the time of infusion, two with IL-4/7-grown EBVSTs and 6 with IL-15/7 grown EBVSTs remain in remission. Of patients with disease at the time of infusion, one receiving IL-4/7-grown EBVSTs had stable disease and 3 had progressive disease, while of 5 patients with IL-15/7-grown EBVSTs, one had stable disease, one had a partial response, one had a complete response and two are too early to assess. We will continue to modify our manufacturing process in an attempt to further increase the specificity and function of EBVSTs from patients with relapsed or refractory lymphoma.
723. Oncolytic Adenovirus Breaks Tumor Tolerance To Adoptive T-Cell Therapy By Recruiting Immune Cells To and Promoting Their Activity in the Tumor
Siri Tähtinen,1 Susanna Grönberg-Vähä-Koskela,1 Dave Lumen,3 Maiju Merisalo-Soikkeli,1 Mikko Siurala,1,2 Anu J. Airaksinen,3 Markus Vähä-Koskela,1 Akseli Hemminki.1,2 1 Cancer Gene Therapy Group, Department of Pathology and Transplantation Laboratory, Haartman Institute, University of Helsinki, Helsinki, Finland; 2TILT Biotherapeutics Ltd, Helsinki, Finland; 3Laboratory of Radiochemistry, Department of Chemistry, University of Helsinki, Helsinki, Finland. Background: Despite the fast growing rate in the development of novel adoptive T-cell therapies (ACT), the clinical benefit in established solid tumors has remained modest. Roadblocks to effective immune attack by infiltrating tumor-specific T-cells include several immune evasion mechanisms, which contribute to tumor tolerance. Oncolytic virotherapy on the other hand is the use of cancer cell specific, conditionally replicative viruses in the treatment of cancer. Oncolytic virotherapy has been suggested to enhance epitope spreading by direct oncolysis of tumor cells, leading to enhanced antigen presentation by dendritic cells and increased immunogenicity of the established tumor. Here, we studied the combination of these two established forms of immunotherapy in a highly resistant and poorly immunogenic B16.OVA mouse melanoma model. Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy