652. Culture and Characterization of Tumor-Infiltrating Lymphocytes of ...

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Finland, 2TILT Biotherapeutics Ltd, Helsinki, Finland. Adoptive transfer of tumor-infiltrating lymphocytes (TIL) has developed into a promising treatment modality ...
Cancer-Oncolytic Viruses II 652. Culture and Characterization of TumorInfiltrating Lymphocytes of the Syrian Hamster

Mikko Siurala1, Markus Vähä-Koskela1, Riikka Havunen1, Siri Tähtinen1, Simona Bramante1, Suvi Parviainen2, Akseli Hemminki1 1 Cancer Gene Therapy Group, University of Helsinki, Helsinki, Finland, 2TILT Biotherapeutics Ltd, Helsinki, Finland Adoptive transfer of tumor-infiltrating lymphocytes (TIL) has developed into a promising treatment modality for metastatic cancer. However, preclinical animal models for studying TIL therapy are limited to mice and therefore we set out to culture and characterize TIL from another rodent species, the Syrian hamster. First, we examined the presence of CD4+ and CD8+ cells in six implantable syngeneic hamster tumors representing various histologies. A tumor type-dependent pattern of lymphocyte infiltration was observed, with CD8+ cells ranging from 0.08% to 0.93% (of all tumor cells) and CD4+ cells ranging from 0.17% to 3.25%. To establish TIL cultures, excised tumor fragments were cultured in high-dose human interleukin-2 (IL-2) for 10 days. Flow cytometric analyses of Day 10 TIL cultures revealed a predominance of CD4+ cells over CD8+ cells in all tumor types studied. In effector/target assays the TIL cultured from HapT1 (pancreatic adenocarcinoma) and RPMI 1846 (melanoma) exhibited tumor-specific cytolytic activity. In addition, MHC Class I blocking abrogated the cell killing of RPMI 1846 TIL, suggesting that cytotoxic CD8+ T-cells were responsible for the observed cytolytic activity. Only partial abrogation was seen with HapT1 TIL suggesting the presence of cytotoxic CD4+ T-cells in the TIL population, which is supported by the observation that HapT1 tumor cells express MHC Class II. To our knowledge, this is the first time tumor-infiltrating lymphocytes of the Syrian hamster have been cultured and characterized. Thorough immunophenotyping of hamster tumors and TIL cultures requires the development of new hamster-specific antibodies in addition to the currently available ones used in this study. In conclusion, our data supports the use of the Syrian hamster as a novel platform for testing TIL protocols in a species other than mouse or human.

653. Sialyl Glycolipid Stage-Specific Embryonic Antigen 4 (SSEA4) - A Novel Target for CAR T Cell Therapy of Solid Cancers

Rita Pfeifer1, Dominik Lock1, Andrea Aloia2, Andreas Bosio1, Andrew Kaiser1, Olaf Hardt1, Ian C. D. Johnston1 1 Miltenyi Biotec GmbH, Bergisch Gladbach, Germany, 2Institute of Molecular Health Sciences, ETH, Zurich, Switzerland Owing to their capacity to eradicate tumors, T cells represent an attractive means for immunomodulation in cancer immunotherapy. In this context, chimeric antigen receptor (CAR) - based therapies are receiving increasing attention. The combination of antibody-derived specificity with T cell effector function renders the immune cells MHC-independent and even enables targeting of antigens for which there is immunological tolerance. T cells, genetically modified with CAR, have shown impressive success in the treatment of leukemia. However, the application of CAR T cells to the treatment of solid tumors remains challenging due to the lack of truly cancer-specific targets and an immunosuppressive tumor microenvironment hostile to T cells. We have identified the sialyl glycolipid stage-specific embryonic antigen 4 (SSEA4) as an epitope whose expression strongly correlates with metastasis and chemoresistence in triple negative breast cancer cells (TNBC). Single chain antibody fragments (scFv) were derived from an antibody that specifically recognizes this sialyl-glycolipid and were cloned into a lentiviral expression vector encoding a CAR containing an IgG1 spacer domain with CD137 and CD3z signaling domains. Healthy donor T cells were enriched by magnetic cell sorting and activated with TransActTM Reagent, a colloidal Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy

nanomatrix-based activation reagent, before lentiviral transduction of the anti-SSEA4 CAR expression construct. Engagement of SSEA4 by CAR expressing T cells induced T cell degranulation, secretion of inflammatory cytokines and resulted in an effective killing of SSEA4 expressing target cells. As TNBC patients are exposed to multiple rounds of chemotherapy and SSEA4 expression is found enriched in residual tumor cells surviving chemotherapy, a combinatorial approach using chemotherapy followed with CAR T cell therapy holds great promise to improve treatment outcome and overall survival of TNBC patients. Having assessed the performance of different anti-SSEA4 CAR constructs in vitro, current studies are focusing on in vivo functionality using mouse models.

Cancer-Oncolytic Viruses II 654. Oncolytic Vesicular Stomatitis Virus Retargeted to the Tumor Stem Cell Marker CD133

Dina Kleinluetzum1, Alexander Muik1, Julia Hanauer1, SarahKatharina Kays1, Camilo Ayala-Breton2, Kah-Whye Peng2, Christian J. Buchholz1 1 Paul-Ehrlich-Institut, Langen, Germany, 2Mayo Clinic, Rochester, MN Therapy resistance and tumor recurrence are often linked to cancer stem cells (CSCs) which represent a minor proportion of highly refractory cancer-propagating cells within a tumor. A putative marker of CSCs is CD133 (prominin-1) which has been suggested to define the tumorigenic population in glioma and hepatocellular cancer (HCC). Moreover, poor prognosis correlates with CD133 expression in cancer biopsies. CD133 is therefore a prime target for novel anti-tumoral strategies. We have previously developed a strategy by which the envelope protein complex of oncolytic measles virus (MV) can be engineered to use CD133 as receptor for cell entry. Notably, MV-CD133 was more effective in killing tumors than non-targeted MV when treating tumors with a high percentage of CD133-positive cells (Bach et al., 2013; Can Res 73, 865). We have meanwhile extended the receptor usage of MV-CD133 to CD46 to effectively infect both CD133-negative and -positive tumor cells. Moreover, to take advantage of the faster replication kinetics and superior cytotoxic activity of the vesicular stomatitis virus (VSV), we used the MV envelope glycoproteins to develop hybrid viruses. The resulting oncolytic virus VSV-CD133 is deleted for its glycoprotein gene G and instead equipped with the mutated MV hemagglutinin (H) and fusion (F) glycoproteins displaying a single-chain antibody (scFv) specific for CD133. The data show that VSV-CD133 infects CD133-positive tumor cells as selectively as MV-CD133, while MVCD133/CD46 in addition infected CD133-/CD46+ tumor cells. We further studied the cytotoxic activity of VSV-CD133 compared to its MV counterpart. Even though both were effective at killing tumor cells, VSV-CD133 induced cell death more rapidly and at lower MOI than MV-CD133. In a mouse tumor model with subcutaneously transplanted HuH7 cells VSV-CD133 reduced the tumor burden more efficiently than MV-CD133 or MV-CD133/CD46. Most importantly, this increase in efficacy did not come at the expense of safety, as CD133+ human hematopoietic stem cells (HSCs) were neither susceptible to infection nor impaired in growth and differentiation into hematopoietic lineages. Ongoing studies will determine whether the superior oncolytic effects of VSV-CD133 translate into prolonged survival in a mouse model with orthotopically transplanted human primary glioblastoma cells.

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