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625. Intratumoral Chemo Gene Therapy with Interleukin 12: Results of a Clinical Trial in Canines
CANCER-IMMUNOTHERAPY III
625. Intratumoral Chemo Gene Therapy with Interleukin 12: Results of a Clinical Trial in Canines
Jeffry J. Cutrera,1 Glenn King,2 Pamela Jones,2 Elias Gumpel,2 Kristin Kicenuik,2 Xueqing Xia,1 Shulin Li.1 1 Pediatrics-Research, The University of Texas MD Anderson Cancer Center, Houston, TX; 2Radiation Oncology, Gulf Coast Veterinary Specialists, Houston, TX.
Electroporation can increase the anti-tumor efficacy of both chemotherapeutics and interleukin 12 (IL12) gene therapies. Combining these treatments can further enhance the efficacy to treat refractory and treatment-resistant tumors. Based on the results of a successful trial with a single cycle of IL12 pDNA and bleomycin in canines, this new trial investigates the safety and efficacy of repeated cycles on recurrent tumors for long-term management of aggressive, refractory lesions. Furthermore this study investigates the potential for alternating the chemotherapeutic agent to overcome treatment resistance. Thirteen (13) canine subjects with naturally-occurring, accessible tumors were enrolled in this study and received multiple cycles of electro-chemo-gene therapy (ECGT) with IL-12 pDNA and either bleomycin or gemcitabine. ECGT treatments are very effective for inducing tumor regression via an antitumor immune response in most histotypes; however, sarcomas were not responsive. These treatments can quickly eradicate or debulk large squamous cell carcinomas, and the versatility of ECGT allows for response-based modifications which can overcome treatment resistance. Importantly, not a single severe adverse event was noted even in animals receiving the highest doses of chemotherapeutics and IL12 pDNA over multiple treatment cycles. The data reveal the importance of inducing a strong anti-tumor response for successfully affecting not only the treated tumors, but also non-treated metastatic tumors. ECGT with IL12 pDNA plus chemotherapy is an effective strategy for treating multiple types of spontaneous cancers including large, refractory, and/or multiple tumor burdens.
626. Tethered IL-15 on CD19-Specific T Cells Sustains Long-Term Persistence and Promotes a Stem Cell Memory-Like Phenotype
Lenka V. Hurton,1 Kirsten Switzer,1 Harjeet Singh,1 Marie-Andree Forget,1,2 Tiejuan Mi,1 Sourindra Maiti,1 Simon Olivares,1 Richard E. Champlin,3 Dean A. Lee,1 Laurence J. N. Cooper.1 1 Pediatrics-Research, MD Anderson Cancer Center, Houston, TX; 2Melanoma Medical Oncology, MD Anderson Cancer Center, Houston, TX; 3Stem Cell Transplantation and Cellular Therapy, MD Anderson Cancer Center, Houston, TX. Clinical responses after adoptive immunotherapy are associated with in vivo expansion and persistence of transferred antigen-specific T cells. Chimeric antigen receptor (CAR)-modified T cells receive activation, survival, and proliferative signaling via the introduced S242
immunoreceptor when docked with a tumor-associated antigen. Recipients that therapeutically benefit from CAR+ T cells experience persistence of the infused product. Sub-groups of T cells with potential for long-lived persistence include a stem-cell memory (TSCM) population. These clinically-appealing T cells represent a minority (23%) of peripheral blood (PB) and thus pose a challenge to isolate and manufacture for human application. We report an approach to render TSCM suitable for expression of CAR+ T cells based on our approach using Sleeping Beauty (SB) to stably insert the CAR transgene and selectively propagate genetically modified T cells on artificial antigen presenting cells (aAPC). It is known that interleukin (IL)-15 exerts a preferential effect on the homeostasis of CD8 memory T cells with recent work showing that IL-15 contributes to the generation of TSCM. We hypothesized that enforced IL-15 signaling may improve the engraftment of genetically modified CD19-specific CAR+ T cells with the preservation of a TSCM-like phenotype. To mimic physiologic cytokine signaling, we generated a tethered IL-15 expressed as a membrane-bound fusion protein of IL-15 and IL-15 receptor a (mIL15) on CAR+ T cells. This construct was electro-transferred with a 2nd generation CD19-specific CAR into primary human T cells. Clinically relevant numbers of mIL15+CAR+ T cells were generated and exhibited enhanced STAT5 activation. Engraftment and persistence of mIL15+CAR+ T cells in vivo was observed for 48 days without evidence of aberrant T-cell growth. In vitro assays showed that mIL15+CAR+ T cells surviving antigen withdrawal for >65 days (WD-mIL15+CAR+ T cells) maintained pSTAT5, aquired the capability to produce IL-2, and up-regulated gene expression associated with a less differentiated state (Tcf7, Lef1, Eomes, Bcl6, Bcl2, IL7ra, and Ccr7). Subset composition revealed that 33% of WD-mIL15+CAR+ T cells were CD45ROnegCCR7+CD95+CD122+, markers that are associated with TSCM. The in vitro WD-mIL15+CAR+ T-cell subset composition was recapitulated in vivo, where the PB and spleen contained the greatest proportion of CD45ROnegCCR7+ T cells. These data provide evidence that CAR+ T cells: (i) have the potential to persist long-term in an early differentiation state and (ii) can sustain this state with signaling induced solely by mIL15. Thus, it appears that mIL15 can sustain not only memory potential, but a TSCM-like state. The mIL15+CAR+ T cells may have clinical application to maintain T-cell persistence when tumor antigen is limiting such as when treating minimal residual disease or malignant disease in sanctuary sites.
627. Adenovirus Encoded Helicobacter pylori Neutrophil Activating Protein Promotes Maturation of DCs with Th-1 Polarization, Improved Antigen Presentation and Migration
Mohanraj Ramachandran,1 Chuan Jin,1 Di Yu,1 Fredrik Eriksson,1 Magnus Essand.1 1 Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
Helicobacter Pylori (H. Pylori) Neutrophil Activating Protein (HPNAP) is used as an adjuvant for vaccination and immunotherapeutic gene to enhance oncolytic virus efficacy. It is a chemoattractant for innate immune cells like neutrophils and monocytes and activates them to produce reactive oxygen species, chemokines (IL-8, MIP1 and MIP-1) and cytokines (IL-12 and IL-23). However, little is known about its mode of action to boost antigen specific immune activation. Dendritic cell (DC) is a key player in bridging innate and adaptive immune responses and here we aim to evaluate the effect of HP-NAP on DC maturation, migration and induce activation of adaptive immune system. Maturation markers CD80, CD86, HLA-DR, CD40, and CCR7 were upregulated on human DCs after treatment with supernatants from HP-NAP adenovirus-infected cells. HP-NAP-activated DCs had a Th1 cytokine secretion profile, with high IL-12 and low IL-10 secretion. Furthermore, HP-NAPMolecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
625. Intratumoral Chemo Gene Therapy with Interleukin 12: Results of a Clinical Trial in Canines
Jeffry J. Cutrera,1 Glenn King,2 Pamela Jones,2 Elias Gumpel,2 Kristin Kicenuik,2 Xueqing Xia,1 Shulin Li.1 1 Pediatrics-Research, The University of Texas MD Anderson Cancer Center, Houston, TX; 2Radiation Oncology, Gulf Coast Veterinary Specialists, Houston, TX.
Electroporation can increase the anti-tumor efficacy of both chemotherapeutics and interleukin 12 (IL12) gene therapies. Combining these treatments can further enhance the efficacy to treat refractory and treatment-resistant tumors. Based on the results of a successful trial with a single cycle of IL12 pDNA and bleomycin in canines, this new trial investigates the safety and efficacy of repeated cycles on recurrent tumors for long-term management of aggressive, refractory lesions. Furthermore this study investigates the potential for alternating the chemotherapeutic agent to overcome treatment resistance. Thirteen (13) canine subjects with naturally-occurring, accessible tumors were enrolled in this study and received multiple cycles of electro-chemo-gene therapy (ECGT) with IL-12 pDNA and either bleomycin or gemcitabine. ECGT treatments are very effective for inducing tumor regression via an antitumor immune response in most histotypes; however, sarcomas were not responsive. These treatments can quickly eradicate or debulk large squamous cell carcinomas, and the versatility of ECGT allows for response-based modifications which can overcome treatment resistance. Importantly, not a single severe adverse event was noted even in animals receiving the highest doses of chemotherapeutics and IL12 pDNA over multiple treatment cycles. The data reveal the importance of inducing a strong anti-tumor response for successfully affecting not only the treated tumors, but also non-treated metastatic tumors. ECGT with IL12 pDNA plus chemotherapy is an effective strategy for treating multiple types of spontaneous cancers including large, refractory, and/or multiple tumor burdens.
626. Tethered IL-15 on CD19-Specific T Cells Sustains Long-Term Persistence and Promotes a Stem Cell Memory-Like Phenotype
Lenka V. Hurton,1 Kirsten Switzer,1 Harjeet Singh,1 Marie-Andree Forget,1,2 Tiejuan Mi,1 Sourindra Maiti,1 Simon Olivares,1 Richard E. Champlin,3 Dean A. Lee,1 Laurence J. N. Cooper.1 1 Pediatrics-Research, MD Anderson Cancer Center, Houston, TX; 2Melanoma Medical Oncology, MD Anderson Cancer Center, Houston, TX; 3Stem Cell Transplantation and Cellular Therapy, MD Anderson Cancer Center, Houston, TX. Clinical responses after adoptive immunotherapy are associated with in vivo expansion and persistence of transferred antigen-specific T cells. Chimeric antigen receptor (CAR)-modified T cells receive activation, survival, and proliferative signaling via the introduced S242
immunoreceptor when docked with a tumor-associated antigen. Recipients that therapeutically benefit from CAR+ T cells experience persistence of the infused product. Sub-groups of T cells with potential for long-lived persistence include a stem-cell memory (TSCM) population. These clinically-appealing T cells represent a minority (23%) of peripheral blood (PB) and thus pose a challenge to isolate and manufacture for human application. We report an approach to render TSCM suitable for expression of CAR+ T cells based on our approach using Sleeping Beauty (SB) to stably insert the CAR transgene and selectively propagate genetically modified T cells on artificial antigen presenting cells (aAPC). It is known that interleukin (IL)-15 exerts a preferential effect on the homeostasis of CD8 memory T cells with recent work showing that IL-15 contributes to the generation of TSCM. We hypothesized that enforced IL-15 signaling may improve the engraftment of genetically modified CD19-specific CAR+ T cells with the preservation of a TSCM-like phenotype. To mimic physiologic cytokine signaling, we generated a tethered IL-15 expressed as a membrane-bound fusion protein of IL-15 and IL-15 receptor a (mIL15) on CAR+ T cells. This construct was electro-transferred with a 2nd generation CD19-specific CAR into primary human T cells. Clinically relevant numbers of mIL15+CAR+ T cells were generated and exhibited enhanced STAT5 activation. Engraftment and persistence of mIL15+CAR+ T cells in vivo was observed for 48 days without evidence of aberrant T-cell growth. In vitro assays showed that mIL15+CAR+ T cells surviving antigen withdrawal for >65 days (WD-mIL15+CAR+ T cells) maintained pSTAT5, aquired the capability to produce IL-2, and up-regulated gene expression associated with a less differentiated state (Tcf7, Lef1, Eomes, Bcl6, Bcl2, IL7ra, and Ccr7). Subset composition revealed that 33% of WD-mIL15+CAR+ T cells were CD45ROnegCCR7+CD95+CD122+, markers that are associated with TSCM. The in vitro WD-mIL15+CAR+ T-cell subset composition was recapitulated in vivo, where the PB and spleen contained the greatest proportion of CD45ROnegCCR7+ T cells. These data provide evidence that CAR+ T cells: (i) have the potential to persist long-term in an early differentiation state and (ii) can sustain this state with signaling induced solely by mIL15. Thus, it appears that mIL15 can sustain not only memory potential, but a TSCM-like state. The mIL15+CAR+ T cells may have clinical application to maintain T-cell persistence when tumor antigen is limiting such as when treating minimal residual disease or malignant disease in sanctuary sites.
627. Adenovirus Encoded Helicobacter pylori Neutrophil Activating Protein Promotes Maturation of DCs with Th-1 Polarization, Improved Antigen Presentation and Migration
Mohanraj Ramachandran,1 Chuan Jin,1 Di Yu,1 Fredrik Eriksson,1 Magnus Essand.1 1 Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
Helicobacter Pylori (H. Pylori) Neutrophil Activating Protein (HPNAP) is used as an adjuvant for vaccination and immunotherapeutic gene to enhance oncolytic virus efficacy. It is a chemoattractant for innate immune cells like neutrophils and monocytes and activates them to produce reactive oxygen species, chemokines (IL-8, MIP1 and MIP-1) and cytokines (IL-12 and IL-23). However, little is known about its mode of action to boost antigen specific immune activation. Dendritic cell (DC) is a key player in bridging innate and adaptive immune responses and here we aim to evaluate the effect of HP-NAP on DC maturation, migration and induce activation of adaptive immune system. Maturation markers CD80, CD86, HLA-DR, CD40, and CCR7 were upregulated on human DCs after treatment with supernatants from HP-NAP adenovirus-infected cells. HP-NAP-activated DCs had a Th1 cytokine secretion profile, with high IL-12 and low IL-10 secretion. Furthermore, HP-NAPMolecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy