Engineered Immune Cells and Synthetic Immunotherapy最新文献

{"title":"Abstract A53: Mechanistic model predicts effects of altering CD3ζ immuno-tyrosine activating motif (ITAM) structure in chimeric antigen receptor- (CAR-) engineered T cells","authors":"Jennifer A. Rohrs, Dongqing Zheng, N. Graham, Pin Wang, Stacey D. Finley","doi":"10.1158/2326-6074.TUMIMM17-A53","DOIUrl":"https://doi.org/10.1158/2326-6074.TUMIMM17-A53","url":null,"abstract":"Chimeric antigen receptor- (CAR-) engineered T cells (CART cells) have emerged as a promising treatment for B cell lymphoma, but their success has not transferred to other cancer types. This is, in part, due to a fundamental lack of understanding of the mechanistic signaling events initiated by the CAR intracellular domains, which prevents us from being able to engineer an optimal CAR. Traditional CARs consist of intracellular signaling domains derived from CD3ζ, the main activating domain in the endogenous T cell receptor (TCR), and a co-stimulatory domain, such as CD28. The immuno-tyrosine activating motifs (ITAMs) on CD3ζ are able to induce T cell cytotoxicity on their own, while CD28 and other co-stimulatory signaling domains augment particular aspects of the response. To provide the quantitative insight needed to engineer a CAR structure that produces a desired level of T cell activation, we have developed a predictive mechanistic computational model that describes the signaling events that occur upon CAR activation. We are applying the model to improve our understanding of how CAR structure influences activation and to develop new hypotheses for the optimal design of CAR-engineered T cell systems. The computational model predicts T cell signaling mediated by CARs containing CD3ζ with and without the CD28 co-stimulatory domain, beginning with CAR activation and proceeding downstream to the activation of the transcription factors ERK and AKT. We include the key kinase, LCK, since it has been shown to play a significant role in the activation of the endogenous TCR and CD28. To account for the many species that emerge from phosphorylation of the six tyrosine sites on CD3ζ and four on CD28, we constructed the model in BioNetGen, a rule-based formalism that generates the ordinary differential equations (ODEs) for each phosphorylation permutation based on a minimal set of rules that describes the interactions between molecular species. The ODEs are implemented in MATLAB, where the different kinetic parameters can be fit to experimental data. We used phospho-proteomic mass spectrometry to quantify the site-specific phosphorylation kinetics of the CAR tyrosine sites. We expressed a variety of recombinant CAR proteins, with or without CD28 and with a series of tyrosine to phenylalanine mutations, and measured site specific phosphorylation by LCK over time. This data was used to fit a minimal model of CAR-specific phosphorylation mediated by LCK. We then extended the model to include ten additional proteins downstream of the CAR and validated it using data from the literature [1]. Our model is able to provide new insights into how the structure of the CAR intracellular signaling domains influences the rate of phosphorylation. For example, the model predicts that the order of CD28 and CD3ζ on the CAR greatly affects the overall phosphorylation rate of all sites by LCK. The model has also generated new hypotheses about how to control the overall level o","PeriodicalId":323684,"journal":{"name":"Engineered Immune Cells and Synthetic Immunotherapy","volume":"23 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134446827","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 A61: Engineered antigen presenting T cells for treatment of solid tumor cancers","authors":"Lee A. Talarico, Ildefonso Vicente‐Suarez, Blagovic Katarina, Eritza Chong-Ng, L. Jones, Lucas Pomerance, H. Bernstein","doi":"10.1158/2326-6074.TUMIMM17-A61","DOIUrl":"https://doi.org/10.1158/2326-6074.TUMIMM17-A61","url":null,"abstract":"Ex vivo manipulation of primary cells has shown immense clinical potential with the advent of adoptive T cell therapies to stimulate CD8 cytotoxic T lymphocyte (CTL) responses for the treatment of cancer. CTLs stimulated by tumor-associated antigens can target and clear solid tumors, however ex vivo manipulation methods for adoptive T cell transfer can be prohibitively time intensive. SQZ’s approach harnesses the endogenous T cell expansion mechanisms stimulated by antigen presentation to produce a relevant CTL dose. Previous efforts using antigen presenting cells (APCs) to induce this response have failed due to the difficulty inherent in delivering antigen to the APC cytosol, a necessary step for CTL activation. Within an APC, antigen location in the cytosol or endosome dictates how antigens are processed, presented, and the resultant adaptive immune system reaction. For an effective CTL response the antigens must be presented on MHC class I (MHC-I) molecules, which only occurs for antigens located in the cytosol. Antigen delivery methods, such as endocytosis, electroporation, and nanoparticle-based systems, can result in low efficiency, accumulation of material in endosomal compartments, cytotoxicity, and/or off-target effects. Furthermore, these processes are not amenable to scalable deployment, limiting the number of patients able to be treated. To circumvent such issues, we can achieve direct delivery of antigens into the APC cytosol with CellSqueeze®, resulting in MHC-I antigen presentation and effective stimulation of CTL activity. CellSqueeze® is a vector-free microfluidic platform that causes temporary membrane disruption by rapid mechanical deformation, enabling delivery of cell-engineering materials to diffuse into the cytosol without disrupting normal cell function. The CellSqueeze® platform, developed at MIT, has demonstrated efficacious delivery of various challenging materials, such as peptides and proteins, to patient-derived cells including stem cells and primary immune cells. We are developing our platform to employ primary human T cells as APCs. Previous CTL stimulation efforts have attempted to deliver antigenic material to dendritic cells (DCs); however, they are much less numerous in the blood and differentiation from monocytes is time consuming. This work uses primary human T cells, which are highly abundant in the blood, as APCs. We have demonstrated that delivery of antigenic material to T cells with the CellSqueeze® technology effectively enables the T cell to present the antigen to stimulate a targeted CTL response. In vitro we have demonstrated effective CTL activation using human T cells as APCs. In vivo we have demonstrated effective prophylactic and therapeutic treatment of murine tumors using CellSqueeze®-processed murine T cells in combination with multiple adjuvant strategies. We have also been investigating checkpoint inhibitors in combination with our T cell APCs. We believe that the unique ability to deliver ","PeriodicalId":323684,"journal":{"name":"Engineered Immune Cells and Synthetic Immunotherapy","volume":"157 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"124387598","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 A57: Adoptive T cell therapy for ovarian cancer: Application of a surgically relevant model","authors":"C. Morse, K. Anderson, Breanna M. Bates, E. Chiu, Nicolas M. Garcia, P. Greenberg","doi":"10.1158/2326-6074.TUMIMM17-A57","DOIUrl":"https://doi.org/10.1158/2326-6074.TUMIMM17-A57","url":null,"abstract":"Ovarian cancer is the most lethal gynecologic cancer, with more than 20,000 new diagnoses and 15,000 cancer-related deaths annually. Current standard therapy for advanced ovarian cancer incorporates upfront surgical cytoreduction followed by cytotoxic chemotherapy. But even with advances in surgical technique, chemotherapeutics and targeted delivery methods, the overall five-year survival rate remains ~50%, highlighting the need for novel treatment strategies. We have focused our efforts on immunotherapy by adoptive transfer of engineered T cells that can target proteins uniquely overexpressed in ovarian cancer. Recent work has identified mesothelin (MSLN) as a potential target, as this protein is overexpressed in malignant ovarian tissue, promotes the oncogenic phenotype, and has limited expression in healthy cells. Preclinical studies have demonstrated that mouse and human T cells engineered to express T cell receptors (TCR) with high-affinity for MSLN kill respectively murine and human ovarian tumor cells in vitro. Furthermore, in vivo studies in mice treating metastatic disease developing after intraperitoneal introduction of mouse ID8 ovarian cancer cells have demonstrated that engineered, adoptively transferred T cells preferentially accumulate in ID8 ovarian cancers, reduce tumor burden and prolong survival. However, our results also revealed that the tumor microenvironment (TME) can limit the persistence and killing capacity of engineered T cells. Immunosuppressive cells, inhibitory ligands that reduce T cell function, and cell death-inducing ligands are abundant within ID8 tumors, and cellular and molecular analyses of human ovarian cancer specimens showed that human T cell therapy will face similar TME-mediated obstacles. The ovarian cancer TME is also a nutrient- and oxygen-deprived milieu, and the adaptive metabolic responses required for survival by infiltrating T cells have protean effects on T cell function. Thus, strategies that modulate T cell responses to inhibitory signals, including metabolic pathways, have the potential to influence activity in the TME, and to enhance T cell function and improve anti-tumor efficacy. Ongoing studies exploring strategies to overcome elements common to the human and murine TME, including both direct modulation of the microenvironment and engineering of T cells to overcome critical components of immune evasion by solid tumors and to promote T cell survival and function, will be discussed. Building upon this work, we have been developing a novel model that more directly reflects the clinical setting requiring intervention. Following orthotopic injection of ID8 ovarian cancer cells directly beneath the ovarian bursa, tumorigenesis occurs initially within the ovary/fallopian tube microenvironment, and is followed by development of small volume metastatic disease. This orthotopic model, which results in formation of intraperitoneal metastatic implants originating from the ovary, replicates the develo","PeriodicalId":323684,"journal":{"name":"Engineered Immune Cells and Synthetic Immunotherapy","volume":"217 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"132724608","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 A50: Trivalent CAR T cells mitigate CD19-negative relapse and improve tumor control in primary pre-B cell acute lymphoblastic leukemia (B-ALL)","authors":"Kristen Fousek, Junji Watanabe, Xingyue An, Ann George, H. Samaha, Shoba A. Navai, Tiara T. Byrd, Jonathan Kirzner, Hye Na Kim, Albert Jang, S. Joseph, M. Baker, M. Hegde, N. Varadarajan, N. Heisterkamp, H. Abdel-Azim, N. Ahmed","doi":"10.1158/2326-6074.TUMIMM17-A50","DOIUrl":"https://doi.org/10.1158/2326-6074.TUMIMM17-A50","url":null,"abstract":"B-acute lymphoblastic leukemia (B-ALL) is the most common malignancy in children, and limited treatment options exist for patients with relapsed or refractory disease. Cellular immunotherapy, specifically chimeric antigen receptor (CAR) T cells targeting CD19 have demonstrated remarkable efficacy in treating B-ALL. However, recent reports show that up to 40% of patients who relapse after CD19 CAR T cell therapy have CD19-negative disease, justifying a need to expand CAR T cell therapy for B-ALL to include additional tumor-associated antigens (TAAs). Here, we hypothesize that targeting three distinct leukemia antigens CD19, CD20, and CD22 will improve B-ALL therapy outcomes and control disease progression during CD19-negative relapse. We designed two trivalent CAR T cell products with exodomains derived from single chain variable fragments (ScFv) targeting CD19 (FMC63 ScFv), CD20 (Rituximab ScFv), and CD22 (m971 ScFv) fused to the intracellular signaling domains of the co-stimulatory molecule 4-1BB and the T-cell receptor zeta chain (2nd generation). Using viral 2A intervening sequences for near equal expression, the first T cell product expresses the three CARs individually on the surface of a single T cell (TriCAR). The second T cell product expresses a traditional single CAR targeting CD19 and a second bi-specific CAR targeting CD20 and CD22 through a tandem arrangement (SideCAR). Donor T cells were successfully engineered to express the CARs using a retroviral system and the surface expression of these CAR molecules was confirmed by flow cytometry. Using a target expression validated panel of patient derived B-ALL cells (US7 CD19/CD20/CD22 +++/++/++, LAX-56 +++/+/+, TXL-2 +++/++/+++), we observed that TriCAR and SideCAR T cells killed ALL cells more robustly than CD19 CAR T cells at low effector to target ratios (E:T) in a 51Cr release cytotoxicity assay. TriCAR and SideCAR T cells secreted similar levels of IFN-gamma; when compared to CD19 CAR T cells demonstrating a safety profile very similar to the CD19 CAR T cells, but with enhanced killing. Further, we tested the efficacy of TriCAR and SideCAR T cells against primary CD19-negative relapsed bone marrow samples and CRISPR CD19 knockouts of the three primary ALL samples. Using these models of CD19 escape we demonstrated that trivalent CAR T cells effectively mitigated CD19 negative relapse, producing IFN-gamma; and killing CD19-negative primary ALL, while CD19 CAR T cells remained ineffective. In conclusion, trivalent CAR T cells are effective at targeting primary ALL cells of varying antigen profiles and mitigating CD19 negative relapse. This strategy has potential for use as a front-line therapy for primary ALL as well as a salvage therapy for patients with CD19-negative disease relapse. Citation Format: Kristen Fousek, Junji Watanabe, Xingyue An, Ann George, Heba Samaha, Shoba Navai, Tiara T. Byrd, Jonathan Kirzner, Hye Na Kim, Albert Jang, Sujith Joseph, Matthew Baker, Meenakshi Hegde, ","PeriodicalId":323684,"journal":{"name":"Engineered Immune Cells and Synthetic Immunotherapy","volume":"85 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"116080434","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 PR01: Targeting multiple myeloma with universal SLAMF7-specific CAR T-cells","authors":"R. Mathur, Zheng Zhang, Jin He, R. Galetto, A. Gouble, I. Chion-Sotinel, Stéphanie Filipe, Annabelle Gariboldi, Tanooha Veeramachaneni, E. Manasanch, S. Thomas, Hans C. Lee, K. Patel, D. Weber, R. Davis, R. Orlowski, J. Smith, Jing Yang, S. Neelapu","doi":"10.1158/2326-6074.TUMIMM17-PR01","DOIUrl":"https://doi.org/10.1158/2326-6074.TUMIMM17-PR01","url":null,"abstract":"Background: Recent studies with autologous chimeric antigen receptor (CAR)-redirected T-cells against CD19 have demonstrated long-term durable remissions in patients with B-cell leukemia and lymphoma, indicating that CAR T-cell therapy is a promising approach for refractory malignancies. Signaling lymphocytic activation molecule F7 (SLAMF7, also called CS1) is highly expressed on multiple myeloma (MM) tumor cells and is present in only a subset of hematopoietic cells among normal tissues. Because of its high expression on MM tumor cells and restricted expression in normal cells, SLAMF7 is a potential target for CAR T-cell therapy approach in MM. We had previously demonstrated that allogeneic “off-the-shelf” CAR T-cells lacking the ability to induce graft versus host disease could be generated for universal use by inactivating the TCRα constant (TRAC) gene using TALEN® gene editing technology. We also demonstrated that to minimize the risk of fratricide of SLAMF7-specific CAR+ T-cells, SLAMF7 could be inactivated by TALEN® in the T-cells prior to introduction of the CAR construct (Galetto et al., ASH 2015, Abstract 116). Here, we report the efficacy of these double KO (TRAC and SLAMF7) SLAMF7-specific universal CAR T cells (UCARTCS1) against MM in in vitro and in vivo studies. Methods: We tested the efficacy of UCARTCS1 cells against MM cell lines and primary tumor cells from MM patients for their capacity to specifically a) degranulate when co-cultured with MM tumor cells as determined by CD107a assay, b) lyse MM cell lines and primary MM cells in in vitro cytotoxicity assay, c) produce cytokines in culture supernatants, d) proliferate in presence of MM cells as determined by CFSE proliferation assay, and e) eradicate primary MM tumors in a patient-derived xenograft model as determined by serum tumor immunoglobulin (M-protein) levels and survival analysis. Results: UCARTCS1 but not control double KO T-cells (lacking SLAMF7 CAR) specifically lysed the MM cell line, MM.1S (median, 93% lysis; range, 78-98% with UCARTCS1 vs. median, 17%; range, 15-47% with control T-cells; n=10). UCARTCS1 cells similarly induced significant lysis of tumor cells from primary MM samples (n=10) (median 59%; range, 20-90%) compared to control T cells (median 9%; range, 0-36%). In agreement with this, we observed specific degranulation in both CD4+ and CD8+ UCARTCS1 cells but not control T-cells in presence of MM.1S cells and primary MM tumor cells. In addition, significant and specific proliferation of both CD4+ and CD8+ UCARTCS1 cells but not control T-cells was observed when they were co-cultured with MM.1S or primary MM tumor samples (n=8). Analysis of culture supernatants for ten cytokines (IFN-γ, GM-CSF, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, and TNF-α) showed that UCARTCS1 cells primarily produced IFN-γ and GM-CSF in presence of primary MM tumor cells (n=6), indicating a Th1/Tc1 response. To test the efficacy of UCARTCS1 cells in vivo, we injected 1x106 prima","PeriodicalId":323684,"journal":{"name":"Engineered Immune Cells and Synthetic Immunotherapy","volume":"43 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"116402028","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 A51: High-throughput method identifies rare, high-affinity, thymus-vetted T cell receptors (TCRs) for clinical translation","authors":"Thomas M. Schmitt, M. McAfee, H. Robins, L. Kropp, P. Greenberg, A. Chapuis","doi":"10.1158/2326-6074.TUMIMM17-A51","DOIUrl":"https://doi.org/10.1158/2326-6074.TUMIMM17-A51","url":null,"abstract":"Adoptive T cell therapy is a promising therapeutic approach with the potential to eradicate otherwise incurable cancers. Through T cell receptor (TCR) gene therapy, donor- or patient-derived T cells can be reprogrammed to recognize tumor-associated antigens (Ags); the ability of these engineered T cells to eliminate tumor cells is largely dictated by the affinity of the transferred TCR. As thymic selection eliminates T cells with high avidity for self, most tumor-reactive T cells in the native repertoire have limited antitumor efficacy. While TCRs can be mutated to enhance affinity, such TCRs carry the clinically-validated risk of off-target or on-target/off-tissue toxicity. Therefore, one of the key challenges of T cell-based immunotherapies is to identify TCRs with sufficient affinity to mediate effective anti-tumor activity without compromising safety. Based on the premise that a wide variability in thymus-vetted, tumor/self antigen-specific T cell avidity exists among individuals, we established a high-throughput method to simultaneously affinity rank and quickly retrieve the highest affinity TCRs from any number of pooled matched donors. We hypothesized high-affinity clonotypes would be preferentially enriched by binding to limiting concentrations of antigen-specific peptide/major histocompatibility complex (pMHC) multimers, such that TCR clonotype enrichment under increasingly stringent conditions would identify the highest affinity TCRs from within heterogenous T cell populations. Using this approach, we isolated TCRs specific for HLA A2-restricted epitopes of over-expressed self Ags (Wilms’ Tumor Antigen 1 [WT1], Mesothelin, Melanoma Antigen Recognized by T cells 1 [MART1]), cancer testis (CT) Ags (Melanoma-associated antigen 1 [MAGE A1]), as well as for viruses, including HIV and the Merkel cell polyoma virus. High-throughput TCRα/β chain pairing was obtained by PairedSeq (Adaptive Biotechnologies) or by single-cell sequencing (10X Genomics). Our results show that 1) clonotype Enrichment Ratios positively correlated with TCR affinity, thus validating our method; 2) the highest affinity T cell clones targeting self-Ags (but not viral Ags) were present at very low frequencies in native TCR repertoires, suggesting impaired antigen-driven expansion in vitro and precluding detection by conventional cloning methods; 3) many of the highest-affinity TCRs targeting viral, CT or self Ags bound pMHC complexes independent of CD8 co-receptor, confirming that tumor/self Ag-specific T cells are not entirely deleted by negative selection in healthy donors; and 4) select class I TCRs thus identified activated CD4+ (class II HLA-restricted) T cells to produce cytokines and lyse cells expressing cognate antigen. This method is poised to rapidly (~6 weeks) identify for clinical translation rare, native, high-affinity tumor-reactive TCRs with the potential to engage CD4+ T cells and enhance anti-tumor CD8+ T cell activity, but that have a low likelihood of o","PeriodicalId":323684,"journal":{"name":"Engineered Immune Cells and Synthetic Immunotherapy","volume":"60 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134122603","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 A52: Vaccine cells derived from cancer stem cells expressing interleukin-15 and its receptor inhibit tumor growth","authors":"D. K. Toukam, J. Steel, Christian Carwell, Ihab Eldessouki, J. Morris","doi":"10.1158/2326-6074.TUMIMM17-A52","DOIUrl":"https://doi.org/10.1158/2326-6074.TUMIMM17-A52","url":null,"abstract":"Background: Interleukin-15 (IL-15) is a powerful activator and inducer of NK cells and cytolytic CD8+ T cells. IL-15 also activates and expands CD8+ memory T cells without stimulating immunosuppressive CD4+CD25+ T regulatory cells. As such, IL-15 may be useful as an immunotherapy for cancer. In an effort to enhance antitumor activity and reduce systemic side effects, we studied an approach using a tumor vaccine enriched for cancer stem cells (CSCs) expressing murine (m) IL-15 and its receptor (mIL-15Rα). Methods: Lentiviral vectors expressing the wild type or optimized (opt) cDNA sequences for mIL-15 and/or mIL-15Rα under the control of the human EF-1 promoter were generated and used to transduce TC1 mouse lung cancer cells. The TC1 cells were cultured under low serum conditions to generate tumor spheroids enriched for CSCs. Results: The transduced TC1 cells demonstrated the expected mRNA transcripts. On flow cytometry only the cells transduced with mIL15Rα in combination with mIL-15 showed surface expression of mIL-15, while cells transduced with mIL-15 or mIL-15Rα constructs did not. When co-cultured with the transduced tumor spheroids or incubated with supernatants from these TC1 cells, CTLL-2 murine T cells demonstrated proliferation indicating that the cloned cDNAs expressed functional proteins. The vector demonstrating the greatest stimulation of CTLL-2 cells expressed both the mIL-15Rα and mIL-15opt sequences and demonstrated suppressed TC1 tumor growth in vivo. Conclusion: CSCs expressing mIL-15Rα and mIL-15 stimulated proliferation of T cells in vitro and demonstrated inhibited tumor growth in mice. In vivo tumor vaccination studies are in progress. Citation Format: Donatien Kamdem Toukam, Jason C. Steel, Christian Carwell, Ihab Eldessouki, John C. Morris. Vaccine cells derived from cancer stem cells expressing interleukin-15 and its receptor inhibit tumor growth [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A52.","PeriodicalId":323684,"journal":{"name":"Engineered Immune Cells and Synthetic Immunotherapy","volume":"138 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"127340540","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 A62: NKG2D RNA CAR is effective in treating peritoneal carcinomatosis in a mouse model","authors":"Zhendong Li, S. Zha, Shu Wang","doi":"10.1158/2326-6074.TUMIMM17-A62","DOIUrl":"https://doi.org/10.1158/2326-6074.TUMIMM17-A62","url":null,"abstract":"Peritoneal carcinomatosis (PC) is widely shown in end-stage neoplastic disease, especially in recurrent colorectal cancer, and has become a significant threat to the overall survival of patients. Since NKG2D ligands are commonly up-regulated on cell surface of carcinomas but rarely present on most healthy tissues, they can be the ideal targets in the treatment of NKG2D ligand-overexpressing colorectal PC. In this study, we developed a chimeric antigen receptor (CAR)-T cell therapy approach by arming human T lymphocytes with mRNA coding a chimeric NKG2D receptor to target NKG2D ligands. We constructed first, second, and third generation CARs specific for NKG2D ligands by linking the extracellular domain of the human NKG2D receptor to the CD3-zeta signaling domain and costimulatory moieties. mRNAs of those chimeric NKG2D CARs were electroporated into human T lymphocytes and the anti-tumor activities of engineered T cells were examined afterwards. The NKG2D CAR expression was detectable for at least 6 days and the modified T cells exhibited efficient cytotoxic immune activity against NKG2D ligand-positive tumor cells, but not NKG2D ligand-negative cells. Multiple infusions of the RNA CAR modified T cells in immunodeficient mice bearing established peritoneal human colorectal tumors led to a significant reduction in the tumor burden. Here, we showed that T cells engineered with NKG2D CAR by RNA electroporation can efficiently control the colorectal peritoneal carcinomatosis in the mouse model. Transfection of mRNA encoding a CAR is an economical way to benefit the test of new CARs and it holds great promise in controlling on-target/off-tumor toxicity and cytokine storms. The possibility of using NKG2D ligand-specific RNA CARs to treat colorectal PC warrants further investigation. Citation Format: Zhendong Li, Shijun Zha, Shu Wang. NKG2D RNA CAR is effective in treating peritoneal carcinomatosis in a mouse model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A62.","PeriodicalId":323684,"journal":{"name":"Engineered Immune Cells and Synthetic Immunotherapy","volume":"43 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"131577276","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 A66: Analysis of dendritic cell derived exosomes that suppressed tumor growth","authors":"M. Takanashi, K. Sudo, Shinobu Ueda, Shin-ichiro Ohno, M. Kuroda","doi":"10.1158/2326-6074.TUMIMM17-A66","DOIUrl":"https://doi.org/10.1158/2326-6074.TUMIMM17-A66","url":null,"abstract":"Immunotherapy is expected as the fourth cancer therapy subsequently to the three pillars of cancer treatment which are surgery, radiation, and chemotherapy. Cancer immunotherapy enhances existing antitumor responses and includes the use of antibodies, lymphocytes and cytokines. Dendritic cells (DCs)-based immunotherapy is one of the cancer immunotherapies. Because DCs play a key role for immune reactions to activate T cells against cancer cells by cancer antigen presentation at cellular membrane, DCs have been used in clinical trials as cellular mediators for therapeutic vaccination of patients with cancer. The cells such as DCs and cancer cells secrete exosomes which are nano-sized extracellular microvesicles. It has reported that the exosomes released from peptide-vaccinated DCs are responsible for the persistence of antigen presentation. On the other hands, cancer cells derived exosomes play an immunosuppressive. So, we considered that whether DCs-derived exosomes could induce suppress cancer cells and more effective response of immune system against cancer with control for the cancer cells-derived exosomes. Luciferase gene transfferd-3LL cells (murine lung cancer cell line derived C57BL/6) were injected to C57BL/6J mice by intraperitoneal administration. And then, DCs, DCs-exosomes or 3LL-exosmes were weekly administrated to lung cancer bearing mice. The exosomes derived from DCs decreased lung cancer cell growth, on the other hand, lung cancer derived-exosomes increased in compared with DCs, DCs-exosomes and non-treated. For cancer immunotherapy, DC-exosomes and controlled cancer-exosomes play important roles. Currently, we are going on analyze immunosuppressive molecules possessing cancer cell-derived exosomes, and immune activation molecules in DCs-exosomes. Citation Format: Masakatsu Takanashi, Katsuko Sudo, Shinobu Ueda, Shinichiro Ohno, Masahiko Kuroda. Analysis of dendritic cell derived exosomes that suppressed tumor growth [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A66.","PeriodicalId":323684,"journal":{"name":"Engineered Immune Cells and Synthetic Immunotherapy","volume":"41 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"133660101","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 A55: Vector-free genome editing of immune cells for cell therapy","authors":"L. Cassereau, Tia DiTommaso, S. Loughhead, J. Gilbert, H. Bernstein, A. Sharei","doi":"10.1158/2326-6074.TUMIMM17-A55","DOIUrl":"https://doi.org/10.1158/2326-6074.TUMIMM17-A55","url":null,"abstract":"The ex vivo manipulation of primary cells is critical to an emerging generation of cell-based therapies, such as chimeric antigen receptor systems and CRISPR mediated genomic editing. However, the limitations of existing methods for delivering desired material to cells of interest could dramatically hinder the development and impact of these therapies. To overcome the challenges associated with conventional cell delivery and engineering systems, we have developed a microfluidic approach, CellSqueeze®, where cells are mechanically deformed as they pass through constricting channels. This process disrupts the cell membrane resulting in the diffusion of material from the surrounding buffer directly into the cytosol. The CellSqueeze® system has demonstrated efficacy in patient-derived cells, such as stem cells and immune cells and with a variety of target molecules that are difficult to address with alternative methods. Moreover, by eliminating the need for electrical fields or exogenous materials such as viral vectors and plasmids, it minimizes the potential for cell toxicity and off-target effects. Here, we present evidence detailing our ability to deliver functional material for gene editing to primary human T cells via membrane deformation with little detectable perturbation in baseline gene expression, cell function, and viability. To determine the effect of membrane deformation on gene expression and to compare to other delivery systems, human T cells were subjected to membrane deformation or electroporation and gene expression changes were compared to unmanipulated control cells using microarray analysis. We performed differential gene expression analysis and found that 6 hours post transfection, electroporation induced statistically significant changes in 33% (7944/23786) of all genes as compared to untreated control cells, whereas cell squeeze treatment significantly changed expression of 0% (0/23786) of genes (FDR q Subsequently, we designed a series of experiments to manipulate gene expression with the CRISPR-CAS9 system using membrane deformation to deliver CAS9 ribonucleoproteins (RNPs; recombinant CAS9 protein complexed with a single-guide RNA). Here, we show efficacious editing of several clinically relevant loci (including B2M-up to 50% editing, CCR5-up to 80% editing, and checkpoint proteins-up to 60% editing) Taken together, these data suggest that membrane deformation is a viable delivery method for genetic engineering of primary human cells with little off target effects on baseline gene expression. Indeed, the ability to deliver structurally diverse materials to difficult-to-transfect primary cells indicate that this method could potentially enable many novel clinical applications. Citation Format: Luke Cassereau, Tia DiTommaso, Scott Loughhead, Jonathan Gilbert, Howard Bernstein, Armon Sharei. Vector-free genome editing of immune cells for cell therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunolog","PeriodicalId":323684,"journal":{"name":"Engineered Immune Cells and Synthetic Immunotherapy","volume":"362 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"122807444","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}