411 CD5 CAR for the Treatment of T Cell Malignancies Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy S162 CanCer – Immunotherapy, CanCer VaC[.]
Cancer – Immunotherapy, Cancer Vaccines II 409 Expression of Mouse CD47 in Human Cancer Cells Profoundly Increases Tumor Metastasis in Murine Models Armando Rivera,1 Xinping Fu,1 Lihua Tao,1 Xiaoliu Zhang.1 Biology & Biochemistry, University of Houston, Houston, TX Although there are many well established tumor cell lines that have been widely used as xenografts in murine models, many of them not efficiently metastasize to other organ tissues after local inoculation Lack of reliable metastatic human cancer models is hindering many aspects of cancer research, in particular, for drug development The underlying reason for the inability of human xenografts to metastasize in immunodeficient mice is currently unclear We hypothesize that innate immune cells such as macrophages play a pivotal role in dictating cancer cell metastasis Specifically, macrophages can limit cancer cell metastasis by clearing tumor cells that not express the correct CD47, the “don’t eat me” signal To test our hypothesis, we transduced the murine CD47 (mCD47) gene into the human prostate cancer cell line PC-3, to establish PC-3-CD47-GFP-Luc A control cell line transduced with GFP-Luc only, PC-3-GFP-Luc, was also established We then compared PC-3CD47-GFP-Luc with PC-3-GFP-Luc and with PC-3M-Pro4, a more metastatic line established by repeatedly inoculating and harvesting PC-3 cells from prostates of athymic nude mice, for their metastatic efficiency in mice with different severity of immunodeficiency In CB17Scid mice, which have an intact macrophage activity but lack functional T and B cells, tumor metastasis was detected in both sentinel lymph node and lung from subcutaneously implanted PC-3-CD47-GFP-Luc cells but not from the other two cell lines In NOD-Scid and NSG mice, which have similar deficiency in T and B cells but have a diminished macrophage activity as compared to CB17Scid, tumor metastasis to the sentinel lymph node, lung and liver was detected from all the three cells implanted subcutaneously (in NOD-Scid mice) or orthotopically (in NSG mice) However, PC-3-CD47-GFP-Luc cells produced 2-3 times more tumor nodules than other two cells The metastatic nodules from PC-3-CD47-GFPLuc cell implantation were also visibly larger than in the other two groups When quantified by luciferase activity, radiance from the metastatic tumor nodules in PC-3-CD47-GFP-Luc group was up to 11 times higher than that in the other two groups Together, these data demonstrate that CD47 plays an important role in dictating tumor cell metastatic potential by providing the “don’t eat me” signal to host’s macrophages 410 The Development of HLAnull K562 Cells Expressing CD1d and Co-Stimulatory Molecules as Artificial Antigen-Presenting Cells for Clinical Scale Expansion of NKT Cells With Superior In Vivo Persistence and Anti-Tumor Potential Gengwen Tian,1 Bipulendu Jena,3 Daofeng Liu,1 Andras Heczey,1 Hiroki Torikai,3 Dean Lee,3 Gianpietro Dotti,2 Laurence Cooper,3 Leonid S Metelitsa.1 Department of Pediatrics, Baylor College of Medicine, Houston, TX; 2Department and Medicine, Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX; 3Cell Therapy, Department of Immunology, MD Anderson Cancer Center, Houston, TX CD1d-restricted type-I Natural Killer T cells (NKTs) have been shown to mediate antitumor responses in mouse models and are associated with improved outcome in several types of cancer However, the therapeutic application of NKTs has been limited by low numbers and functional defects of these cells in patients with cancer Herein we present an effective method for ex-vivo NKT-cell numeric expansion for adoptive immunotherapy The method is based S162 on engineered properties of K562 cells to function as artificial antigenpresenting cells (aAPC) K562 cells express a single endogenous HLA allele, HLA-Cw3, an NK-cell activating ligand which favors the expansion of NK cells that compete with NKTs in culture We rendered K562 cells HLAnull by eliminating HLA-C gene from K562 cell genome using a HLA-C-specific zinc finger nuclease We then transduced parental and HLAnull K562 cells with CD1d cDNA followed by single cell sorting and clonal expansion The clones were pulsed with αGalactosylceramide and tested as irradiated aAPC for NKTs using CFSE proliferation assay We found that in contrast to HLA-Cw3+ K562/CD1d, HLAnullK562/CD1d clones selectively expanded NKTs when added to primary PBMC, and clones with an intermediate level of CD1d expression induced the highest rate of NKT-cell proliferation Next, a selected HLAnullCD1dmed clone was further modified to express CD86 alone or in combination with 4-1BBL and/or OX40L followed by single cell sorting and clonal expansion, producing 250 clones One clone with the phenotype HLAnullCD1dmedCD86high4-1BBmedOX40Lmed (designated B-8-2) consistently induced the highest rate of NKT-cell expansion B-8-2 aAPC were able to efficiently expand both primary NKTs as well as NKTs modified to express a chimeric antigen receptor (CAR) specific for CD19 antigen, a clinically validated therapeutic target for B-cell malignancies CAR expression in NKTs rendered them highly cytotoxic against CD19+ Raji leukemia cells Compared with CAR-NKTs expanded with autologous PBMC, those expanded with B-8-2 cells exhibited a Th-1-skewed cytokine profile, prolonged in vivo persistence, and superior therapeutic activity in Raji leukemia model Gene expression analysis of NKTs expanded with B-8-2 cells vs those expanded with autologous PBMC revealed a significant upregulation of genes associated with T- and NK-cell memory (CCR7, CD45RA, KLRG1, KLRD1, IL-21R), a mixture of Th17- and Th-1associated molecules (IL-17A, IL-17F, STAT1, IL12RB1, GZMA, GZMB GNLY) and downregulation of a particular pro-apoptotic gene from the BCL2 family, BCL2L11 Therefore, the engineered K562-derived B-8-2 cells can be used as a highly efficient aAPC for ex vivo propagation of fully functional primary as well as genemodified NKTs amenable for cancer immunotherapy 411 CD5 CAR for the Treatment of T-Cell Malignancies Maksim Mamonkin,1 Malcolm K Brenner.1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX Chimeric antigen receptors (CARs) have emerged as a powerful therapeutic tool by redirecting patients’ own T cells to tackle hematologic malignancies – in particular, those of B cell origin However, T cell malignancies remain a more challenging task for CAR T cells owing to the shared expression of targetable tumor-associated antigens between normal and cancerous T cells, potentially invoking fratricide of CAR T cells and/or profound immunodeficiency We designed a novel CAR targeting CD5, a common marker expressed in ∼80% of T-cell leukemia / lymphoma blasts as well as in normal T cells Upon transduction with CD5 CAR, more than 95% of T cells expressed the CAR while producing limited and transient fratricide Fratricide of CD5 CAR T cells was mainly directed against naïve phenotype and central memory cells, eliminating more than 50% of cells in these subsets and resulting in preferential expansion of effector memory and effector T cells CD5 CAR T cells acquired resistance to self-killing by days post-transduction and could then expand normally, up to 100,000-fold Expansion of CD5 CAR T cells coincided with downregulation of CD5 protein from the cell surface while preserving CAR expression CD5 CAR T cells mounted an effective anti-tumor response, selectively killing five CD5-positive T-ALL and T lymphoma cell lines in 4-hour chromium release assays while displaying no activity Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy Cancer – Immunotherapy, Cancer Vaccines II against CD5-negative cell lines Upon longer-term coculture, CD5 CAR T cells eliminated >95% of leukemia cells from T-ALL lines within 48h and 100% by day We also observed the ability of CD5 CAR T cells to eliminate leukemia cells in sequential killing assays where we recurrently replenished fresh target cells for at least iterations Lack of functional exhaustion in sequential killing assays supports the fitness of CD5 CAR T cells for eradicating large numbers of tumor cells in vivo CD5 CAR T cells dramatically suppressed systemic in vivo disease progression in different xenograft mouse models, doubling median survival Importantly, CD5 CAR T cells demonstrated significant cytokine production and cytotoxicity against primary T-ALL blasts (n=6), highlighting the therapeutic potential of CD5 CAR for patients with T cell malignancies Overall, we demonstrated for the first time that CD5 CAR redirects T cells to eliminate CD5-positive malignant T cells in vitro and in vivo while producing only limited fratricide of the normal T cell population 412 Development of GD2-Specific Immunoliposomes for Immunotherapy of Neuroblastoma Wei Huang,1 Daofeng Liu,1 Linjie Guo,1 Gianpietro Dotti,2 Leonid S Metelitsa.1 Baylor College of Medicine, Houston, TX; 2Baylor College of Medicine, Houston, TX Tumor growth creates a highly immunosuppressive tumor microenvironment (TME) that impairs T-cell localization, persistence, or the execution of their effector function This represents a major and as yet unsolved critical challenge to the development of effective adoptive immunotherapy of solid tumors To selectively target TME in neuroblastoma and make it permissible for survival and function of tumor-specific T cells, we have developed a novel nanoparticle (NP) delivery platform which consists of 150 nm immunoliposomes rendered specific for neuroblastoma cells using the Fab fragment obtained from the anti-GD2 mAb clone 14g2a To ensure high density surface coverage and correct orientation of anti-GD2 Fab on the NPs, we synthesized a fusion protein consisting of 14g2a Fab and folate receptor (Fab14g2a-FR) and attached it on the outer layer of the immunoliposomes enriched with folic acid Rhodamine-labeled GD2specific but not control NPs could specifically bind GD2-positive CHLA-255 neuroblastoma cells but not GD2-negative LA-N-6 neuroblastoma cells in vitro as determined by FACS To examine the in vivo biodistribution of NPs, DiR-labeled GD2-specific or non-specific NPs were injected to NOD/SCID mice implanted with CHLA-255 cells Tumor tissues and normal organs were imaged after 72 hours using ex vivo fluorescence imaging Up to 58% of GD2specific NPs accumulated at the tumor sites The only other organ with significant accumulation of NPs was the liver Minor traceable portions were detected in the spleen and lung To utilize the observed targeting capabilities of GD2-specific NPs to achieve antitumor effects, we are now loading NPs with recombinant human (rhIL-7) and will test whether the preferential delivery of rhIL-7 to the tumor site and liver, a major site of NB metastasis, will increase the survival and anti-tumor activity of T and NKT cells engineered to express a GD2-specific chimeric antigen receptor with IL-7Rα The results of this study will inform design of immunotherapy of neuroblastoma and other tumors in combination with TME-modifying NPs 413 Pre-Clinical Preparation and Validation of Tumor Cell-Based IL-12 Immunotherapy for Acute Myeloid Leukemia Bryan C Au,1 Yuanfeng Liu,1 Ju Huang,1 Megan Nelles,1 Andrea Arruda,1 Michael Rothe,2 Gabi Paul,2 Axel Schambach,2 Dwayne L Barber,1 Mark D Minden,1 Christopher J Paige,1 Jeffrey A Medin.1 Ontario Cancer Institute, University Health Network, Toronto, ON, Canada; 2Institut für Experimentelle Hämatologie, Medizinische Hochschule Hannover, Hannover, Germany Interleukin(IL)-12 is a potent pro-inflammatory cytokine that stimulates a variety of effector cells involved in anti-tumor immunity Systemic administration of IL-12 has associated toxicities, however Various strategies are being developed to reduce such toxicities by restricting IL-12 distribution Options here include generating fusions with tumor-targeting molecules and directing gene delivery to specific cells First - we used lentivirus vectors (LVs) to engineer expression of murine IL-12 in tumor cells ex vivo and subsequently infused such modified cells into recipient mice This strategy restricts IL-12 to the local tumor microenvironment thereby promoting immune activation in the context of tumor-associated antigens (TAAs) Using mouse models of both leukemia and solid tumors, we found that this cellbased approach generated effective anti-tumor protection when as little as 1% of the tumor burden expressed IL-12 as long as threshold expression levels on a per cell basis were reached Second - our groups showed that anti-tumor mechanisms here involve CD4+ killer T cells, dendritic cells, and direct cell-cell contact with effectors Clinical translation of this cell-based IL-12 therapy is in progress in Toronto for AML using a novel LV to modify patients’ own blast cells Patient AML cells collected to date (n = 21) were stratified based on in vivo growth kinetics and transduced with a near-GMP-grade bicistronic LV that encodes the human IL-12 cDNA as a p40-p70 fusion, as well as a mutant thymidylate kinase (tmpK) fused to the ectodomain of LNGFR (trLNGFR) as a suicide (cell-fate control) cassette The trLNGFR/tmpK element allows selection and also selective ablation of transduced cells by administration of AZT Furthermore, it also allows tracking of transduced cells and quantification of transduction frequencies/transgene expression levels With our current protocol, functional transduction efficiencies of primary patient AML blasts ranged from 20% to 70% (n=17) Transduced AML cells displayed a strong correlation between vector copy number and trLNGFR/ tmpK + IL-12 levels and dose-dependent sensitivity to AZT In vitro immortalization (IVIM) assays determined that the near-GMP LV/ IL-12 vector displayed minimal genotoxic risk in transduced lin- cells; insertion site analyses carried out on expanded clones displayed polyto oligo-clonality patterns Pre-clinical data on toxicity and scale-up considerations are being accumulated in preparation for a Clinical Trial Application to Health Canada targeting AML This LV/IL-12 immunotherapy platform targeting tumor cells themselves thus holds potential to be effective against a wide variety of cancers 414 CAR Spacers Including NGFR Domains Allow Efficient T-Cell Tracking and Mediate Superior Antitumor Effects Monica Casucci,1 Laura Falcone,1 Barbara Camisa,1,2 Chiara Bonini,2 Attilio Bondanza.1 Innovative Immunotherapies Unit, San Raffaele Hospital Scientific Institue, Milano, Italy; 2Experimental Hematology Unit, San Raffaele Hospital Scientific Institute, Milano, Italy Introduction Chimeric antigen receptors (CARs) frequently include an IgG1-CH2CH3 spacer conferring optimal flexibility for antigen engagement and allowing the selection and tracking of CARexpressing T cells A serious drawback of CH2CH3-spaced CARs Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy S163 ... highlighting the therapeutic potential of CD5 CAR for patients with T cell malignancies Overall, we demonstrated for the first time that CD5 CAR redirects T cells to eliminate CD5- positive malignant T cells... test whether the preferential delivery of rhIL-7 to the tumor site and liver, a major site of NB metastasis, will increase the survival and anti-tumor activity of T and NKT cells engineered to... cassette The trLNGFR/tmpK element allows selection and also selective ablation of transduced cells by administration of AZT Furthermore, it also allows tracking of transduced cells and quantification