- Email: [email protected]
191. Amelioration of Colitis and Its Associated Colorectal Tumors by CEA — Redirected Tregs
CANCER – IMMUNOTHERAPY I of mature T-cells has yielded clinical response, the failure of this approach to achieve consistent, complete response is thought to lie in the detrimental ex vivo expansion required to attain sufficient numbers of cells for efficacy. Cell expansion with high dose IL-2 results in the biased amplification of effector T-cells with the exhaustion of earlier, more replication competent central memory T-cells. We propose that the addition of a tumor-specific TCR into hematopoietic stem cells should provide patients with a supply of earlier T-cell progenitors which, due to their increased replicative capacity, are capable of populating the periphery with anti-tumor T-cells for the life of the patient. We have utilized the humanized NSG mouse model as a platform to study the feasibility of gene therapy to treat melanoma. Neonatal NSG mice were transplanted with human CD34+ cells purified from umbilical cord blood (CB) or GM-CSFmobilized peripheral blood (PBSC) transduced with lentivirus encoding a TCR that recognizes the human melanocyte MART1 antigen (TCR was cloned from a patient that had autoreactivity to melanoma). Transplanted mice were monitored monthly for immune reconstitution by polychromatic flow cytometry. We observed that PBSC were approximately 5 times less efficient at reconstitution of the lymphoid compartment compared with CB, consistent with previously published findings. Human B-cells were present as early as 2 months post transplant, while T-cells were present in the periphery at approximately 3 months. T-cells were further characterized for CD4 and CD8 phenotype as well as the markers of naïve (CD45RA) or experienced (CD45RO) cells. While mice were housed in a pathogenfree environment, we were surprised to find that the majority of CD4 and CD8 cells expressed a CD45RO phenotype. To rule out that these cells simply resulted from an expansion of human T-cells contaminating the original CB or PBSC graft, we assayed for T-cell recombinant excision circles (TREC). TREC were detectable in mouse spleens, indicating de novo generation of T-cells is occurring in this mouse model. We observed MART1-TCR expressing T-cells, indicated by MART-1 tetramer staining, represented approximately 1-10% of total CD4 and CD8 cells. To test the function of these T-cells, mice challenged with subcutaneous human tumor cell lines expressing MART1 antigen showed a decreased tumor mass that correlated with the total percentage of MART1 expressing T-cells. Our future work will be to more completely characterize T-cell phenotype in these mice and determine the implications of these populations on tumor clearance. This work was funded by NIH NCI CA132681.
189. CD40-Ligand Armed Vaccinia Virus for Induction of Anti-Tumor Immune Response
Marko Ahonen,1 Suvi Parviainen,1 Iulia Diaconu,1 Vincenzo Cerullo,1 Akseli Hemminki.1 1 Molecular Cancer Biology Program, University of Helsinki, Finland. Immunotherapy of cancer has resulted in recent clinical successes validating the potential of the approach. A key realization has been that induction of an anti-tumor immune response is not enough, but reduction of tumor immune suppressiveness is also required. Oncolytic vaccinia virus is an attractive platform for immunotherapy because virus replication is immunogenic and not subject to tolerance. Oncolysis releases tumor antigens and provides costimulatory danger signals. However, arming the virus can improve efficacy further. CD40 ligand (CD40L, CD154) can induce apoptosis of tumor cells and it also triggers several immune mechanisms. One of these is a T-helper type 1 (Th1) response which leads to activation of cytotoxic T-cells and reduction of immune suppression. Therefore, we constructed a vaccinia oncolytic virus expressing hCD40L (vvDD-CD40L), which in addition features a cDNA expressing the tdtomato fluorochrome for an easy identification of the virus in vitro and in vivo and a deletion of the TK region to completely avoid the possibility of recombination to generate a wild type TK gene. We Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
show effective expression of functional CD40L both in vitro and in vivo. In a xenograph model of bladder carcinoma sensitive to CD40L treatment, we show that growth of tumors was significantly inhibited by the oncolysis and apoptosis exerted by vv-DD-CD40L following both intravenous and intratumoral administration of the virus. The advantage of the expression of CD40L was reduced if not abolished in tumor model not particularly sensitive to CD40L treatment such as M4A4-LM3 breast tumors. To deeply dissect the immune system component of vvdd-CD40L we generated a new version of it coding for the murine version of CD40L (vvdd-mCD40L). With this reagents we were able to study the different effects of the transgene on the composition of tumor stroma for what concerns tumor-specific T cells, B cells, NK cells and ultimately dendritic cells. In summary, oncolytic vaccinia virus coding for CD40L mediate multiple antitumor effects including oncolysis, apoptosis and induction of T-cell responses through upregulation of Th1 cytokines.
190. Analysis of Gene Expression in Adoptively Transferred TCR-Engineered Lymphocytes Daniel Abate-Daga,1 Steven A. Rosenberg,1 Richard A. Morgan.1 Surgery Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD.
1
In recent years it has been demonstrated that adoptive transfer of highly activated autologous T cells, engineered ex vivo to express tumor-targeting T cell receptors (TCR), can mediate objective tumor regressions. Although significant progress has been made concerning the selection of TCR and manufacturing of cellular products, little is known about the fate of these cells after infusion. In the present study we analyzed the expression of 84 genes relevant to T cell function in T lymphocytes isolated one month after infusion, and compared it to their expression in matched infusion preparations. Engineered T cells were purified, using FACS-sorting, from peripheral blood of ten patients that received gp100- or MAGE A3-specific TCR-transduced lymphocytes. Transcriptomic analysis was performed by Q-RT-PCR, allowing for the identification of 37 differentially expressed genes (fold change >2, p<0.05) including: 1) Anergy-related genes encoding for ubiquitin-ligases ITCH and CBLB and kinase DGKA, that were upregulated in post-infusion lymphocytes 2) FoxP1, a transcription factor involved in the maintenance of T cell quiescence, that was dramatically down regulated in infusion samples, but up-regulated in post-infusion cells both at mRNA and protein levels. 3) A group of functionally related genes (TNFRSF14, TNFSF14 and LTA), known to modulate T cell signaling, whose expression was tightly regulated at the RNA. Co-stimulatory genes LTA and TNFSF14 were under-expressed in post-infusion cells, whereas their binding partner TNFRSF14 was over-expressed. Another functionally related gene, CD160, which encodes for a gpi-anchored binding partner of TNFRSF14 was also found to be over-expressed in post-infusion cells, and its surface expression was associated with a reduced production of IFN-gamma by T cells when stimulated with relevant targets. These results may contribute to a more thorough understanding of the molecular processes governing the fate of adoptively transferred T cells and their implications for treatment efficacy and potential toxicities. These may also open an opportunity for interventions based on modulation of gene expression, as a potential method to improve adoptive cell therapy for cancer.
191. Amelioration of Colitis and Its Associated Colorectal Tumors by CEA – Redirected Tregs
Zelig Eshhar,1 Ehud Zigmond,1 Dan Blat.1 Immunology, The Weizmann Institute of Science, Rehovot, Israel.
1
Ulcerative colitis is a prominent example of a chronic inflammatory disease associated with high risk for cancer development. Our previous animal studies have shown that adoptive transfer of S75
CANCER – IMMUNOTHERAPY I chimeric, antibody-type receptor (CAR) redirected CD4+CD25 + regulatory T cells (Treg T-bodies) can prevent, ameliorate and even cure acute colitis. Here we used carcinoembryonic antigen (CEA), a well known human tumor marker which also exhibits tumor-like expression patterns in inflammatory bowel disease patients and in CEA-transgenic (Tg) mice that were induced to develop colitisassociated colorectal cancer by dextran sodium sulfate-azoxymethane (DSS-AOM). We have now demonstrated that adoptive transfer of CEA-specific Treg T-bodies could ameliorate colitis and/or hinder the subsequent appearance of colorectal tumors. Moreover, while systemic administration of CEA-specific CD4+ Teff T-bodies to naïve, CEA Tg mice caused colitis with a chronic manifestation, colonoscopically-assisted intratumoral administration of CD8+ Teff T-bodies into the colorectal tumors showed a potent, antigen-specific anti-tumor effect.
192. Naive-Derived Memory Stem T Cells: A Novel Promising Platform for Cancer ImmuneGene Therapy
Nicoletta Cieri,1 Barbara Camisa,1 Fabienne Cocchiarella,2 Mattia Forcato,3 Elena Provasi,1 Anna Mondino,4 Attilio Bondanza,1 Claudio Bordignon,5 Fulvio Mavilio,2 Silvio Bicciato,3 Alessandra Recchia,2 Chiara Bonini.1 1 Experimental Hematology Unit, Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, Milan, Italy; 2Center for Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy; 3 Center for Genome Research, Department of Biomedical Sciences, University of Modena and Reggio Emilia, Milan, Italy; 4Lymphocyte Activation Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy; 5MolMed SpA, Milan, Italy. Memory stem T cells (TSCM) with the ability to self renew and the plasticity to differentiate into potent effector cells represent ideal weapons to treat cancer. Nonetheless, clinical-grade procedures to specifically target this T-cell population remain elusive. Here we report that it is possible and feasible to differentiate in vitro, vigorously expand and genetically engineer TSCM lymphocytes in clinically compliant conditions starting from naïve precursors. Requirements for the generation of this T-cell subset, best described as CD62L+ CCR7+ CD45RA+ CD45R0+ IL-7Rα+ CD95+, are CD3/CD28 engagement and culture with IL-7 and IL-15. In contrast with other proposed protocols to generate TSCM, our method is able to sustain a significant expansion of this cell population, an essential prerequisite for any immune-gene therapy approach. The gene expression profile and functional phenotype validate this cell population as a distinct memory T lymphocyte subset, hierarchically superior to central memory (TCM) and effector memory lymphocytes. To better define naive-derived TSCM function and potential in a clinically relevant context, we evaluated their ability to mediate xenogenic GvHD in vivo. Indeed, naïve-derived and gene-modified TSCM prove superior in vivo expansion and persistence than TCM manipulated with the same protocol, and are able to differentiate into effector cells, mediating a lethal xenogeneic GvHD with the same kinetics and intensity of unmanipulated lymphocytes. Furthermore, gene-modified TSCM are the only T-cell subset able to engraft and mediate GvHD upon serial transplantation, thus demonstrating that self-renewal capacity is preserved even after ex vivo expansion and genetic manipulation. Finally, we identified and functionally validated the natural counterpart of our gene-modified TSCM population in healthy donors as a rare fraction of naive T cells, recapitulating the CD62L+ CCR7+ CD45RA+ CD45R0+ IL-7Rα+ CD95+ phenotype. These findings pave the way for a rapid clinical translation of cancer adoptive immunotherapy approaches based on ex vivo generated TSCM lymphocytes. S76
193. Engineering CD20-Specific Chimeric Receptor-Redirected T Cells with Inducible CoExpression of a Caspase-9 Based Suicide Switch for Adoptive Immunotherapy of CD20 Positive Lymphoma
Lihua E. Budde,1 Carolina Berger,1 Yukang Lin,1 Jinjuan Wang,1 Stan R. Riddell,1 Oliver W. Press,1 David Spencer.2 1 Clinical Research Division, Fred Hutchinson Cancer Research, Seattle, WA; 3Departments of Pathology & Immunology and Urology, Baylor College of Medicine, Houston, TX.
Non-Hodgkin’s Lymphoma (NHL) is the sixth most common cancer. Majority of NHL are of B cell origin and express surface CD20. Works from our laboratory using genetically engineered CD20-specific chimeric receptor-redirected T cells via plasmidbased transfect ion in murine model and phase I clinical trial, have demonstrated the promise of this approach. However, limitations including low transfection efficiency, low surface expression of chimeric T cell receptor (cTCR), and risk of insertional mutagenesis hinder the further exploitation of such an approach. Here we describe a new immunotherapeutic approach for treatment of NHL using autologous T lymphocytes that have been genetically modified with a lentiviral vector to express both a cTCR recognizing the human CD20 antigen, a “suicide gene” relying on inducible activation of caspase 9 and a truncated CD19 selectable marker. The cTCR gene was designed to encode a SP163 translational enhancer, a 1F5scFvFc anti-CD20 recognition domain, CD28 and CD137 co-stimulatory domains, and a CD3ζ signaling region for maximal expression, activation and cytolytic activity. We have been able to achieve transduction efficiency ranging from 10% to 70% of primary human T cells. These transduced T cells executed highly effective cytolytic function when cultured together with 51Cr-labeled CD20+ lymphoma cell lines including EL4-CD20, Daudi and Granta. They had no effect on CD20- cell lines. This demonstrates the high specificity of these modified T cells. When exposed to chemical inducers of dimerization (CID) AP1903 or AP20187, the T cells transduced with both cTCR and iCas-9 gene underwent CID-induced caspase-mediated apoptosis. The same duration of exposure to CID had no effect on T cells transduced with cTCR alone. To expand these T cells, we generated NIH3T3 based artificial antigen presenting cells carrying CD20 antigen and CD80 co-stimulatory molecules (NIH3T3-CD20/80). Coculture transduced T cells and NIH3T3-CD20/80 led to rapid expansion of transduced T cells. The efficacy of these expanded anti-CD20 cTCR T cells in eliminating tumor cells and the ability of CID to delete transferred T cells were tested in vivo using a mouse xenograft tumor model. SCID/NOD mice received firefly luciferasecontaining Raji lymphoma cells injected i.v. They were then treated with either anti-CD20 cTCR T cells or non-transduced T cells. We observed a 70% overall survival in the treatment group (n=16), while no survival of over 25 days was seen in the control group (n=11) that received non-transduced T cell treatment. Moreover, more than 90% of these CD20 cTCR T cells were eliminated from the mice after 2 doses of CID treatment. Our work demonstrates the feasibility and the great promise of this approach in treating relapsed CD20 bearing malignancies in a more efficient and safer way. A phase I clinical trial in patients with relapsed indolent B-NHL using this lentiviral vector is being considered.
Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
189. CD40-Ligand Armed Vaccinia Virus for Induction of Anti-Tumor Immune Response
Marko Ahonen,1 Suvi Parviainen,1 Iulia Diaconu,1 Vincenzo Cerullo,1 Akseli Hemminki.1 1 Molecular Cancer Biology Program, University of Helsinki, Finland. Immunotherapy of cancer has resulted in recent clinical successes validating the potential of the approach. A key realization has been that induction of an anti-tumor immune response is not enough, but reduction of tumor immune suppressiveness is also required. Oncolytic vaccinia virus is an attractive platform for immunotherapy because virus replication is immunogenic and not subject to tolerance. Oncolysis releases tumor antigens and provides costimulatory danger signals. However, arming the virus can improve efficacy further. CD40 ligand (CD40L, CD154) can induce apoptosis of tumor cells and it also triggers several immune mechanisms. One of these is a T-helper type 1 (Th1) response which leads to activation of cytotoxic T-cells and reduction of immune suppression. Therefore, we constructed a vaccinia oncolytic virus expressing hCD40L (vvDD-CD40L), which in addition features a cDNA expressing the tdtomato fluorochrome for an easy identification of the virus in vitro and in vivo and a deletion of the TK region to completely avoid the possibility of recombination to generate a wild type TK gene. We Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
show effective expression of functional CD40L both in vitro and in vivo. In a xenograph model of bladder carcinoma sensitive to CD40L treatment, we show that growth of tumors was significantly inhibited by the oncolysis and apoptosis exerted by vv-DD-CD40L following both intravenous and intratumoral administration of the virus. The advantage of the expression of CD40L was reduced if not abolished in tumor model not particularly sensitive to CD40L treatment such as M4A4-LM3 breast tumors. To deeply dissect the immune system component of vvdd-CD40L we generated a new version of it coding for the murine version of CD40L (vvdd-mCD40L). With this reagents we were able to study the different effects of the transgene on the composition of tumor stroma for what concerns tumor-specific T cells, B cells, NK cells and ultimately dendritic cells. In summary, oncolytic vaccinia virus coding for CD40L mediate multiple antitumor effects including oncolysis, apoptosis and induction of T-cell responses through upregulation of Th1 cytokines.
190. Analysis of Gene Expression in Adoptively Transferred TCR-Engineered Lymphocytes Daniel Abate-Daga,1 Steven A. Rosenberg,1 Richard A. Morgan.1 Surgery Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD.
1
In recent years it has been demonstrated that adoptive transfer of highly activated autologous T cells, engineered ex vivo to express tumor-targeting T cell receptors (TCR), can mediate objective tumor regressions. Although significant progress has been made concerning the selection of TCR and manufacturing of cellular products, little is known about the fate of these cells after infusion. In the present study we analyzed the expression of 84 genes relevant to T cell function in T lymphocytes isolated one month after infusion, and compared it to their expression in matched infusion preparations. Engineered T cells were purified, using FACS-sorting, from peripheral blood of ten patients that received gp100- or MAGE A3-specific TCR-transduced lymphocytes. Transcriptomic analysis was performed by Q-RT-PCR, allowing for the identification of 37 differentially expressed genes (fold change >2, p<0.05) including: 1) Anergy-related genes encoding for ubiquitin-ligases ITCH and CBLB and kinase DGKA, that were upregulated in post-infusion lymphocytes 2) FoxP1, a transcription factor involved in the maintenance of T cell quiescence, that was dramatically down regulated in infusion samples, but up-regulated in post-infusion cells both at mRNA and protein levels. 3) A group of functionally related genes (TNFRSF14, TNFSF14 and LTA), known to modulate T cell signaling, whose expression was tightly regulated at the RNA. Co-stimulatory genes LTA and TNFSF14 were under-expressed in post-infusion cells, whereas their binding partner TNFRSF14 was over-expressed. Another functionally related gene, CD160, which encodes for a gpi-anchored binding partner of TNFRSF14 was also found to be over-expressed in post-infusion cells, and its surface expression was associated with a reduced production of IFN-gamma by T cells when stimulated with relevant targets. These results may contribute to a more thorough understanding of the molecular processes governing the fate of adoptively transferred T cells and their implications for treatment efficacy and potential toxicities. These may also open an opportunity for interventions based on modulation of gene expression, as a potential method to improve adoptive cell therapy for cancer.
191. Amelioration of Colitis and Its Associated Colorectal Tumors by CEA – Redirected Tregs
Zelig Eshhar,1 Ehud Zigmond,1 Dan Blat.1 Immunology, The Weizmann Institute of Science, Rehovot, Israel.
1
Ulcerative colitis is a prominent example of a chronic inflammatory disease associated with high risk for cancer development. Our previous animal studies have shown that adoptive transfer of S75
CANCER – IMMUNOTHERAPY I chimeric, antibody-type receptor (CAR) redirected CD4+CD25 + regulatory T cells (Treg T-bodies) can prevent, ameliorate and even cure acute colitis. Here we used carcinoembryonic antigen (CEA), a well known human tumor marker which also exhibits tumor-like expression patterns in inflammatory bowel disease patients and in CEA-transgenic (Tg) mice that were induced to develop colitisassociated colorectal cancer by dextran sodium sulfate-azoxymethane (DSS-AOM). We have now demonstrated that adoptive transfer of CEA-specific Treg T-bodies could ameliorate colitis and/or hinder the subsequent appearance of colorectal tumors. Moreover, while systemic administration of CEA-specific CD4+ Teff T-bodies to naïve, CEA Tg mice caused colitis with a chronic manifestation, colonoscopically-assisted intratumoral administration of CD8+ Teff T-bodies into the colorectal tumors showed a potent, antigen-specific anti-tumor effect.
192. Naive-Derived Memory Stem T Cells: A Novel Promising Platform for Cancer ImmuneGene Therapy
Nicoletta Cieri,1 Barbara Camisa,1 Fabienne Cocchiarella,2 Mattia Forcato,3 Elena Provasi,1 Anna Mondino,4 Attilio Bondanza,1 Claudio Bordignon,5 Fulvio Mavilio,2 Silvio Bicciato,3 Alessandra Recchia,2 Chiara Bonini.1 1 Experimental Hematology Unit, Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, Milan, Italy; 2Center for Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy; 3 Center for Genome Research, Department of Biomedical Sciences, University of Modena and Reggio Emilia, Milan, Italy; 4Lymphocyte Activation Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy; 5MolMed SpA, Milan, Italy. Memory stem T cells (TSCM) with the ability to self renew and the plasticity to differentiate into potent effector cells represent ideal weapons to treat cancer. Nonetheless, clinical-grade procedures to specifically target this T-cell population remain elusive. Here we report that it is possible and feasible to differentiate in vitro, vigorously expand and genetically engineer TSCM lymphocytes in clinically compliant conditions starting from naïve precursors. Requirements for the generation of this T-cell subset, best described as CD62L+ CCR7+ CD45RA+ CD45R0+ IL-7Rα+ CD95+, are CD3/CD28 engagement and culture with IL-7 and IL-15. In contrast with other proposed protocols to generate TSCM, our method is able to sustain a significant expansion of this cell population, an essential prerequisite for any immune-gene therapy approach. The gene expression profile and functional phenotype validate this cell population as a distinct memory T lymphocyte subset, hierarchically superior to central memory (TCM) and effector memory lymphocytes. To better define naive-derived TSCM function and potential in a clinically relevant context, we evaluated their ability to mediate xenogenic GvHD in vivo. Indeed, naïve-derived and gene-modified TSCM prove superior in vivo expansion and persistence than TCM manipulated with the same protocol, and are able to differentiate into effector cells, mediating a lethal xenogeneic GvHD with the same kinetics and intensity of unmanipulated lymphocytes. Furthermore, gene-modified TSCM are the only T-cell subset able to engraft and mediate GvHD upon serial transplantation, thus demonstrating that self-renewal capacity is preserved even after ex vivo expansion and genetic manipulation. Finally, we identified and functionally validated the natural counterpart of our gene-modified TSCM population in healthy donors as a rare fraction of naive T cells, recapitulating the CD62L+ CCR7+ CD45RA+ CD45R0+ IL-7Rα+ CD95+ phenotype. These findings pave the way for a rapid clinical translation of cancer adoptive immunotherapy approaches based on ex vivo generated TSCM lymphocytes. S76
193. Engineering CD20-Specific Chimeric Receptor-Redirected T Cells with Inducible CoExpression of a Caspase-9 Based Suicide Switch for Adoptive Immunotherapy of CD20 Positive Lymphoma
Lihua E. Budde,1 Carolina Berger,1 Yukang Lin,1 Jinjuan Wang,1 Stan R. Riddell,1 Oliver W. Press,1 David Spencer.2 1 Clinical Research Division, Fred Hutchinson Cancer Research, Seattle, WA; 3Departments of Pathology & Immunology and Urology, Baylor College of Medicine, Houston, TX.
Non-Hodgkin’s Lymphoma (NHL) is the sixth most common cancer. Majority of NHL are of B cell origin and express surface CD20. Works from our laboratory using genetically engineered CD20-specific chimeric receptor-redirected T cells via plasmidbased transfect ion in murine model and phase I clinical trial, have demonstrated the promise of this approach. However, limitations including low transfection efficiency, low surface expression of chimeric T cell receptor (cTCR), and risk of insertional mutagenesis hinder the further exploitation of such an approach. Here we describe a new immunotherapeutic approach for treatment of NHL using autologous T lymphocytes that have been genetically modified with a lentiviral vector to express both a cTCR recognizing the human CD20 antigen, a “suicide gene” relying on inducible activation of caspase 9 and a truncated CD19 selectable marker. The cTCR gene was designed to encode a SP163 translational enhancer, a 1F5scFvFc anti-CD20 recognition domain, CD28 and CD137 co-stimulatory domains, and a CD3ζ signaling region for maximal expression, activation and cytolytic activity. We have been able to achieve transduction efficiency ranging from 10% to 70% of primary human T cells. These transduced T cells executed highly effective cytolytic function when cultured together with 51Cr-labeled CD20+ lymphoma cell lines including EL4-CD20, Daudi and Granta. They had no effect on CD20- cell lines. This demonstrates the high specificity of these modified T cells. When exposed to chemical inducers of dimerization (CID) AP1903 or AP20187, the T cells transduced with both cTCR and iCas-9 gene underwent CID-induced caspase-mediated apoptosis. The same duration of exposure to CID had no effect on T cells transduced with cTCR alone. To expand these T cells, we generated NIH3T3 based artificial antigen presenting cells carrying CD20 antigen and CD80 co-stimulatory molecules (NIH3T3-CD20/80). Coculture transduced T cells and NIH3T3-CD20/80 led to rapid expansion of transduced T cells. The efficacy of these expanded anti-CD20 cTCR T cells in eliminating tumor cells and the ability of CID to delete transferred T cells were tested in vivo using a mouse xenograft tumor model. SCID/NOD mice received firefly luciferasecontaining Raji lymphoma cells injected i.v. They were then treated with either anti-CD20 cTCR T cells or non-transduced T cells. We observed a 70% overall survival in the treatment group (n=16), while no survival of over 25 days was seen in the control group (n=11) that received non-transduced T cell treatment. Moreover, more than 90% of these CD20 cTCR T cells were eliminated from the mice after 2 doses of CID treatment. Our work demonstrates the feasibility and the great promise of this approach in treating relapsed CD20 bearing malignancies in a more efficient and safer way. A phase I clinical trial in patients with relapsed indolent B-NHL using this lentiviral vector is being considered.
Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy