- Email: [email protected]
418. Effective Treatment of Oligometastatic Melanoma Using Combination VSV Viroimmunotherapy and Stereotactic Radiotherapy
Cancer – Immunotherapy, Cancer Vaccines II
The 2nd and 3rd generation GPC3 CARs induced superior killing, cytokine release and T cell expansion compared to 1st generation GPC3 CAR. Importantly, 2nd and 3rd generation GPC3 CAR T cells demonstrated curative antitumor activity by eliminating established orthotopic GPC3-positive Huh-7 xenografts in NOD/SCID/IL2gnull (NSG) mice.
cancer immune therapeutic, or as a unique line of defense against tumor recurrence. There remains an urgent need to develop more potent hTERT vaccines. Here, we developed a synthetic highly optimized hTERT DNA vaccine (phTERT) to express full-length hTERT. When delivered by electroporation, phTERT elicited robust hTERT-specific CD8 response (the total response in phTERT-immunized mice was 1817 ± 211 SFU/106splenocytes). Epitope mapping results showed that 9 out of 26 matrix pools exhibited more than 50 spots, indicating phTERT could elicit a broad range of T-cell immune responses. The average percentage of CD107a, IFN-γ and TNF-α secreting cells from the total CD8+ T cell population were approximately 0.8%, 1.2% and 1.8%, respectively. Moreover, vaccination with phTERT slows the tumor growth in HPV16-associated tumor-bearing mice. Six out of ten phTERT-immunized mice still survived thirty-nine days post tumor implantation while all mice in naïve group were dead or euthanized. In vivo cytotoxicity assay confirmed that phTERTinduced CD8 cells exhibited specific CTL activity and were capable of eliminating hTERT-pulsed target cells. In addition, the ability of phTERT to overcome tolerance was evaluated in a NHP model, whose TERT protein is 96% homologous to that of hTERT. Immunized monkeys exhibited robust (average of 1834 SFU/106PBMCs), diverse (multiple immunodominant epitopes) IFN-γ responses and antigen-specific perforin release (average of 332 SFU/106 PBMCs), suggesting phTERT breaks tolerance and induces potent cytotoxic responses in this human relevant model. Lastly, we demonstrated that co-immunization with a rhesus macaque IL-12 plasmid as an adjuvant could further increase the vaccine-induced responses for about two fold in NHP. Taken together, these findings support that this synthetic EP-delivered DNA phTERT may have a role as a broad therapeutic cancer vaccine candidate.
418. Effective Treatment of Oligometastatic Melanoma Using Combination VSV Viroimmunotherapy and Stereotactic Radiotherapy
The results provide justification for testing safety and efficacy of GPC3 CAR T cells in a Phase 1/2a clinical trial in HCC patients.
417. Induction of Potent Cytotoxic and Antitumor Activity By a Highly Optimized hTERT DNA Vaccine
Jian Yan,1 Jaemi S. Chu,2 Nyamekye Obeng-Adjei,2 Matthrew P. Morrow,1 Kimberly Kraynyak,1 Anna M. Slager,1 Maria Yang,1 Amir S. Khan,1 Kate E. Broderick,1 Niranjan Y. Sardesai,1 David B. Weiner.2 1 Inovio Pharmaceuticals Inc., Plymouth Meeting, PA; 2 Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA. Immunotherapy of cancer through induction of anti-tumor cellular immunity has recently re-emerged as an important therapy for the treatment of nonresponsive cancers. Human telomerase reverse transcriptase (hTERT) is highly expressed in more than 85% of human tumors, with little or no expression in normal somatic cells. Expression of hTERT correlates well with telomerase activity and is associated with tumor cell growth and contributes crucially to the long-term survival of tumor cells. In addition, targeting hTERT may have the potential to eliminate cancer stem cells as recent studies have suggested that cancer stem or stem-like cells express hTERT. Immunological analysis supports that hTERT is a widely applicable target recognized by T cells and can be potentially studied as a broad Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
Kevin G. Shim,1 Rosa Diaz,1 Shane Zaidi,1 Miran Blanchard,2 Michael Grams,2 Timothy Kottke,1 Jill Thompson,1 Svetomir N. Markovic,3 Richard G. Vile.1 1 Molecular Medicine, Mayo Clinic, Rochester, MN; 2Radiation Oncology, Mayo Clinic, Rochester, MN; 3Medical Oncology, Mayo Clinic, Rochester, MN.
Oligometastatic (OM) disease represents an intermediate stage of progression between fully systemic metastasized disease and localized tumor burden. Clinical data is emerging that patients with OM are potentially curable, in part because of advances in the precision of radiation therapy treatment, and in part because of advances in the efficacy of immunotherapy on low burden systemic disease. We developed a model of OM melanoma by challenging mice both locally (subcutaneous injection) and systemically (intravenous injection) with B16 melanoma cells, resulting in both local tumor burden and a small number of prominent, systemically distributed lung metastases. We used this model to investigate whether systemic viroimmunotherapy, using Vesicular Stomatitis Virus (VSV) expressing tumor associated antigens to treat low level metastatic tumor burden, could be combined with clinically relevant stereotactic body radiation therapy (SBRT) to treat advanced local disease. In this respect, mice treated with only one treatment modality (systemic VSV-TAA, or local SBRT) needed to be sacrificed reproducibly due to progression of the untreated disease (local tumor or systemic metastatic disease, respectively). In contrast, mice treated with a combination of VSV-TAA and SBRT entered long-term remission resulting in apparent cure of tumor in up to 100% of mice depending upon the experiment. Increased lymphocyte, and CD45+ leukocyte infiltration was observed in tumors following both SBRT and VSV therapy 6-10 days following therapy. Experiments S165
Cancer – Immunotherapy, Cancer Vaccines II to delineate the role of immune cell subsets in the successful therapy are underway. In a significant proportion of mice, systemic VSVTAA therapy was able to control local tumor, provided it was small at the start of therapy. SBRT was rarely able to have an impact upon the treatment of metastatic disease suggesting that, in this model, radiotherapy was not the dominant immunotherapy. However, treating mice with VSV-TAA was able to stimulate a detectable anti-tumor T-cell IFN-γ memory response. We plan to further investigate the immunogenicity of VSV-TAA treatment in the context of SBRT as part of a prime-boost immunotherapy strategy for OM disease. Therefore, we have developed a successful therapeutic regime to treat oligometastic disease that combines both radiotherapy to treat local disease, and systemic viroimmunotherapy to treat low-level metastatic disease and further studies to potentiate the anti-tumor effector response in vivo are in progress.
419. Allogenic CAR T-Cells Targeting CD123 for Adoptive Immunotherapy of Acute Myeloid Leukemia (AML)
Roman Galetto,1 Céline Lebuhotel,1 Patricia Françon,1 Agnès Gouble,1 Julianne Smith.1 1 Cellectis, Paris, France.
Chimeric antigen receptor (CAR)-redirected T-cells have given rise to long-term durable remissions and remarkable objective response rates in patients with refractory leukemia, raising hopes that a wider application of CAR technology may lead to a new paradigm in cancer treatment. A limitation of the current autologous approach is that CAR T-cells must be manufactured on a “per patient basis”. We have developed a standardized platform for manufacturing T-cells from third-party healthy donors to generate allogeneic “off-theshelf” engineered CAR+ T-cell–based frozen products. This platform utilizes Transcription Activator-Like Effector Nuclease (TALEN) gene editing technology to inactivate the TCRα constant (TRAC) gene, eliminating the potential for T-cells bearing alloreactive TCR’s to mediate Graft versus Host Disease (GvHD). We have previously demonstrated that editing of the TRAC gene can be achieved at high frequencies, obtaining up to 80% of TCRα negative cells. This allows us to efficiently produce TCR-deficient T-cells that have been shown to no longer mediate alloreactivity in a xeno-GvHD mouse model. In addition to CAR expression, our T-cells are engineered to co-express the RQR8 gene as a safety feature, with the aim of rendering them sensitive to the monoclonal antibody rituximab. In this work we present the adaptation of this allogeneic platform to the production of T cells targeting CD123, the transmembrane alpha chain of the interleukin-3 receptor, which is expressed in tumor cells from the majority of patients with Acute Myeloid Leukemia (AML). To identify an effective CAR targeting CD123, we have screened multiple antigen recognition domains in the context of several different CAR architectures to identify candidates displaying activity against cell lines expressing variable levels of the CD123 antigen. Furthermore, experiments in an AML mouse model using anti-CD123 CAR T cells demonstrate important anti-tumor activity in vivo. The ability to carry out large scale manufacturing of allogeneic, non alloreactive CD123 specific T cells from a single healthy donor will thus offer the possibility of an off-the-shelf treatment that would be immediately available for administration to a large number of AML patients.
S166
420. A Promising Neo-Adjuvant ImmunoTherapeutic Agent for Preventing Cancer Recurrence and Metastasis After Surgery
Jahangir Ahmed,1 Ming Yuan,1 Yuenan Li,2 Yongchao Chu,2 Zhongxian Zhang,2 Dongling Gao,2 Louisa S. Chard,1 Gus Alusi,1 Nicholas R. Lemoine,1,2 Yaoh Wang.1,2 1 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, United Kingdom; 2SinoBritish Research Centre for Molecular Oncology, Zhengzhou University, Zhengzhou, China. Local recurrence and remote metastasis are major challenges to overcome for improving the survival of cancer patients after surgery. The main source for these is the minimal residual disease (MRD) that remains in situ after resection in microscopic deposits beyond the clearance margins and in micrometastases (lymphatic or haematogenous). There is increasing evidence that surgical intervention can actually promote tumour recurrence and distant metastass by several mechanisms including suppression of NK cells and prominent Th2 polarisation. Tumour-targeted oncolytic viruses (TOVs) are attractive therapeutics for cancer because they selectively amplify through replication and spread the input dose of virus in the target tumour. TOVs not only kill the tumorr cells by direct lysis but also attack them through multiple other mechanisms of action, especially breaking down the immuno-suppressive tumor microenvironment and inducing a long-lasting tumour-specific immunity. In addition, TOVs can specifically deliver therapeutic proteins into tumours at increasing levels following viral replication within the malignant cells. We have recently created a novel tumourtargeted oncolytic vaccinia virus (VVDTKDN1L). Treatment with this virus resulted in strong NK cell activation and potent tumourspecific immunity, and dramatically reduced lung metastasis after surgery in several cancer models when used as a neo-adjuvant therapeutic agent. To improve its anti-tumour efficacy further, we armed VVDTKDN1L with several immunomodulatory genes. Here we demonstrated that VVDTKDN1L-IL12 is the most effective therapeutic agent and resulted in complete eradication of tumours in 86% of mice bearing subcutaneous pancreatic cancer tumours. Strikingly, five daily treatments with the VVDTKDN1L-IL12 before surgery dramatically improved animal survival and even cured most animals whereas the GM-CSF-armed virus did not show benefit compared to the unarmed backbone virus. These data provide proof of concept for translation of the regime into clinical trials. This new approach may significantly improve the survival of cancer patients in the future.
421. A Multidrug Resistant Engineered CAR T Cell for Allogeneic Combination Immunotherapy
Julien Valton,1 Valerie Guyot,1 Alan Marechal,1 Jean-Marie Filhol,1 Alexandre Juillerat,1 Aymeric Duclert,1 Philippe Duchateau,1 Laurent Poirot.1 1 Cellectis SA, Paris, France. The adoptive transfer of CAR T cell represents a highly promising strategy to fight against multiple cancers. The clinical outcome of such therapies is intimately linked to the ability of effector cells to engraft, proliferate and specifically kill tumor cells within patients. When allogeneic CAR T cell infusion is considered, host versus graft and graft versus host reactions must be avoided to prevent rejection of adoptively transferred cells, host tissue damages and to elicit significant antitumoral outcome. This work proposes to address these three requirements through the development of multidrug resistant TCRαΒ-deficient CAR T cells. We demonstrate that these engineered T cells displayed efficient antitumor activity and proliferated in the presence of drugs, currently used in clinic as preconditioning Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
The 2nd and 3rd generation GPC3 CARs induced superior killing, cytokine release and T cell expansion compared to 1st generation GPC3 CAR. Importantly, 2nd and 3rd generation GPC3 CAR T cells demonstrated curative antitumor activity by eliminating established orthotopic GPC3-positive Huh-7 xenografts in NOD/SCID/IL2gnull (NSG) mice.
cancer immune therapeutic, or as a unique line of defense against tumor recurrence. There remains an urgent need to develop more potent hTERT vaccines. Here, we developed a synthetic highly optimized hTERT DNA vaccine (phTERT) to express full-length hTERT. When delivered by electroporation, phTERT elicited robust hTERT-specific CD8 response (the total response in phTERT-immunized mice was 1817 ± 211 SFU/106splenocytes). Epitope mapping results showed that 9 out of 26 matrix pools exhibited more than 50 spots, indicating phTERT could elicit a broad range of T-cell immune responses. The average percentage of CD107a, IFN-γ and TNF-α secreting cells from the total CD8+ T cell population were approximately 0.8%, 1.2% and 1.8%, respectively. Moreover, vaccination with phTERT slows the tumor growth in HPV16-associated tumor-bearing mice. Six out of ten phTERT-immunized mice still survived thirty-nine days post tumor implantation while all mice in naïve group were dead or euthanized. In vivo cytotoxicity assay confirmed that phTERTinduced CD8 cells exhibited specific CTL activity and were capable of eliminating hTERT-pulsed target cells. In addition, the ability of phTERT to overcome tolerance was evaluated in a NHP model, whose TERT protein is 96% homologous to that of hTERT. Immunized monkeys exhibited robust (average of 1834 SFU/106PBMCs), diverse (multiple immunodominant epitopes) IFN-γ responses and antigen-specific perforin release (average of 332 SFU/106 PBMCs), suggesting phTERT breaks tolerance and induces potent cytotoxic responses in this human relevant model. Lastly, we demonstrated that co-immunization with a rhesus macaque IL-12 plasmid as an adjuvant could further increase the vaccine-induced responses for about two fold in NHP. Taken together, these findings support that this synthetic EP-delivered DNA phTERT may have a role as a broad therapeutic cancer vaccine candidate.
418. Effective Treatment of Oligometastatic Melanoma Using Combination VSV Viroimmunotherapy and Stereotactic Radiotherapy
The results provide justification for testing safety and efficacy of GPC3 CAR T cells in a Phase 1/2a clinical trial in HCC patients.
417. Induction of Potent Cytotoxic and Antitumor Activity By a Highly Optimized hTERT DNA Vaccine
Jian Yan,1 Jaemi S. Chu,2 Nyamekye Obeng-Adjei,2 Matthrew P. Morrow,1 Kimberly Kraynyak,1 Anna M. Slager,1 Maria Yang,1 Amir S. Khan,1 Kate E. Broderick,1 Niranjan Y. Sardesai,1 David B. Weiner.2 1 Inovio Pharmaceuticals Inc., Plymouth Meeting, PA; 2 Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA. Immunotherapy of cancer through induction of anti-tumor cellular immunity has recently re-emerged as an important therapy for the treatment of nonresponsive cancers. Human telomerase reverse transcriptase (hTERT) is highly expressed in more than 85% of human tumors, with little or no expression in normal somatic cells. Expression of hTERT correlates well with telomerase activity and is associated with tumor cell growth and contributes crucially to the long-term survival of tumor cells. In addition, targeting hTERT may have the potential to eliminate cancer stem cells as recent studies have suggested that cancer stem or stem-like cells express hTERT. Immunological analysis supports that hTERT is a widely applicable target recognized by T cells and can be potentially studied as a broad Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
Kevin G. Shim,1 Rosa Diaz,1 Shane Zaidi,1 Miran Blanchard,2 Michael Grams,2 Timothy Kottke,1 Jill Thompson,1 Svetomir N. Markovic,3 Richard G. Vile.1 1 Molecular Medicine, Mayo Clinic, Rochester, MN; 2Radiation Oncology, Mayo Clinic, Rochester, MN; 3Medical Oncology, Mayo Clinic, Rochester, MN.
Oligometastatic (OM) disease represents an intermediate stage of progression between fully systemic metastasized disease and localized tumor burden. Clinical data is emerging that patients with OM are potentially curable, in part because of advances in the precision of radiation therapy treatment, and in part because of advances in the efficacy of immunotherapy on low burden systemic disease. We developed a model of OM melanoma by challenging mice both locally (subcutaneous injection) and systemically (intravenous injection) with B16 melanoma cells, resulting in both local tumor burden and a small number of prominent, systemically distributed lung metastases. We used this model to investigate whether systemic viroimmunotherapy, using Vesicular Stomatitis Virus (VSV) expressing tumor associated antigens to treat low level metastatic tumor burden, could be combined with clinically relevant stereotactic body radiation therapy (SBRT) to treat advanced local disease. In this respect, mice treated with only one treatment modality (systemic VSV-TAA, or local SBRT) needed to be sacrificed reproducibly due to progression of the untreated disease (local tumor or systemic metastatic disease, respectively). In contrast, mice treated with a combination of VSV-TAA and SBRT entered long-term remission resulting in apparent cure of tumor in up to 100% of mice depending upon the experiment. Increased lymphocyte, and CD45+ leukocyte infiltration was observed in tumors following both SBRT and VSV therapy 6-10 days following therapy. Experiments S165
Cancer – Immunotherapy, Cancer Vaccines II to delineate the role of immune cell subsets in the successful therapy are underway. In a significant proportion of mice, systemic VSVTAA therapy was able to control local tumor, provided it was small at the start of therapy. SBRT was rarely able to have an impact upon the treatment of metastatic disease suggesting that, in this model, radiotherapy was not the dominant immunotherapy. However, treating mice with VSV-TAA was able to stimulate a detectable anti-tumor T-cell IFN-γ memory response. We plan to further investigate the immunogenicity of VSV-TAA treatment in the context of SBRT as part of a prime-boost immunotherapy strategy for OM disease. Therefore, we have developed a successful therapeutic regime to treat oligometastic disease that combines both radiotherapy to treat local disease, and systemic viroimmunotherapy to treat low-level metastatic disease and further studies to potentiate the anti-tumor effector response in vivo are in progress.
419. Allogenic CAR T-Cells Targeting CD123 for Adoptive Immunotherapy of Acute Myeloid Leukemia (AML)
Roman Galetto,1 Céline Lebuhotel,1 Patricia Françon,1 Agnès Gouble,1 Julianne Smith.1 1 Cellectis, Paris, France.
Chimeric antigen receptor (CAR)-redirected T-cells have given rise to long-term durable remissions and remarkable objective response rates in patients with refractory leukemia, raising hopes that a wider application of CAR technology may lead to a new paradigm in cancer treatment. A limitation of the current autologous approach is that CAR T-cells must be manufactured on a “per patient basis”. We have developed a standardized platform for manufacturing T-cells from third-party healthy donors to generate allogeneic “off-theshelf” engineered CAR+ T-cell–based frozen products. This platform utilizes Transcription Activator-Like Effector Nuclease (TALEN) gene editing technology to inactivate the TCRα constant (TRAC) gene, eliminating the potential for T-cells bearing alloreactive TCR’s to mediate Graft versus Host Disease (GvHD). We have previously demonstrated that editing of the TRAC gene can be achieved at high frequencies, obtaining up to 80% of TCRα negative cells. This allows us to efficiently produce TCR-deficient T-cells that have been shown to no longer mediate alloreactivity in a xeno-GvHD mouse model. In addition to CAR expression, our T-cells are engineered to co-express the RQR8 gene as a safety feature, with the aim of rendering them sensitive to the monoclonal antibody rituximab. In this work we present the adaptation of this allogeneic platform to the production of T cells targeting CD123, the transmembrane alpha chain of the interleukin-3 receptor, which is expressed in tumor cells from the majority of patients with Acute Myeloid Leukemia (AML). To identify an effective CAR targeting CD123, we have screened multiple antigen recognition domains in the context of several different CAR architectures to identify candidates displaying activity against cell lines expressing variable levels of the CD123 antigen. Furthermore, experiments in an AML mouse model using anti-CD123 CAR T cells demonstrate important anti-tumor activity in vivo. The ability to carry out large scale manufacturing of allogeneic, non alloreactive CD123 specific T cells from a single healthy donor will thus offer the possibility of an off-the-shelf treatment that would be immediately available for administration to a large number of AML patients.
S166
420. A Promising Neo-Adjuvant ImmunoTherapeutic Agent for Preventing Cancer Recurrence and Metastasis After Surgery
Jahangir Ahmed,1 Ming Yuan,1 Yuenan Li,2 Yongchao Chu,2 Zhongxian Zhang,2 Dongling Gao,2 Louisa S. Chard,1 Gus Alusi,1 Nicholas R. Lemoine,1,2 Yaoh Wang.1,2 1 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, United Kingdom; 2SinoBritish Research Centre for Molecular Oncology, Zhengzhou University, Zhengzhou, China. Local recurrence and remote metastasis are major challenges to overcome for improving the survival of cancer patients after surgery. The main source for these is the minimal residual disease (MRD) that remains in situ after resection in microscopic deposits beyond the clearance margins and in micrometastases (lymphatic or haematogenous). There is increasing evidence that surgical intervention can actually promote tumour recurrence and distant metastass by several mechanisms including suppression of NK cells and prominent Th2 polarisation. Tumour-targeted oncolytic viruses (TOVs) are attractive therapeutics for cancer because they selectively amplify through replication and spread the input dose of virus in the target tumour. TOVs not only kill the tumorr cells by direct lysis but also attack them through multiple other mechanisms of action, especially breaking down the immuno-suppressive tumor microenvironment and inducing a long-lasting tumour-specific immunity. In addition, TOVs can specifically deliver therapeutic proteins into tumours at increasing levels following viral replication within the malignant cells. We have recently created a novel tumourtargeted oncolytic vaccinia virus (VVDTKDN1L). Treatment with this virus resulted in strong NK cell activation and potent tumourspecific immunity, and dramatically reduced lung metastasis after surgery in several cancer models when used as a neo-adjuvant therapeutic agent. To improve its anti-tumour efficacy further, we armed VVDTKDN1L with several immunomodulatory genes. Here we demonstrated that VVDTKDN1L-IL12 is the most effective therapeutic agent and resulted in complete eradication of tumours in 86% of mice bearing subcutaneous pancreatic cancer tumours. Strikingly, five daily treatments with the VVDTKDN1L-IL12 before surgery dramatically improved animal survival and even cured most animals whereas the GM-CSF-armed virus did not show benefit compared to the unarmed backbone virus. These data provide proof of concept for translation of the regime into clinical trials. This new approach may significantly improve the survival of cancer patients in the future.
421. A Multidrug Resistant Engineered CAR T Cell for Allogeneic Combination Immunotherapy
Julien Valton,1 Valerie Guyot,1 Alan Marechal,1 Jean-Marie Filhol,1 Alexandre Juillerat,1 Aymeric Duclert,1 Philippe Duchateau,1 Laurent Poirot.1 1 Cellectis SA, Paris, France. The adoptive transfer of CAR T cell represents a highly promising strategy to fight against multiple cancers. The clinical outcome of such therapies is intimately linked to the ability of effector cells to engraft, proliferate and specifically kill tumor cells within patients. When allogeneic CAR T cell infusion is considered, host versus graft and graft versus host reactions must be avoided to prevent rejection of adoptively transferred cells, host tissue damages and to elicit significant antitumoral outcome. This work proposes to address these three requirements through the development of multidrug resistant TCRαΒ-deficient CAR T cells. We demonstrate that these engineered T cells displayed efficient antitumor activity and proliferated in the presence of drugs, currently used in clinic as preconditioning Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy