- Email: [email protected]
207. Variable Lymphocyte Receptors Enable Development of Chimeric Antigen Receptors for the Treatment of T-Cell Malignancies
Cancer-Targeted Gene and Cell Therapy I BBζ CARs demonstrate lower induction of the exhaustion marker PD-1 and proliferate better compare with 28ζ CARs. Both HER2CARs similarly lead to tumor eradication and prolonged survival. Based on these data, we have successfully developed HER2-specific CAR T cells, and plan to clinically develop these CARs for the treatment of HER2+ metastatic disease.
205. Cytotoxic and Immunotherapeutic Effects of Toca 511 and 5-Fluorocytosine in an Intraperitoneal Model of Metastatic Colorectal Cancer
Kader Yagiz, Daniel Mendoza, Maria Rodriguez-Aguirre, Fernando Lopez Espinoza, Carlos E. Ibañez, Harry E. Gruber, Douglas J. Jolly, Joan M. Robbins Tocagen Inc., San Diego, CA Toca 511 (vocimagene amiretrorepvec), a gamma retroviral replicating vector encoding an optimized yeast cytosine deaminase (CD) gene, selectively replicates and spreads in tumors. In infected cells, CD enzyme is expressed and converts 5-FC (5-fluorocytosine, an orally-available anti-fungal drug) to 5-FU (5-fluorouracil), leading to both direct tumor cytotoxicity and extended immunotherapeutic effects in preclinical models. Toca 511 in combination with Toca FC (extended-release 5-FC) recently entered a Phase 2/3 trial (NCT02414165) in patients with recurrent HGG (high grade glioma). Toca 511 is also under investigation delivered via intravenous (IV) infusion followed by injection into the wall of the resection cavity (NCT01985256), followed by Toca FC, in patients with recurrent HGG. IV administration of vectors is minimally invasive, can easily be repeated if desired, and may be applicable to other tumor types including metastatic colorectal cancer (mCRC). Previously we have shown that IV delivery of Toca 511 in a mouse syngeneic mCRC liver model resulted in expression of CD in tumor foci, but not in adjacent normal liver, and followed by courses of 5-FC resulted in direct tumor response, improved survival, and a systemic anti-tumor immune response. Elevated circulating myeloid-derived suppressor cells (MDSC) are associated with advanced disease stages in CRC patients and failure of immunotherapeutic strategies. We further investigated the effect of intralesional administration of Toca 511 with 5-FC treatment on tumor recurrence and immune infiltrates in a model of CRC brain metastases. A significant decrease in MDSC in spleens and tumors was observed with Toca 511 and 5-FC compared to controls, via in situ production of 5-FU (p=0.03, p<0.0001; respectively). Currently, we are investigating the efficacy of Toca 511 and 5-FC in a mouse syngeneic intraperitoneal (IP) model. We identified the optimal delivery method for treatment of IP metastases, and evaluated survival after IV and IP vector delivery followed by courses of 5-FC. Treatment with Toca 511 and 5-FC led to an improved median survival compared to control. Systemic 5-FU has hematological toxicity even at low doses (20 mg/kg) in both naïve and CRC tumor-bearing mice which could have an adverse effect on anti-tumor immune responses. We further evaluated Toca 511 and 5-FC treatment compared to systemic 5-FU in terms of efficacy, hematologic toxicity, and induction of anti-tumor immune responses in the IP mCRC model. The effect of Toca 511 and 5-FC compared to systemic 5-FU on MDSC and other immune cell populations will be presented. Our data provides support for the development of Toca 511 and 5-FC as a unique approach targeting both the tumor and the immune system for the treatment of metastatic cancers such as mCRC. A phase 1 study of IV Toca 511 and Toca FC in solid tumors, including mCRC, is planned (NCT02576665).
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206. Determining the Effect of Endogenous PD-1 Expression on the Co-Stimulatory Potential of the PD1:CD28 Chimera Megan E. Keys, Jonathan Arroyo, Bridgette Kielhack, Megan Prosser Department of Biology and Chemistry, Azusa Pacific University, Azusa, CA
Adoptive immunotherapy is an emerging field of study involving genetic alteration of the immune system to enhance its effectiveness in eliminating cancer. A previous study created a novel chimera (PD1:CD28) containing the extracellular portion of an inhibitory molecule, Programmed Death 1 (PD-1), and the intracellular portion of a co-stimulatory molecule, CD28, resulting in tumor induced costimulation of T cells. Given that endogenous PD-1 expression is likely to up-regulate on T cells in the tumor microenvironment, the current study aims to determine whether the presence of PD-1 on the cell surface negatively impacts T cell stimulation by the PD1:CD28 chimera. Endogenous PD1 was tagged with a red fluorescent protein, mCherry, and PD1:CD28 with a green fluorescent protein, GFP. Fusion proteins were transfected into H9 cells and positive expression was detected via flow cytometric analysis and fluorescence microscopy. Stable cell lines expressing both PD1:mCherry and PD1:CD28:GFP fusion proteins are being examined for downstream signaling markers, proliferation, and cytokine expression following exposure to Programmed Death Ligand 1 (PD-L1), as compared to cells engineered to express only PD1:mCherry or PD1:CD28:GFP. This study will provide further insight into the ability of the PD1:CD28 chimera to overcome PD-1 mediated tumor immune evasion.
Cancer-Targeted Gene and Cell Therapy I 207. Variable Lymphocyte Receptors Enable Development of Chimeric Antigen Receptors for the Treatment of T-Cell Malignancies
Sunil S. Raikar1, Daniel Tylawsky1, Robert Moot2, Lauren Fleischer2, David McCarty1, Christopher Doering1, H. Trent Spencer1 1 Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Emory University, Atlanta, GA, 2Graduate Program in Molecular and Systems Pharmacology, Emory University, Atlanta, GA Relapsed T-cell leukemia has a poor prognosis. Chimeric antigen receptor (CAR) therapy could be an effective treatment modality, although targeting T-cell disease without a T-lymphoblast specific antigen is difficult. The use of natural killer (NK) cells as the effector cell and CD5 as the target antigen provides a potential solution. While traditionally CARs use an immunoglobulin (Ig) based single chain variable fragment (scFV) to target tumor cells, we designed a novel CAR structure using a variable lymphocyte receptor (VLR) as our antigen recognition sequence. VLRs represent the functional unit of the adaptive immune system in jawless vertebrates (lamprey and hagfish) and are analogous but not homologous to immunoglobulins. VLRs have a fundamentally different structure and geometry compared to Ig-based antibodies while still demonstrating high degrees of specificity and avidity. Additionally, VLRs exist naturally as single chain structures that allows for rapid creation of CAR cassettes. In the current study our objective was to develop a VLR-CAR-NK cell against T-cell leukemia using an anti-CD5 VLR sequence. We constructed a second generation CAR using a VLR sequence targeting CD5 as our antigen recognition sequence. The CAR structure consisted of the anti-CD5 VLR sequence, the CD28 co-stimulatory transmembrane domain, and the intracellular CD3ζ signaling domain. Using this construct, high titer CD5 VLR Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
cANceR-tARgeted geNe ANd cell theRAPy i CAR self-inactivating lentivirus was produced at titers >10^8 TU/ ml. To test the functionality of the CAR construct, CD5 expressing Jurkat cells were transduced with various doses of lentivirus, and activation was measured by expression of CD69. For cytotoxicity studies, NK-92 cells were used as the effector cell with CCRF-CEM cells being the target cell. Cytotoxicity was measured at different effector: target ratios using a flow cytometry based assay. Activation positively correlated with lentiviral copy numbers and CAR expression in Jurkat cells. Both copy number and activation decreased over time, which was anticipated as we show that highly activated cells had a growth disadvantage. NK-92 cells expressing the CD5 VLR CAR showed approximately two fold increase in cytotoxicity towards CCRF-CEM cells when compared with naïve NK-92 cells; however, low transduction efficiency and copy numbers (<0.5) were problematic, similar to previous data showing that NK-92 cells are resistant to lentiviral transduction. In order to improve upon these results, we created a bicistrionic vector co-expressing GFP and the CD5-VLR-CAR. Jurkat cells genetically modified to express the new construct showed a direct correlation between GFP expression and activation, thus confirming dual expression and function of both proteins. Transduced NK-92 cells were sorted and expanded. These cells expand robustly, demonstrate CAR expression and have the functional characteristics required for targeting T-cell disease. Our studies show the utility of VLR-derived CARs and provide the foundation to progress these studies into preclinical testing as a treatment for relapsed T-cell leukemia.
tumor-lysate viral titration assays, respectively. Results. The results suggest that the viral payload does not interfere with the NSC tumortropism or penetration (Fig 1). NSC-mediated viral distribution was confirmed via immunohistochemistry, and the presence of active virus within lysed peritoneal tumors was assessed. Significant tumor killing was observed in both OVCAR8 and SKOV3 ovarian cancer cells after 5 days of culture with diluted tumor lysates (1:10 and 1:100) derived from mice that received NSC.CRad-S-pk7 injections (Fig 2). No killing was observed when cancer cells were cultured with tumor lysates derived from mice that received parental NSCs. These results demonstrate that NSC.CRad-S-pk7 can selectively penetrate peritoneal ovarian metastases and produce CRad-S-pk7 adenovirus following IP administration. Studies underway include free virus distribution and pharmacokinetic comparisons.
208. Neural Stem Cell Mediated Oncolytic Virotherapy for Ovarian Cancer
Asma Abdul Majid1, Rachael Mooney1, Megan Gilchrist1, Maciej Lesniak2, Karen Aboody1 1 Developmental and Stem Cell Biology, City of Hope, Duarte, CA, 2 Neuro-Oncology, Northwestern University, Evanston, IL Intro. Oncolytic virotherapy is a promising novel cancer treatment that uses replication-competent viruses to induce cancer cell death. While clinical trials are underway for a variety of solid tumors; success has been hampered by rapid immune-mediated clearance/ neutralization of the viral vectors, and poor viral distribution to tumor satellites dispersed throughout normal tissue. Neural stem cells (NSCs) are ideal cell carriers that could overcome viral delivery hurdles due to their intrinsic tumor-tropism and penetration capabilities. Our lab has established a well-characterized, nonimmunogenic human NSC line that can selectively distribute to many different solid tumors. Most recently, we observed impressive selectivity and penetration of peritoneal ovarian cancer metastases after intraperitoneal NSC administration. We have engineered our NSCs to produce a conditionally replication-competent adenovirus, CRad-Survivin-pk7 (NSC.CRad-S-pk7). This virus has two notable genetic modifications: (1) a polylysine fiber addition that enables high affinity binding to cell-surface proteoglycans, thus promoting viral entry into the target cell; and (2) a E1A transcriptional modification which prevents viral replication in the the absence of the surviving promoter, a gene over-expressed in many cancers. Clinical grade equivalent research banks of the NSC.CRad-S-pk7 cells have demonstrated safety and efficacy in orthotopic glioma models, but have not yet been tested in a metastatic ovarian cancer model. We hypothesize that NSCs are able to selectively distribute this virus to ovarian metastases, and provide protection from immune-mediated clearance and neutralization. Our long-term goal is to demonstrate efficacy and safety of NSC.CRad-S-pk7 for targeted selective tumor killing in patients suffering from stage III ovarian cancer. Methods. Two million NSC and NSC.CRad-S-pk7 cells were injected IP into nude mice containing established human peritoneal ovarian (OVCAR8 and SKOV3) metastases. After 7 days, tissue was assessed for viral distribution and activity using standard immunological and Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
209. Stem Cell/Nanoparticle Constructs for Targeted Ovarian Cancer Therapy
Pengpeng Cao1, Revathiswari Tirughana2, Ugochi Nwokafor2, Soraya Aramburo3, Linda Flores3, Jacob Berlin1, Karen S. Aboody3 1 Molecular Medicine, City of Hope, Duarte, CA, 2Develpmental and Stem Cell Biology, City of Hope, Duarte, CA, 3Developmental and Stem Cell Biology, City of Hope, Duarte, CA Intro. Targeted drug delivery is a critical goal for effective cancer therapy. Nanoparticles (NPs) have shown promise as platforms for S81
205. Cytotoxic and Immunotherapeutic Effects of Toca 511 and 5-Fluorocytosine in an Intraperitoneal Model of Metastatic Colorectal Cancer
Kader Yagiz, Daniel Mendoza, Maria Rodriguez-Aguirre, Fernando Lopez Espinoza, Carlos E. Ibañez, Harry E. Gruber, Douglas J. Jolly, Joan M. Robbins Tocagen Inc., San Diego, CA Toca 511 (vocimagene amiretrorepvec), a gamma retroviral replicating vector encoding an optimized yeast cytosine deaminase (CD) gene, selectively replicates and spreads in tumors. In infected cells, CD enzyme is expressed and converts 5-FC (5-fluorocytosine, an orally-available anti-fungal drug) to 5-FU (5-fluorouracil), leading to both direct tumor cytotoxicity and extended immunotherapeutic effects in preclinical models. Toca 511 in combination with Toca FC (extended-release 5-FC) recently entered a Phase 2/3 trial (NCT02414165) in patients with recurrent HGG (high grade glioma). Toca 511 is also under investigation delivered via intravenous (IV) infusion followed by injection into the wall of the resection cavity (NCT01985256), followed by Toca FC, in patients with recurrent HGG. IV administration of vectors is minimally invasive, can easily be repeated if desired, and may be applicable to other tumor types including metastatic colorectal cancer (mCRC). Previously we have shown that IV delivery of Toca 511 in a mouse syngeneic mCRC liver model resulted in expression of CD in tumor foci, but not in adjacent normal liver, and followed by courses of 5-FC resulted in direct tumor response, improved survival, and a systemic anti-tumor immune response. Elevated circulating myeloid-derived suppressor cells (MDSC) are associated with advanced disease stages in CRC patients and failure of immunotherapeutic strategies. We further investigated the effect of intralesional administration of Toca 511 with 5-FC treatment on tumor recurrence and immune infiltrates in a model of CRC brain metastases. A significant decrease in MDSC in spleens and tumors was observed with Toca 511 and 5-FC compared to controls, via in situ production of 5-FU (p=0.03, p<0.0001; respectively). Currently, we are investigating the efficacy of Toca 511 and 5-FC in a mouse syngeneic intraperitoneal (IP) model. We identified the optimal delivery method for treatment of IP metastases, and evaluated survival after IV and IP vector delivery followed by courses of 5-FC. Treatment with Toca 511 and 5-FC led to an improved median survival compared to control. Systemic 5-FU has hematological toxicity even at low doses (20 mg/kg) in both naïve and CRC tumor-bearing mice which could have an adverse effect on anti-tumor immune responses. We further evaluated Toca 511 and 5-FC treatment compared to systemic 5-FU in terms of efficacy, hematologic toxicity, and induction of anti-tumor immune responses in the IP mCRC model. The effect of Toca 511 and 5-FC compared to systemic 5-FU on MDSC and other immune cell populations will be presented. Our data provides support for the development of Toca 511 and 5-FC as a unique approach targeting both the tumor and the immune system for the treatment of metastatic cancers such as mCRC. A phase 1 study of IV Toca 511 and Toca FC in solid tumors, including mCRC, is planned (NCT02576665).
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206. Determining the Effect of Endogenous PD-1 Expression on the Co-Stimulatory Potential of the PD1:CD28 Chimera Megan E. Keys, Jonathan Arroyo, Bridgette Kielhack, Megan Prosser Department of Biology and Chemistry, Azusa Pacific University, Azusa, CA
Adoptive immunotherapy is an emerging field of study involving genetic alteration of the immune system to enhance its effectiveness in eliminating cancer. A previous study created a novel chimera (PD1:CD28) containing the extracellular portion of an inhibitory molecule, Programmed Death 1 (PD-1), and the intracellular portion of a co-stimulatory molecule, CD28, resulting in tumor induced costimulation of T cells. Given that endogenous PD-1 expression is likely to up-regulate on T cells in the tumor microenvironment, the current study aims to determine whether the presence of PD-1 on the cell surface negatively impacts T cell stimulation by the PD1:CD28 chimera. Endogenous PD1 was tagged with a red fluorescent protein, mCherry, and PD1:CD28 with a green fluorescent protein, GFP. Fusion proteins were transfected into H9 cells and positive expression was detected via flow cytometric analysis and fluorescence microscopy. Stable cell lines expressing both PD1:mCherry and PD1:CD28:GFP fusion proteins are being examined for downstream signaling markers, proliferation, and cytokine expression following exposure to Programmed Death Ligand 1 (PD-L1), as compared to cells engineered to express only PD1:mCherry or PD1:CD28:GFP. This study will provide further insight into the ability of the PD1:CD28 chimera to overcome PD-1 mediated tumor immune evasion.
Cancer-Targeted Gene and Cell Therapy I 207. Variable Lymphocyte Receptors Enable Development of Chimeric Antigen Receptors for the Treatment of T-Cell Malignancies
Sunil S. Raikar1, Daniel Tylawsky1, Robert Moot2, Lauren Fleischer2, David McCarty1, Christopher Doering1, H. Trent Spencer1 1 Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Emory University, Atlanta, GA, 2Graduate Program in Molecular and Systems Pharmacology, Emory University, Atlanta, GA Relapsed T-cell leukemia has a poor prognosis. Chimeric antigen receptor (CAR) therapy could be an effective treatment modality, although targeting T-cell disease without a T-lymphoblast specific antigen is difficult. The use of natural killer (NK) cells as the effector cell and CD5 as the target antigen provides a potential solution. While traditionally CARs use an immunoglobulin (Ig) based single chain variable fragment (scFV) to target tumor cells, we designed a novel CAR structure using a variable lymphocyte receptor (VLR) as our antigen recognition sequence. VLRs represent the functional unit of the adaptive immune system in jawless vertebrates (lamprey and hagfish) and are analogous but not homologous to immunoglobulins. VLRs have a fundamentally different structure and geometry compared to Ig-based antibodies while still demonstrating high degrees of specificity and avidity. Additionally, VLRs exist naturally as single chain structures that allows for rapid creation of CAR cassettes. In the current study our objective was to develop a VLR-CAR-NK cell against T-cell leukemia using an anti-CD5 VLR sequence. We constructed a second generation CAR using a VLR sequence targeting CD5 as our antigen recognition sequence. The CAR structure consisted of the anti-CD5 VLR sequence, the CD28 co-stimulatory transmembrane domain, and the intracellular CD3ζ signaling domain. Using this construct, high titer CD5 VLR Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
cANceR-tARgeted geNe ANd cell theRAPy i CAR self-inactivating lentivirus was produced at titers >10^8 TU/ ml. To test the functionality of the CAR construct, CD5 expressing Jurkat cells were transduced with various doses of lentivirus, and activation was measured by expression of CD69. For cytotoxicity studies, NK-92 cells were used as the effector cell with CCRF-CEM cells being the target cell. Cytotoxicity was measured at different effector: target ratios using a flow cytometry based assay. Activation positively correlated with lentiviral copy numbers and CAR expression in Jurkat cells. Both copy number and activation decreased over time, which was anticipated as we show that highly activated cells had a growth disadvantage. NK-92 cells expressing the CD5 VLR CAR showed approximately two fold increase in cytotoxicity towards CCRF-CEM cells when compared with naïve NK-92 cells; however, low transduction efficiency and copy numbers (<0.5) were problematic, similar to previous data showing that NK-92 cells are resistant to lentiviral transduction. In order to improve upon these results, we created a bicistrionic vector co-expressing GFP and the CD5-VLR-CAR. Jurkat cells genetically modified to express the new construct showed a direct correlation between GFP expression and activation, thus confirming dual expression and function of both proteins. Transduced NK-92 cells were sorted and expanded. These cells expand robustly, demonstrate CAR expression and have the functional characteristics required for targeting T-cell disease. Our studies show the utility of VLR-derived CARs and provide the foundation to progress these studies into preclinical testing as a treatment for relapsed T-cell leukemia.
tumor-lysate viral titration assays, respectively. Results. The results suggest that the viral payload does not interfere with the NSC tumortropism or penetration (Fig 1). NSC-mediated viral distribution was confirmed via immunohistochemistry, and the presence of active virus within lysed peritoneal tumors was assessed. Significant tumor killing was observed in both OVCAR8 and SKOV3 ovarian cancer cells after 5 days of culture with diluted tumor lysates (1:10 and 1:100) derived from mice that received NSC.CRad-S-pk7 injections (Fig 2). No killing was observed when cancer cells were cultured with tumor lysates derived from mice that received parental NSCs. These results demonstrate that NSC.CRad-S-pk7 can selectively penetrate peritoneal ovarian metastases and produce CRad-S-pk7 adenovirus following IP administration. Studies underway include free virus distribution and pharmacokinetic comparisons.
208. Neural Stem Cell Mediated Oncolytic Virotherapy for Ovarian Cancer
Asma Abdul Majid1, Rachael Mooney1, Megan Gilchrist1, Maciej Lesniak2, Karen Aboody1 1 Developmental and Stem Cell Biology, City of Hope, Duarte, CA, 2 Neuro-Oncology, Northwestern University, Evanston, IL Intro. Oncolytic virotherapy is a promising novel cancer treatment that uses replication-competent viruses to induce cancer cell death. While clinical trials are underway for a variety of solid tumors; success has been hampered by rapid immune-mediated clearance/ neutralization of the viral vectors, and poor viral distribution to tumor satellites dispersed throughout normal tissue. Neural stem cells (NSCs) are ideal cell carriers that could overcome viral delivery hurdles due to their intrinsic tumor-tropism and penetration capabilities. Our lab has established a well-characterized, nonimmunogenic human NSC line that can selectively distribute to many different solid tumors. Most recently, we observed impressive selectivity and penetration of peritoneal ovarian cancer metastases after intraperitoneal NSC administration. We have engineered our NSCs to produce a conditionally replication-competent adenovirus, CRad-Survivin-pk7 (NSC.CRad-S-pk7). This virus has two notable genetic modifications: (1) a polylysine fiber addition that enables high affinity binding to cell-surface proteoglycans, thus promoting viral entry into the target cell; and (2) a E1A transcriptional modification which prevents viral replication in the the absence of the surviving promoter, a gene over-expressed in many cancers. Clinical grade equivalent research banks of the NSC.CRad-S-pk7 cells have demonstrated safety and efficacy in orthotopic glioma models, but have not yet been tested in a metastatic ovarian cancer model. We hypothesize that NSCs are able to selectively distribute this virus to ovarian metastases, and provide protection from immune-mediated clearance and neutralization. Our long-term goal is to demonstrate efficacy and safety of NSC.CRad-S-pk7 for targeted selective tumor killing in patients suffering from stage III ovarian cancer. Methods. Two million NSC and NSC.CRad-S-pk7 cells were injected IP into nude mice containing established human peritoneal ovarian (OVCAR8 and SKOV3) metastases. After 7 days, tissue was assessed for viral distribution and activity using standard immunological and Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
209. Stem Cell/Nanoparticle Constructs for Targeted Ovarian Cancer Therapy
Pengpeng Cao1, Revathiswari Tirughana2, Ugochi Nwokafor2, Soraya Aramburo3, Linda Flores3, Jacob Berlin1, Karen S. Aboody3 1 Molecular Medicine, City of Hope, Duarte, CA, 2Develpmental and Stem Cell Biology, City of Hope, Duarte, CA, 3Developmental and Stem Cell Biology, City of Hope, Duarte, CA Intro. Targeted drug delivery is a critical goal for effective cancer therapy. Nanoparticles (NPs) have shown promise as platforms for S81