Abstracts J Immunother Cancer: first published as 10.1136/jitc-2020-ITOC7.82 on 1 October 2020. Downloaded from of SAR-T cells and directed tumour cell lysis with specificity (https://www.addgene.org/104349/) was expressed and purified towards two TYRP-expressing murine melanoma and two from Expi293 cells. MCSP-expressing human melanoma cancer models. In vivo, Results Overexpression of MCL1 in both Jurkat T cells and anti-tumoural activity was mediated by the co-administration primary human T cells protected cells against mitochondria of SAR-T cells and BiAb, in an A375 melanoma xenograft depolarization as well as the loss of cell viability in response model. Further, overexpression of IDO (a key immunosuppres- to CD95L-triggering. Expression of miR429 downregulated sive enzyme implicated in the suppression of function TIA1, TCAIM and MFF. A HER2-CAR construct with either in the tumor microenvironment) in a melanoma model did MCL1 or miR429 in a lentiviral system was successfully not influence the killing kinetics of SAR T cells. designed and transduced into primary T cells. Mitochondria in Conclusions Here we apply the SAR x BiAb approach in transduced T demonstrated enlarged and fusion morphology - efforts to deliver specific and conditional activation of syn- a classic feature of memory T cells. thetic agonistic receptor transduced T cells, and targeted Conclusions Overexpressing MCL1 or miR429 significantly tumour cell lysis. The modularity of our platform is key for a improves mitochondrial function in T cells. This approach will targeting approach in a tumor entity with a high mutational be used to increase persistence of adoptively transferred CAR load such as melanoma and is fundamental in our drive T cells. towards personalised immunotherapies. Further, the SAR Disclosure Information A. Hosseini Rad: None. G. Min Yi approach has demonstrated resistance to IDO-mediated inhibi- Tan: None. A. Poudel: None. A. McLellan: None. tion in the context of melanoma, an interesting axis that requires further investigation. Disclosure Information M. Benmebarek: None. J. Keyl: None. F. Märkl: None. M. Geiger: A. Employment (full or part- P06.03 C-C CHEMOKINE RECEPTOR 8 TUMOR-DIRECTED time); Significant; Roche. C. Karches: None. S. Rausch: None. RECRUITMENT ENABLES CAR T CELLS TO REJECT A. Gottschlich: None. A. Öner: None. M. Feinendegen: SOLID TUMORS None. J. Dörr: None. B. Cadilha: None. S. Endres: None. C. 1BL Cadilha*, 1KDorman,1DHuynh,1TLorenzini,1MVänttinen,1M-R Benmebarek, Klein: A. Employment (full or part-time); Significant; Roche. 1 1 1 1,2 1 S. Kobold: None. S Stoiber, J Suárez-Gosálvez, S Endres, S Kobold. Center of Integrated Science Munich and Division of Clinical Pharmacology, Department of Medicine IV, Klinikum der Universität München, Munich, Germany, Member of the German Center for Lung Research., Munich, Germany; 2German Center for Translational Cancer Research (DKTK), partner site Munich, Germany, Munich, Germany P06.02 ENHANCING CAR T CELL PERSISTENCE AND MEMORY THROUGH MODULATING MITOCHONDRIAL FUNCTION 10.1136/jitc-2020-ITOC7.82 A Hosseini Rad*, G Min Yi Tan, A Poudel, A McLellan. University of Otago, Dunedin, New Background CAR T cell therapy is remarkably successful in Zealand patients with hematological malignancies, in some cases induc- ing durable remissions. However, it remains ineffective in solid 10.1136/jitc-2020-ITOC7.81 tumors, in part due to poor T cell infiltration into the tumor Background CAR T cell therapy for solid tumours has mass. Determinants of successful T cell infiltration to the http://jitc.bmj.com/ achieved limited success compared to its application to B cell tumor site remain to be defined. In contrast, tumors actively malignancies. One reason for this failure is the low differentia- attract T regulatory (Treg) cells for immune suppression tion rate to memory subsets and low persistence of CAR T through the C-C chemokine receptor 8 (CCR8) - CCL1 axis. cells due to activation-induced cell death (AICD) in lymphoid As this axis is functional across cancer entities, we postulated tissue and the tumour microenvironment. In this study, we that CCR8 could also be used to target tumor-ablating T have expressed the MCL1 within CAR T cells to over- cells to the tumor site. Material and methods Murine and human CCR8 have been come losses by AICD in adoptively transferred T cells. The on September 25, 2021 by guest. Protected copyright. MCL1 gene expresses two isoforms; the long isoform localises cloned in a retroviral expression vector. CCR8 can be expressed to the outer membrane of mitochondria and inhibits the in murine and human T cells upon transduction. A chimeric CD95 signalling death pathway, while the short isoform local- antigen receptor (CAR) targeting the murine epithelial cell adhe- ises to the inner membrane of mitochondria to enhance mito- sion molecule (EpCAM) was used for syngeneic pancreatic tumor chondrial oxidation, and fusion. In addition, models and a CAR targeting human mesothelin was used for a we have also utilized a microRNA (miR) 429 to promote xenograft pancreatic tumor model.Mechanistically, we use flow memory T cell formation through the suppression of cytometry and multi-photon intra-vital microscopy to interrogate such as T-cell-restricted intracellular antigen-1 (TIA-1), T cell infiltration of CCR8-transduced CAR T cells. activation inhibitor, mitochondrial (TCAIM) and mitochondrial Results Here we show that genetically engineering CAR T cells fission factor (MFF). to express CCR8 improves their migration into solid tumors Materials and Methods Overexpression of MCL1 was con- and allows rejection of tumors that are otherwise resistant to firmed at both mRNA and protein level by real time RT-PCR CAR T cell therapy. We demonstrate the capacity of these (qPCR) and western blot. Similarly, overexpression of miR-429 enhanced CAR T cells to stunt solid tumor growth and was measured by qPCR and specific binding of miR-429 to improve survival in both murine syngeneic and human xeno- the 3¢ UTR of target genes was confirmed by luciferase graft tumor models. reporter assay. Mitochondrial depolarization and cell viability Conclusion Our results demonstrate the viability of using were assessed by TMRE mitochondrial membrane potential CCR8 to confer Tregcell trafficking-properties in CAR T cells assay (flow-cytometry) and resazurin assay. The effect of to enable their effectiveness in solid tumors. This receptor MCL1 or miR429 overexpression on HER2-CAR T cells was may be combined with other promising strategies to improve determined by flow cytometry. Soluble leucine-zipper CD95L the efficacy of cellular approaches.

A42 J Immunother Cancer 2020;8(Suppl 2):A1–A67 Abstracts J Immunother Cancer: first published as 10.1136/jitc-2020-ITOC7.82 on 1 October 2020. Downloaded from

Disclosure Information B. L. Cadilha: None. K. Dorman: blocking protein kinase A (PKA) localization. Cancer Immunol Res 2016; 4(6): – None. D. Huynh: None. T. Lorenzini: None. M. Vänttinen: 541 551 2. Hussain M, Shah Z, Abbas N, Javeed A, Mukhtar MM, Zhang J. Targeting None. M. -R. Benmebarek: None. S. Stoiber: None. J. Suárez- tumour-associated immune suppression with selective protein kinase A type I Gosálvez: None. S. Endres: None. S. Kobold: None. (PKAI) inhibitors may enhance cancer immunotherapy. Medical Hypotheses 2016;86:56–59

Disclosure Information A. Poudel: None. S.M. Rad: None. G. P06.04 ENHANCING T CELL FUNCTION FOR CANCER Tan: None. A.D. McLellan: None. IMMUNOTHERAPY BY MICRORNA MEDIATED KNOCKDOWN OF PRKAR1A A Poudel*, SM Rad, G Tan, AD McLellan. University of Otago, Dunedin, New Zealand P06.05 IDO1-DELETED CAR T CELLS SHOW IMPROVED 10.1136/jitc-2020-ITOC7.83 THERAPEUTIC EFFICACY IN MURINE PANCREATIC CANCER MODELS Background Protein Kinase A (PKA) is a heterotetramer hol- oenzyme that consists of two regulatory and catalytic subunits. 1AM Senz*, 1PMetzger,1RK Rubens, 1B Cadilha, 2M Kirmaier, 1S Lesch, 1MR Benmebarek, During T cell activation, one of the regulatory subunits 2S Theurich, 3PMurray,1S Endres, 1S Kobold, 1LM König, 1PDuewell,1M Schnurr. 1Division 2 (PRKAR1A) localizes to the immune synapse, inhibiting several of Clinical Pharmacology, University Hospital, LMU, München, Germany; Gene Center, 3 central in the T-cell signalling cascade and leading to Ludwig-Maximilians-Universität (LMU), München, Germany; Max Planck Institute of Biochemistry, Martinsried, Germany T cell inactivation. Previously, the disruption of localisation of PKA type I R1a (PRKAR1A) to the immune synapse using 10.1136/jitc-2020-ITOC7.84 disruptor has been shown to improve chimeric anti- gen receptor (CAR) T cell function.12However, the effect of Background Indoleamine-2,3-dioxygenase 1 (IDO1) is a cyto- PRKAR1A knockdown in T cells (including CAR T cells) has solic enzyme that catalyzes the rate limiting reaction in the not been studied yet. In this study, we have utilized micro- kynurenine pathway. Dendritic cells, macrophages and several RNAs (miR); miR96/183 or miR155 to knockdown PRKAR1A tumor entities have been described to express IDO1. In the and explored the advantages of PRKAR1A knockdown on T tumor microenvironment IDO1 promotes tryptophan starva- cell activation and function. tion and accumulation of kynurenines which result in T effec- Materials and Methods MicroRNAs (miR); miR96/183 or tor cell proliferation arrest and T regulatory cell induction. miR155 were cloned from human genomic DNA into a sleep- Additionally, IDO1 possesses two immunoreceptor - ing beauty system under a doxycycline inducible promoter based inhibitory motifs (ITIM) that upon phosphorylation can (TCE). Overexpression of miRNA and target knockdown was act as docking sites for the recruitment and activation of the assessed at both transcript level (by real time RT-PCR) and/or tyrosine phosphatases SHP–1 and SHP–2 and ultimately to an protein level (by western blot) respectively while target valida- activation of the non-canonical NF-KB pathway. Whether tion was done by luciferase assay. The fate of PRKAR1A IDO1 is expressed in T cells and its potential function is knockdown on Jurkat T cells activated with anti-CD3 and unknown. anti-CD28 antibodies were determined by measuring IL-2 pro- Materials and Methods Using IDO1-deleted splenocytes from duction (ELISA) and CD69 surface expression (flow cytome- CD4-Cre Ido1fl/fl mice and WT controls, we evaluated the http://jitc.bmj.com/ try). The effect of miR96/183 or miR155 overexpression in induction of IDO1 in T cells, as well as the effect of IDO1 in primary T cells expressing HER2-CAR were also compared. T cell proliferation, differentiation and metabolism. Addition- Results We efficiently overexpressed both miRNAs and down- ally, we compared in vitro and in vivo the cytotoxic activity regulated PRKAR1A expression in HEK293 cells at both of anti-epithelial cell adhesion molecule (EpCAM) chimeric mRNA and protein level. Luciferase assay confirmed miRNA antigen receptor (CAR) T cells using pancreatic tumor cell mediated specific knockdown of PRKAR1A; mutated 3’UTR lines. on September 25, 2021 by guest. Protected copyright. of PRKAR1A was used as negative control. Overexpression of Results IDO1 is inducible in primary mouse T cells upon T miRNAs also downregulated PRKAR1A expression in Jurkat cell activation and type I and type II interferon signaling. cells which resulted in enhanced activation (CD69 expression) Interestingly, the use of IDO1 knockout CAR T cells prolongs and IL-2 production following anti-CD3/CD28 stimulation survival and improves tumor control compared to WT CAR T compared to untransfected controls (with normal PRKAR1A cell treatment in subcutaneous and orthotopic pancreatic can- expression). Additionally, miRNA 96/183 and miRNA155 were cer models. In vitro, T cell proliferation, differentiation and found to target inhibitory proteins of TCR signalling such as cytotoxic function is comparable in WT and IDO1-deleted T CTLA4, Foxo3 and ptpn2 and resulted in superior T cell cells. RNA sequencing, metabolic and in vivo tracking studies function. A third-generation lentiviral system has been opti- are currently being performed to pin down IDO1-intrinsic mised to express either miR96/183 or miR155 and HER2- effects on CAR T cells. CAR in the same vector and currently we are assessing the Conclusions IDO1 is expressed in T cells upon T cell receptor effect of PRKAR1A knockdown on primary CAR T cells. and IFN stimulation and appears to negatively affect tumor Conclusions Overexpressing miRNA for knockdown of inhibi- control mediated by CAR T cells. Specific IDO1 deletion may tory proteins could be an efficient way of enhancing T cell improve therapeutic efficacy of CAR T cells in solid tumors, function against solid tumours. Additionally, co-expressing such as pancreatic cancer. CAR and miRNAs using lentiviral system would benefit such Disclosure Information A.M. Senz: None. P. Metzger: None. approaches for cancer immunotherapy. R.K. Rubens: None. B. Cadilha: None. M. Kirmaier: None. S. Lesch: None. M.R. Benmebarek: None. S. Theurich: None. REFERENCES P. Murray: None. S. Endres: None. S. Kobold: None. L.M. 1. Newick K, O’ Brien S, Sun J, Kappor V, Maceyko S, Lo A, Pure E, Moon E, Albelda SM. Augmentation of CAR T cell trafficking and antitumour efficacy by König: None. P. Duewell: None. M. Schnurr: None.

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