Genetically Engineered T-cells最新文献

{"title":"Abstract A025: Eradication of neuroblastoma by T-cells redirected with an optimized GD2-specific chimeric antigen receptor and IL-15","authors":"Yuhui Chen, Chuang Sun, L. Metelitsa, G. Dotti, B. Savoldo","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A025","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A025","url":null,"abstract":"The treatment with CD19-specific chimeric antigen receptor T-cells (CAR-Ts) has shown remarkable antitumor activity in patients with B-cell malignancies. However, the clinical benefits of CAR-Ts in solid tumors remain unclear. A compelling concern is that in patients with solid tumors CAR-Ts do not immediately encounter their cognate antigen in the circulation and thus lack the appropriate costimulatory signals necessary for full activation. We here sought to explore if expressing IL-15 in CAR-Ts would provide them with sufficient sustained survival until they engage the cognate antigen. Using the GD2-specific CAR and neuroblastoma (NB) as a tumor model, we explored the benefits of incorporating the IL-15 cytokine (and the iCaspase9 suicide gene for safety) within the CAR molecule. CAR-Ts from 12 healthy donors were transduced with an optimized GD2.CAR (encoding the CD28 endodomain) without (GD2-Ts) or with the IL15 (GD2.15.iC9-Ts) and expanded ex vivo with IL-7/IL-15 for 14 days. CAR transduction (82% ± 8% and 82% ± 12%, respectively) and ex vivo antitumor activity in 4 days co-culture at different E:T ratios were comparable. However, upon repetitive stimulation with GD2+ NB tumors (CHLA-255 and LAN-1), only GD2.15.iC9-Ts showed significantly superior expansion and antitumor activity (p Citation Format: Yuhui Chen, Chuang Sun, Leonid Metelitsa, Gianpietro Dotti, Barbara Savoldo. Eradication of neuroblastoma by T-cells redirected with an optimized GD2-specific chimeric antigen receptor and IL-15 [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A025.","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"135 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"127499019","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract A038: Effective rerouting of NK cell cytotoxicity against B-cell malignancies upon TCR gene transfer","authors":"Laura T Morton, A. Wouters, D. Remst, R. Hagedoorn, M. Loenen, R. Boer, J. H. Falkenberg, M. Heemskerk","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A038","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A038","url":null,"abstract":"T-cell receptor (TCR) gene transfer involves ex-vivo introduction of a tumour-reactive TCR into patient-derived CD8 T-cells enabling specific-targeting of tumour cells. Competition for expression with CD3 from the endogenous TCR and the potential for TCR mixed- dimer formation necessitate optimisation of cellular therapeutics with sustained potency and increased safety. NK-cells (CD3-CD56+) are potent short-lived effector cells that lyse abnormal or stressed cells independent of antigen. Efficacy and safety of adoptive NK therapy has been demonstrated in the treatment of hematologic malignancies in both the autologous and allogeneic setting. Here,we aimed to exploit NK-cell cytotoxicity and redirect it toward antigen-specific recognition of tumors without the limitation of TCR mixed- dimer formation and competition for CD3.Firstly, peripheral blood derived NK-cells were expanded and retrovirally transduced to express BOB1-specific TCR, restricted to HLA- B*07:02, in combination with CD3 alongside CD8 co-receptor.BOB1 is a B-cell restricted transcription factor, important for B-cell survival. Purified BOB1-TCR expressing NK-cells demonstrated antigen-specific binding of BOB1-specific pMHC-tetramer and proliferated upon co-culture with HLA-B*07:02 positive B-lymphoblastic cell lines (B-LCL) but not with HLA-B*07:02 negative B-LCL. Furthermore, BOB1-TCR expressing NK-cells demonstrated in vitro cytotoxicity against HLA-B*07:02 positive B-LCL, multiple myeloma and B-ALL cell lines. Conversely, these tumor cell lines remained resistant to NK-cell mediated lysis when co-cultured with mock transduced NK-cells. Finally, NK sensitive cell line K562 was comparably lysed by both BOB1-TCR or mock transduced NK-cells demonstrating retained NK-cell mediated activity.These data demonstrated that NK-cell cytotoxicity can be redirected toward antigen-specific recognition of tumors and is TCR-dependent. Retention of NK-cell function in genetically modified cells allows for a double-hit therapeutic approach that can offer advantages over current cellular approaches. Citation Format: Laura T. Morton, Anne K. Wouters, Dennis F. Remst, Renate S. Hagedoorn, Marleen M. Van Loenen, Renate de Boer, J. H.F. Falkenberg, Mirjam H.M. Heemskerk. Effective rerouting of NK cell cytotoxicity against B-cell malignancies upon TCR gene transfer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A038.","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"22 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"122068016","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract A024: Identifying a T-cell receptor for immunotherapy against a leukemia-associated self-antigen in an allogeneic setting","authors":"Maxi-Lu Böschen, Weiwen Yang, E. Strønen, J. Olweus","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A024","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A024","url":null,"abstract":"Our aim was to identify a T-cell receptor (TCR) that recognizes a leukemia-associated self-antigen that is highly expressed in acute myeloid leukemia and that is restricted by HLA-A2 (expressed in approximately 50% of Caucasians) for future adoptive T-cell therapy. Tumor-associated self-antigens (self-TAA) make attractive targets for adoptive T-cell therapy in cancer. However, autologous T-cells are tolerant to self-TAA expressed on self-HLA if antigen expression is sufficiently high. In contrast, T-cells are not tolerant to self-antigens presented on non-self HLA. By identifying alloreactive T-cells reactive to self-peptide in complex with foreign HLA and subsequently cloning their TCR, it is possible to redirect patient T-cells against self-TAA. The advantage of targeting self-antigens is the potential application of TCR-engineered T-cells in all cancer patients with a certain cancer type and expressing the restricting HLA molecule. We identified a protein that is selectively expressed in normal myeloid cells and highly expressed by leukemic cells in patients with acute myeloid leukemia (AML). HLA-A2 negative CD8 T-cells from healthy donors were stimulated with HLA-A2 positive dendritic cells that were expressing the full-length target antigen. CD8 T-cells recognizing peptides from the target antigen in complex with HLA-A2 were subsequently identified and sorted using fluorescently labeled HLA-A2-peptide-multimers. T-cell clones obtained from sorted multimer-positive T-cells responded to HLA-A2 positive patient leukemia cells and cell lines expressing the antigen, whereas no response was seen to HLA-A2 positive antigen negative targeT-cells, unless loaded with the relevant peptide. Responses were measured as secretion of interferon gamma, expression of the T-cell activation marker CD137 or expression of the degranulation marker CD107. The TCR sequences were identified, and we next evaluated specificity and functionality of TCR-engineered cells. Indeed, TCR-transduced healthy donor T-cells were able to selectively kill HLA-A2 positive AML patient-derived leukemic cells as well as antigen positive cell lines in vitro. We also performed a comprehensive peptide scan to determine potential cross reactivity of the obtained TCR. For this scan, the amino acids (AA) in each position of the target peptide-epitope were individually replaced by every other natural amino acid (19 AA for each position x 9 postions = 171 peptides). In addition, potential responses to other variants of the 9mer target peptide based on the natural antigen protein sequence were evaluated (8mer, 10mers, 11mers and 12mers). Based on the results obtained with TCR-transduced T-cells (interferon gamma ELISA), candidate peptides naturally occurring in the proteome to which our TCR could cross-react were not identified. These data warrant further evaluation of the TCR for in vivo efficacy and specificity. Citation Format: Maxi-Lu Boschen, Weiwen Yang, Erlend Stronen, Johanna Olweus. Id","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"4 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"121334397","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract A023: Highly enriched memory stem T-cell subsets (TSCM) expressing a novel CAR30 have enhanced antitumor effect in Hodgkin lymphoma","authors":"C. Álvarez-Fernández, L. Escribà-García, A. Caballero, J. Sierra, J. Briones","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A023","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A023","url":null,"abstract":"Background: Up to 20% of patients with advanced Hodgkin lymphoma (HL) are not cured with current chemo-immunotherapy treatments. Adoptive immunotherapy (AIT) with mature T-cells modified with a chimeric antigen receptor (CAR) holds promise for the treatment of various types of B cell lymphoma. The CD30 antigen, expressed in virtually all HL tumor cells, is absent in most healthy tissues, thus representing an ideal target for AIT of HL. In contrast to CAR19 for B cell malignancies, efficacy of CAR30 T-cells for Hodgkin and other CD30 lymphomas remains modest. Thus, novel approaches are needed to significantly improve the clinical efficacy of this approach. To this end, we used a novel antigen binding domain of a CD30 mAb, unaffected by soluble CD30 protein, as part of a chimeric antigen receptor (CAR) coupled to the 4.1BB and ζ chain endodomains, and transduced memory stem T-cells (TSCM) in an effort to ensure efficient engraftment, prolonged persistence, and enhancement of in vivo antitumor activity. To evaluate the feasibility of ex vivo expansion and antitumor efficacy of genetically-modified TSCM with a novel CD30-specific CAR that recognizes a membrane-proximal epitope of the CD30 molecule for the treatment of HL. Methods: A second-generation CD30-41BBz-EGFRt CAR was developed using a scFv that recognizes a membrane-proximal epitope of the CD30 protein. TSCM cells were generated with IL-7, IL-15 and IL-21, and transduced on day 2 of culture with a third-generation lentiviral vector encoding the CD30-CAR. The HL derived cell line L540 was used as tumor model. Tumor-specific cytotoxicity and cytokines were analysed at 24 hours. The in vivo antitumor efficacy was tested in a NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mouse model of HL xenografted with 2,5x10^6 L450 cells. Results: After 10 days, TSCM represented the majority of T-cells in culture (50.38±5.47 % for CD4+ and 70.84 ± 3.36 % for CD8+ TSCM, respectively) with a very high expression of CD30-CAR (77 ± 3.64 % in CD4+ and 83.12 ± 4.25 % in CD8+ TSCM, respectively). Expression of CD30 protein in TSCM increased over the culture period, peaking at day 8 in both CD4+ (17.5 ± 4.17 %) and CD8+ TSCM (42.37 ± 5.75 %), respectively. Despite these high levels of CD30 expression, TSCM expanded ex vivo very efficiently (CD4+ CD30-CAR: 97.24 ± 26.2 fold expansion, and CD8+ CD30-CAR: 96.79 ± 22.84 fold expansion, respectively), although lower than TSCM transduced with a CD19-CAR (CD4+ CD19-CAR: 161.96±35.9 fold expansion, and CD8+ CD19-CAR: 163.25 ± 26.51, fold expansion, respectively). Bulky CD30-CAR TSCM mediated very efficient in vitro tumor cytotoxicity (tumor cell death at 5:1 ratio: 85.25% vs. 0% with untransduced TSCM), while control CD30- targeT-cells were not killed. In addition, we evaluated the antitumor efficacy of CD30-CAR TSCM in vivo. Upon adoptive transfer of 10x10^6 bulk (CD4+ and CD8+) TSCM into NSG mice engrafted with L540 (2,5x10^6 subcutaneously), complete eradication of HL was observ","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"45 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"123918587","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract IA15: Advancing CAR T cell therapy for the treatment of brain tumors","authors":"D. Alizadeh, Dongrui Wang, R. Starr, B. Aguilar, Vanessa D. Jonsson, S. Priceman, M. Barish, B. Badie, S. Forman, Christine E. Brown","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-IA15","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-IA15","url":null,"abstract":"Encouraging clinical experience with chimeric antigen receptor (CAR) T cells supports the notion that even immune-privileged sites such as the brain may be amenable to CAR T therapy. In the context of hematologic B cell malignancies, CD19-CARs have been shown to accumulate in the cerebrospinal fluid (CSF) and reduce the incidence of metastatic disease in the central nervous system. However, treatment of solid tumors, including brain tumors, has proven quite challenging due to heterogeneous antigen expression, suboptimal trafficking, and immunosuppressive networks in the tumor microenvironment that limit CAR T cell function and persistence. This presentation will describe our efforts to overcome these challenges for the treatment of glioblastoma (GBM), one of the most common and aggressive primary malignant brain tumors. Tumor antigen selection is a key challenge for CAR T therapy of brain tumors, as on-target/off-tumor toxicities could be life-threatening. Our preclinical studies demonstrate that a glioma-associated protein, interleukin 13 receptor alpha 2 (IL13Rα2), is an attractive CAR T cell target for glioblastoma in part because this tumor-restricted antigen is expressed by the majority of GBM. Initial clinical findings demonstrate that local delivery of IL13Rα2-CAR T cells is feasible and safe, with evidence for clinical activity in patients. In addition, as part of our on-going efforts to expand the repertoire of immunologic targets for GBM, we have optimized a HER2-CAR and developed a novel toxin-based CAR harnessing the selective GBM-binding properties of chlorotoxin (CLTX). To reduce the potential for tumor antigen escape, we are exploring approaches that leverage multiple antigens, including combining CAR T cells specific to different antigens and developing tandem CARs. Another challenge is achieving efficient tumor trafficking and infiltration of CAR T cells to brain tumors. While different routes of delivery present advantages and disadvantages, our preclinical studies using several tumor models suggest that locoregional delivery, into either the tumor or cerebral spinal fluid (CSF), is more effective than systemic delivery for treatment of brain tumors. These studies underlie our clinical approach of infusing T cells into both the resected tumor cavity and CSF. Indeed, we have demonstrated that CAR T cells administered into the CSF induced a complete response in a patient with recurrent multifocal GBM, including metastatic lesions in the spine.Additional efforts are addressing the highly immunosuppressive GBM microenvironment that limits the effectiveness of adoptively transferred T cells. Our studies highlight the impact of the PD-1/PDL-1 immune checkpoint axis in reducing the antitumor activity of CAR T cells, an inhibition that can be overcome by anti-PD-1 checkpoint blockade. In another approach designed to interrogate the interplay between the tumor microenvironment (TME), host immune system, and CAR T cells, we have establish","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"79 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"117252068","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract PR06: Dual-specific T-cells and an indirect vaccine eradicate large solid tumors","authors":"C. Slaney, B. Scheidt, P. Darcy, M. Kershaw","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-PR06","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-PR06","url":null,"abstract":"While immunotherapy can eliminate substantial burdens of some leukemias, the ultimate challenge remains the eradication of large solid tumors and metastases for most cancers. Here we generate dual-specific T-cells expressing a chimeric antigen receptor (CAR) specific for Her2 and a TCR specific for the melanocyte protein (gp100). Injection of T-cells, together with a vaccine that contains a recombinant vaccinia virus expressing gp100, induced durable complete remission of a variety of Her2+ tumors and established metastases, some in excess of 150 mm2, in immunocompetent mice expressing Her2 in normal tissues. Tumor destruction occurred rapidly over seven days and was associated with an extensive infiltrate of T-cells. Mice that had rejected tumors were resistant to rechallenge with the same Her2+ tumor cells, indicating the formation of immune memory. Furthermore, we have established methods to transduce dual-specific T-cells from human peripheral blood with both a TCR specific for gp100 and a CAR for Her2. From as little as 1 ml of human buffy coat, we could generate sufficient numbers of cells for a course of treatment for a patient. The stimulation of gp100 through TCR enhanced the human dual-specific CAR T-cell proliferation, secretion of IFN-γ and killing of Her2+ human cancer cells in vitro. These characteristics were identified to be important for eradicating tumors in the mouse models. Taken together, our data provide valuable information for the development of CAR T-cell therapies for patients with solid cancers and evidence for pursuing a phase I clinical trial. Citation Format: Clare Y. Slaney, Bianca von Scheidt, Phillip K. Darcy, Michael H. Kershaw. Dual-specific T-cells and an indirect vaccine eradicate large solid tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr PR06.","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"1 1-2","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"131686940","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract A032: Chimeric antigen receptor (CAR) targeted epitope determines optimal CAR spacer length for therapy against medulloblastoma","authors":"Adam J. Johnson, Cindy A. Chang, M. Baldwin, J. Yokoyama, M. Jensen","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A032","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A032","url":null,"abstract":"Improved therapeutic outcomes for children with embryonal brain tumors (EBTs) hinge on the development and optimization of novel, targeted therapies able to eradicate tumors without serious treatment-related damage to the central nervous system (CNS). To this end, we describe the design and validation of optimized chimeric antigen receptors (CARs) T-cells that target the EBT-associated antigen Her2. Our studies demonstrate that Her2CAR extracellular spacer domains actively influence T-cell in vitro functional outputs and in vivo responses, with the medium (M-) spacer able to confer the greatest antitumor activity against the most common EBT, medulloblastoma. Furthermore, we show that the juxtamembrane epitope targeted on Her2 precluded short spacer Her2CAR activity; yet, this activity could be rescued when the targeted epitope was expressed in a membrane distal position. Similarly, while modifications that abrogate Fc region interactions in the long spacer Her2CAR rescued in vivo activity, the resultant functional outputs failed to reach the antitumor potency elicited by the M-spacer CAR. Here we also demonstrate the in vitro and in vivo activity of M-spacer CAR T-cells produced by our clinical manufacturing process. Results from this preclinical dataset have directed the implementation of a clinical trial that delivers Her2CAR T-cells locoregionally to patients with EBT tumors, coined BrainChild-01. Collectively, these results reiterate the necessity to tailor CARs to their respective targeted antigen epitope and describe the optimization of Her2-targeted CARs for the treatment of EBT tumors. Citation Format: Adam J. Johnson, Cindy A. Chang, Michael L. Baldwin, Jason K. Yokoyama, Michael C.M. Jensen. Chimeric antigen receptor (CAR) targeted epitope determines optimal CAR spacer length for therapy against medulloblastoma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A032.","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"116 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"133279237","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract A041: Hypoxia-responsive CAR T-cells","authors":"Tina Sarén, Anne Marie Senz, Mohanraj Ramachandran, Di Yu, M. Essand","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A041","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A041","url":null,"abstract":"In recent years, CD19 CAR T-cell therapy has been successfully implemented against therapy-resistant B-cell malignancies. The results are especially striking for acute lymphoblastic leukemia (ALL), while results are somewhat less stunning when it comes to lymphoma. This is in part due to the semi-solid structure of lymphoma with an immune suppressive tumor microenvironment and less oxygenated areas within the tumors. These obstacles become even more pronounced in solid tumors. Furthermore, in the case of solid tumors the lack of tumor-specific antigens represents a major challenge. Instead, overexpressed tumor-associated antigens (TAAs) are often used as targets in CAR T-cell therapy of solid tumors. However, as TAAs are also expressed to some extent in normal tissue, targeting these antigens may result in severe OFF-tumor ON-target toxicity. In the case of CD19 CAR T-cell therapy of B-cell malignancies, OFF-tumor ON-target toxicity causes B-cell aplasia.The aim of this study was to reduce OFF-tumor ON-target toxicity by engineering CAR T-cells that mainly express the CAR molecule in the hypoxic tumor microenvironment. Hypoxia-inducible factor 1 (HIF1) is a transcription factor that is a major mediator of hypoxia-induced gene expression as it binds to hypoxia response elements (HREs) in promoter regions of hypoxia-responsive genes and initiates their expression. In this study, a cassette of HREs was inserted together with a minimal CMV (mCMV) promoter in front of the CD19 CAR cassette to promote expression during hypoxic conditions. Chemically induced hypoxia augmented CD19 CAR expression over time in hypoxia-responsive CD19 CAR T-cells (HRE-mCMV-CD19CAR-GFP), whereas CAR expression was unaffected in CD19 CAR T-cells with constitutively expressed CAR (EF1α-CD19CAR-GFP). Furthermore, hypoxia-responsive CAR T-cells had an increased activation status and killing capacity upon antigen encounter in hypoxic compared to normoxic conditions. This design could be evaluated in the clinic to reduce B-cell aplasia in CD19 CAR T-cell therapy of lymphomas and especially to reduce OFF-target toxicities from CAR T-cells targeting TAAs in solid tumors. Citation Format: Tina Anna Saren, Anne Marie Senz, Mohanraj Ramachandran, Di Yu, Magnus Essand. Hypoxia-responsive CAR T-cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A041.","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"70 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"121786037","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract A037: A novel pharmacologic “ON/OFF” switch to modulate CAR-T-cell function in vitro and in vivo","authors":"K. Mestermann, R. Julian, Frenz Silke, E. Hermann, M. Hudecek","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A037","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A037","url":null,"abstract":"Background: Immunotherapy with CAR-T-cells (CAR-T) is a powerful novel treatment for hematologic malignancies, but also bound with significant acute and chronic side effects, including potentially life-threatening cytokine release syndrome (CRS) and on-target recognition of normal cells expressing the targeted antigen. This toxicity limits clinical utility and is at least in part caused by the inability to effectively control CAR-T function following infusion. Here, we present a novel strategy of pharmacologic “ON/OFF” switch to precisely control CAR-T function in real-time, which we demonstrate to modulate T-cell activity in vitro and in vivo. Methods: We considered that an effective way for controlling CAR-T function was to interfere with signal transduction though the CAR. We assembled a library of clinically approved drug compounds and screened for their ability to reversible block CAR-T function without affecting CAR-T viability. We performed functional testing with CD8+ and CD4+ CAR-T (n=3 donors) in the presence of titrated doses of the lead compound, and employed CD19- and ROR1-specific CARs comprising 4-1BB or CD28 costimulatory moieties. Results: We identified a lead compound, TCI-1, that stood out through its ability to confer a dose-dependent (partial at lower, complete at higher doses) blockade of all CAR-T effector functions, i.e., cytolytic activity, cytokine secretion and proliferation. We confirmed TCI-1 was effective in both CD8+ and CD4+ T-cells, and in each of the 3 CAR constructs. The onset of CAR-T blockade was immediate after exposure to TCI-1 and was caused by interference with early phosphorylation events in the CAR signaling cascade as demonstrated by Western blot, and interference with the induction of transcription factors, as demonstrated with an NFAT-inducible reporter gene. Intriguingly, blockade of CAR-T function was effective for several days if exposure to TCI-1 was sustained and instantaneously and fully reversible after removal of the compound. Short- and long-term exposure to TCI-1 did not induce a reduction of CAR-T viability, and did not hinder the subsequent ability of CAR-T to exert their functions. We considered that in patients with CRS, CAR-T are in an activated state, and performed comprehensive testing to show that TCI-1 was able to arrest CAR-T that are in the process of executing their effector functions. In addition, we employed a xenograft model in immunodeficient mice (NSG/Raji) to determine whether TCI-1 was capable of controlling the function of CD19 CAR-T-cells in vivo. Indeed, we demonstrate that administration of TCI-1 conferred a functional arrest of CAR-T, and that CAR-T resumed their antitumor function once administration of TCI-1 was discontinued. Conclusions: Our data show that TCI-1 is capable to exert real-time, on/off control over CAR-T function, suggesting the potential to prevent or mitigate side effects of CAR-T therapy in a clinical setting. The reversible complete inhibition of ","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"59 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"115943101","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract A039: FAM49B–specific regulatory T-cells recognize and target cancer cells in the context of Qa-1","authors":"H. Nakagawa, Hye-Jung Kim, H. Cantor","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A039","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A039","url":null,"abstract":"The outcome of cancer patients has recently been improved by utilizing novel immunotherapies that inhibit the regulatory immune system. Regulatory T-cells (Treg) contribute not only to suppression of excessive immune responses in the steady state but also to reduction of antitumor immune responses in cancer patients. We have shown that down-regulation of the Helios transcription factor undermines CD4 Treg stability inducing these cells to convert into effector cells that enhance the antitumor immune response. Helios is also the canonical transcription factor of CD8 Treg and essential for CD8 Treg function. CD8 Treg are Qa-1 (HLA-E in human)–restricted CD8 T-cells that mainly regulate Qa-1+ immune cells. The peptide FAM49B(p190-198) is a Qa-1 binding peptide derived from ubiquitously expressed FAM49B. Although FAM49B(p190-198) is regularly degenerated in the endoplasmic reticulum normal cells, we found that FAM49B(p190-198) is stably presented in some tumor cells. Here, we investigate the potential contribution of FAM49B(p190-198)/Qa-1 specific CD8 Treg to cancer.FAM49B(p190-198)/Qa-1 specific CD8 Treg were detected by FAM49B(p190-198)/Qa-1 tetramer after immunization with FAM49B(p190-198) peptide–loaded dendritic cells in CD8 Treg (i.e., CD44+CD122+Ly49+ CD8 T-cells). T-cell receptor cDNAs were amplified from the sorted single cells and inserted into retrovirus expression vectors for transduction in 58α–β– hybridoma cells and bone marrow cells. EL4-Qa-1 KO cells and EL4-FAM49B KO cells were generated using CRISPR/Cas9 technology. These tools allowed validation of the specificity of the TCRs and their Qa-1–restriction. For in vivo studies, TCR retrogenic mice were developed by transferring TCR-transduced Rag2–/– bone marrow hematopoietic stem cells into lethally irradiated CD45.1 mice. Retrogenic T-cells were prepared by CD8 enrichment of retrogenic mouse splenocytes and in vitro culture for 3 days in the presence of IL15c. For in vivo studies, we use an adoptive transfer model of retrogenic T-cells for EL4 tumor bearing mice. Rag2–/–γC–/– mice were inoculated with 0.5 x 10^6 EL4 WT-cells and 0.5 x 10^6 EL4 Qa-1 KO cells on each lateral flank, then intravenously injected with retrogenic T-cells (on day 13 and 17). FAM49B(p190-198)/Qa-1 specific CD8 T-cells preferentially use TCR Vα3.2 (TRAV9N-3) and Vβ5.1/5.2 (TRBV12-1/12-2) chains independent of their expression of Ly49+. We tested 17 pairs obtained from Ly49+ cells using the in vitro system and found that 11 pairs bound strongly to Fam49b(p190-198)/Qa-1 tetramer while 8 out of the 11 pairs exhibited antigen-specific activation. Interestingly, one TCR pair (No.8) responded to WT EL4 cells without antigen peptides, which was abolished in coculture wells with Qa-1 KO EL4 cells and FAM49B KO EL4 cells. This TCR hybridoma was also activated by stimulated WT B6 splenocytes but not by Qa-1 KO splenocytes suggesting its regulatory potential. Adoptive transfer of TCR No.8-expressing retrogenic T-cells de","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"43 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"115658748","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}