Genetically Engineered T-cells最新文献

{"title":"Abstract IA14: Cell transfer immunotherapy targeting unique somatic mutations in cancer","authors":"S. Rosenberg","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-IA14","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-IA14","url":null,"abstract":"Adoptive cell transfer (ACT) immunotherapy using T lymphocytes with antitumor activity is a living therapy that can be highly effective in patients with a variety of metastatic cancers. Naturally occurring tumor infiltrating lymphocytes (TIL) expanded in vitro and administered following a lymphodepleting preparative regimen mediated objective regression of metastatic melanoma in 55% of 194 patients including complete regressions in 24% of patients who remain ongoing disease-free 3 to 10 years later. To determine the antigens recognized by TIL we developed an approach based on deep exomic sequencing of the cancer and immunologic testing of TIL or peripheral lymphocytes to generate T-cells that recognized immunogenic mutations. TIL from 22 patients with metastatic melanoma recognized 54 random somatic mutations none of which were shared among different melanomas. We next extended these studies to patients with common epithelial cancers and showed that 81 of 99 (82%) patients with a variety human cancer types including esophageal, colorectal, bile duct, gastric, pancreatic, ovarian, cervical and lung cancer contained T-cells that recognized 197 neoantigens all of which were unique except for KRAS (2 patients). Targeting unique cancer mutations has extended the reach of ACT immunotherapy and was used to mediate objective regressions in selected patients with chemorefractory metastatic cancers of the bile duct, colon, cervix, and breast. In addition to the use of naturally occurring mutation-reactive cells, we have genetically engineered autologous lymphocytes to express shared antitumor T- cell receptors (TCR) or chimeric antigen receptors (CAR) for use in ACT immunotherapy that can mediate durable cancer regressions in heavily pretreated patients with refractory lymphomas, sarcomas and melanoma. Autologous T- cells can be used to provide a highly personalized immunotherapy for cancer patients refractory to conventional cancer treatments. Citation Format: Steven A. Rosenberg. Cell transfer immunotherapy targeting unique somatic mutations in cancer [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 IA14.","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"2 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":"125149609","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 PR05: T-cells engineered to overcome death signaling within the tumor microenvironment enhance adoptive cancer immunotherapy","authors":"C. Klebanoff","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-PR05","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-PR05","url":null,"abstract":"Across clinical trials, T-cell expansion and persistence following adoptive cell transfer (ACT) has correlated with superior patient outcomes. Herein, we undertook a pan-cancer analysis across human tumors to identify potentially actionable ligand/receptor pairs that might limit T-cell function and persistence following ACT. We discovered that FASLG, the gene encoding the apoptosis-inducing ligand FasL, is overexpressed within the majority of human tumor microenvironments. Further, we uncovered that Fas, the receptor for FasL, is highly expressed on patient-derived T-cells used for ACT. We hypothesized that a cognate Fas-FasL interaction within the tumor microenvironment might limit both T-cell persistence and antitumor efficacy. We discovered that genetic engineering of Fas variants impaired the ability to bind FADD function as dominant negative receptors (DNRs) in Fas-competent mouse and human T-cells, rescuing cells from FasL-induced apoptosis. Fas DNR-engineered T-cells exhibited enhanced persistence within tumors following ACT, resulting in superior cancer regression and overall survival in solid and hematologic malignancies treated with TCR or CAR-modified cells. Despite enhanced longevity, Fas DNR-engineered T-cells did not undergo aberrant clonal expansion, demonstrating the safety of this approach. Thus, cell-intrinsic “insulation” of T-cells from the negative influence of FasL is a potentially universal strategy to enhance ACT efficacy across a broad range of human malignancies. Citation Format: Christopher A. Klebanoff. T-cells engineered to overcome death signaling within the tumor microenvironment enhance adoptive cancer immunotherapy [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 PR05.","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"95 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":"123586697","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 A027: Targeted insertion of an HPV-16 E7-specific engineered T-cell receptor into the TRAC locus","authors":"Alexandra Croft, C. Brandt, Stephen M Burleigh, E. Chadwick, M. Chin, D. Toy, B. Donahue, Clay Patton, Stephen J. Goldfless, Brian J. Belmont, R. Salmon, G. Welstead, Blythe Sather, D. J. Huss","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A027","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A027","url":null,"abstract":"Background: Adoptive T-cell therapy with engineered T-cell receptors (eTCR) has demonstrated promising, yet modest clinical benefit to date. Key obstacles for this technology are 1) competition with the endogenous TCR for CD3 components that are required for surface expression of a functional TCR complex and 2) mispairing of endogenous and exogenous TCRα and TCRβ chains. Substituting murine TCR constant domains has been shown to enhance eTCR expression, but increases the risk of immunologic rejection. Knockout or knockdown of the endogenous TCR has also been shown to improve eTCR expression. Current eTCR delivery approaches use semi-randomly integrating lentivirus or retrovirus to generate an eTCR expressing T-cell product. An alternative approach is to combine TCR knockout with targeted integration of eTCRs into the T-cell receptor alpha constant (TRAC) locus. In this study, we evaluate newly discovered eTCRs specific for human papillomavirus (HPV) type 16 oncogenic protein E7, expressed via these various gene engineering approaches, to optimize engineered T-cell functionality. Methods: HPV E7-specific eTCRs were introduced into primary human T-cells by lentiviral transduction or a dual CRISPR-Cas9/AAV eTCR delivery platform for targeted insertion into the TRAC locus using homology directed repair (HDR). Comparisons were made among TCR sufficient and TCR knockouT-cells with gene delivery by lentivirus or HDR. Engineered T-cell function was assessed both in vitro and in vivo against HPV-16+ head and neck squamous cell carcinoma cell lines. Results: CRISPR/Cas9-mediated TRAC editing eliminated endogenous TCR expression in >85% of T-cells. The impact of TRAC editing on eTCR expression and engineered T-cell function was variable across multiple eTCRαβ sequences. With many eTCRs, TRAC editing in lentivirus-derived populations resulted in increased expression and improved cytokine and killing responses. Targeted insertion of these eTCRs into the TRAC locus by HDR increased engineered cell product homogeneity and enhanced function compared to lentiviral integration. However, there were also examples of eTCRs that were minimally impacted by TRAC locus engineering. Conclusions: Our results demonstrate that elimination of the endogenous TCR, alone or combined with targeted insertion at the TRAC locus, improves eTCR expression and engineered T-cell function for the majority of eTCRs tested. While infrequent, some eTCRs were less impacted by TRAC locus engineering, suggesting a sequence-specific property that enables these eTCRs to out-compete the endogenous TCR for CD3 componentry. The cause for this variable response to TRAC locus engineering is still being explored. Citation Format: Alexandra Croft, Cameron Brandt, Stephen Burleigh, Eric Chadwick, Melissa Chin, Dean Toy, Bailey Donahue, Clay Patton, Stephen Goldfless, Brian Belmont, Ruth Salmon, Grant Welstead, Blythe D. Sather, David J. Huss. Targeted insertion of an HPV-16 E7-specific engineered T-cell","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"5 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":"129719461","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 A028: Memory stem T-cells expressing an optimized CD30-specific chimeric antigen receptor (CAR) efficiently eradicate peripheral T-cell lymphoma in vivo","authors":"L. Escribà-García, C. Álvarez-Fernández, A. Caballero, R. Julian, E. Hermann, J. Sierra, M. Hudecek, J. Briones","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A028","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A028","url":null,"abstract":"Peripheral T-cell lymphomas (PTCL) represent the most aggressive form among non-Hodgkin lymphomas with a very poor prognosis (5-year survival of 30%), demanding innovative novel treatment strategies. Adoptive immunotherapy with chimeric antigen receptor (CAR) T-cells has demonstrated its therapeutic potential in advanced B-cell hematologic malignancies. However, its application to PTCL remains a formidable challenge mainly due to a lack of truly tumor-specific antigens that are not expressed on normal T-cells. Anaplastic large T-cell lymphomas (ALCL) and several other subtypes of PTCL express CD30, which is also expressed by activated normal T-cells but no other healthy tissues. Indeed, brentuximab-vedotin, an anti-CD30 antibody-drug conjugate, has shown some clinical efficacy in PTCL and ALCL patients although duration of responses is short in the majority of cases. Here, we developed a refined CD30-CAR T-cell approach to target CD30+ PTCL as a potential novel therapeutic strategy. We selected a novel targeting domain that is unaffected by soluble CD30 protein (Nagata S et al., Clin Cancer Res 2002) to prevent blockade of the CD30-CAR. Moreover, we optimized the therapy by using memory stem T-cells (TSCM) to promote engraftment and persistence of CD30-CAR T-cells after transfer.TSCM were generated with CD3/CD28 costimulation in presence of IL-7, IL-15 and IL-21 (Alvarez C et al., J Transl Med 2016). On day 2 of culture, cells were transduced with a third-generation lentiviral vector encoding the CD30-41BBz-EGFRt CAR. The cell line Karpas 299 (ALCL) was used as tumor model. Cytotoxicity assay was performed at 24 hours and the tumor cell death was detected by luminiscence. Mice were injected with Karpas 299 tumor cells (2x10^6 cells/mice; iv) and were treated with CD30-CAR TSCM (1x10^7 cells/mice; iv) when the tumor was established (day 10). Mice were followed daily for survival. The presence of CD30-CAR TSCM in lymphoid organs and their expression of immune checkpoint molecules (i.e., TIM-3, LAG-3) were analyzed at the end of the in vivo study by flow cytometry. TSCM were the most prevalent T-cell subset at day 10 of culture (84±3.1% of total cells), and the CD30-CAR expression was 82.8±1.0% in CD4+ TSCM and 85.2±2.0% in CD8+ TSCM. Although CD30 protein was detected in a fraction of activated T-cells in culture, CD30-CAR TSCM could be expanded ex vivo (CD4+ CD30-CAR TSCM: 96.0±3.2 fold expansion; CD8+ CD30-CAR TSCM: 109.0±4.2 fold expansion). CD30-CAR TSCM conferred specific cytolytic activity against Karpas 299 cells in vitro (tumor cell death 5:1 (effector:target) ratio: 70.2±5.1% vs. 0% with untransduced TSCM; p 80% of detected CD4+ and CD8+ T-cells) were still present in lymphoid organs (bone marrow, spleen and lymph nodes) of surviving mice at 50 days after infusion. Tumor-bearing mice also showed CD30-CAR T-cells in their lymphoid organs (80% of total CD4+ and CD8+ T-cells) and in the tumor (67.5% of detected T-cells). Interestingly, a high","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"6 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":"129687118","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 A044: Mutated NPM1 as target for immunotherapy of acute myeloid leukemia","authors":"Dy Lee, Rogier M. Reijmers, M. Honders, R. Hagedoorn, R. D. Jong, M. Kester, D. M. V. D. Steen, A. Ru, C. Kweekel, I. Jedema, H. Veelken, M. Heemskerk, P. Veelen, J. Falkenburg, M. Griffioen","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A044","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A044","url":null,"abstract":"The most frequent subtype of acute myeloid leukemia (AML) is defined by mutations in the nucleophosmin (NPM1) gene. Mutated NPM1 is an attractive target for immunotherapy, since it is an essential driver gene and 4 base pair frameshift insertions in exon 12 occur in 30-35% of AML, resulting in a novel C-terminal alternative reading frame of 11 amino acids. By searching in the HLA class I ligandome of primary AML, we identified multiple peptides derived from mutated NPM1. For one of these peptides, i.e., HLA-A*02:01-presented CLAVEEVSL, we searched for specific T-cells in AML patients and healthy individuals using peptide-MHC tetramers. Tetramer-positive CD8 T-cell clones were isolated and analyzed for reactivity against primary AML with mutated NPM1. From one selected clone with superior antitumor reactivity, we isolated the T-cell receptor (TCR) and demonstrated specific recognition and lysis of HLA-A*02:01-positive AML with mutated NPM1 in vitro after retroviral transfer to CD8 and CD4 T-cells. In vivo antitumor efficacy of TCR-transduced CD8 and CD4 T-cells was confirmed in immunodeficient mice engrafted with a human AML cell line expressing mutated NPM1. These data show that mutated NPM1-derived peptides are presented on AML and that CLAVEEVSL is a neoantigen that can be efficiently targeted on AML with mutated NPM1 by TCR gene transfer in a co-receptor independent fashion. Immunotherapy targeting mutated NPM1 may therefore contribute to treatment of AML. Citation Format: Dyantha I. van der Lee, Rogier M. Reijmers, M. Willy Honders, Renate S. Hagedoorn, Rob. M. de Jong, Michel G.D. Kester, Dirk M. van der Steen, Arnoud H. de Ru, Christiaan Kweekel, Inge Jedema, Hendrik Veelken, Mirjam M. Heemskerk, Peter A. van Veelen, J.H. Frederik Falkenburg, Marieke Griffioen. Mutated NPM1 as target for immunotherapy of acute myeloid leukemia [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 A044.","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"80 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":"126786817","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 A036: Multiplex human T-cell engineering by Cas9 base editor technology","authors":"Cara-Lin Lonetree, Beau R. Webber, Miechaleen D. Diers, Walker S. Lahr, Mitchell G. Kluesner, Mark J. Osborn, Matthew J. Johnson, B. Moriarity","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A036","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A036","url":null,"abstract":"Chimeric antigen receptor (CAR)-engineered T-cells have mediated impressive outcomes in a subset of hematologic malignancies, yet this therapy remains highly personalized and largely ineffective against solid tumors. Genome editing strategies using targeted nucleases could overcome these limitations and have begun to enter clinical application. Multiplex editing strategies to develop off-the- shelf therapies are of high interest but remain limited by concerns of off-target effects and chromosomal translocations formed by simultaneous double-strand break (DSB) induction at multiple loci. The risk of genotoxic side effects is amplified when combining multiplex DSB induction with randomly integrating platforms for antigen-specific receptor delivery. An ideal strategy would allow for multigene disruption and targeted integration of antigen-specific receptors without introduction of multiple genomic DSBs. To this end, we evaluated the application of third- and fourth-generation Cas9 base editor technologies for gene disruption and integration in primary human T-cells. Chemically modified gRNAs and Cas9 base editor mRNA were delivered to stimulated T-cells by electroporation, followed by viral transduction for delivery of a DNA repair template as recombinant adeno-associated virus (rAAV). Base editing efficiencies were determined on the genomic level by PCR amplification, Sanger sequencing, and analysis of resultant traces using the EditR web app. Gene knock-out and knock-in efficiencies were analyzed on the protein level by flow cytometry. Through systematic reagent and dose optimization efforts, we achieved highly efficient C>T base conversion and consequent protein knockout at multiple therapeutically relevant loci including TRAC (KO = 83.6 ± 3.3%), PD-1 (KO = 78.6 ± 2.3%), and B2M (KO = 80 ± 1.8%). We observed that fourth-generation base editor (BE4) achieved consistently higher C>T conversion rates with reduced non-canonical editing (i.e., C>A/G) compared to third-generation base editor (BE3). Targeted disruption of splice acceptor (SA) and splice donor (SD) sites resulted in higher frequency of protein knockout vs. induction of premature stop codons at all loci examined. Importantly, while multiplex editing using Cas9 nuclease resulted in detectable translocations between the targeted sites, we were unable to detect these translocations using BE3 and BE4 as measured by PCR. Finally, we exploited the single-strand nickase function of the base editors in conjunction with rAAV delivery to achieve simultaneous targeted integration of a gene expression cassette at the AAVS1 safe harbor locus. Staphylococcus aureus Cas9 (SaCas9) was also employed as an orthogonal approach to AAVS1 targeting, and yielded higher rates of donor integration. Collectively, we demonstrate that Cas9 base editor technology can be utilized to mediate efficient, multiplex gene disruption and targeted gene integration in primary human T-cells without associated translocations. Th","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"285 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":"116103498","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 IA12: CAR T-cell therapy for lymphoma and multiple myeloma","authors":"J. Kochenderfer","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-IA12","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-IA12","url":null,"abstract":"Chimeric antigen receptors (CARs) are fusion proteins that incorporate antigen-binding domains and T-cell signaling domains. The first CAR T cells to be shown to be clinically effective targeted the B-cell antigen CD19. Anti-CD19 CAR T cells have been effective against all major types of B-cell lymphomas, and anti-CD19 CAR T cells have now obtained Food and Drug Administration (FDA) approval for treatment of the most common type of lymphoma, diffuse large B-cell lymphoma. This approval was based on clinical results that showed an approximately 50% complete remission rate of anti-CD19 CAR T-cell therapy for diffuse large B-cell lymphoma. These responses have been durable. Eleven of twelve patients who obtained complete remissions on a phase 1 trial of anti-CD19 CAR T cells are still progression-free 13 to 36 months after treatment (Kochenderfer et al. Journal of Clinical Oncology, 2017). Even more durable remissions have occurred in a small number of patients in an early cohort of anti-CD19 CAR therapy. Four patients with remissions of 38 to 56 months will be presented. These patients are particularly interesting because 3 of the 4 patients with long remissions have had recovery of normal B cells while the complete remissions have continued. This result shows that remissions of diffuse large B-cell lymphoma can continue after disappearance of a functional anti-CD19 CAR T-cell response. Beyond CD19, active CAR T-cell therapy for multiple myeloma has been developed by targeting B-cell maturation antigen (BCMA). Anti-BCMA CAR T cells have undergone rapid clinical development since first being reported in 2013 (Carpenter et al. Clinical Cancer Research, 2013). Clinical results of the first-in-humans clinical trial of anti-BCMA CARs (Brudno et al. Journal of Clinical Oncology, 2018) will be presented, and future plans for multiple myeloma CAR T-cell therapies will be discussed. Citation Format: James N. Kochenderfer. CAR T-cell therapy for lymphoma and multiple myeloma [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 IA12.","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"365 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":"133384388","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 A040: Hijacking CAR19 T-cells for use in targeting diverse hematopoietic and solid tumors","authors":"P. Rennert, Fay J. Dufort, Lihe Su, Lan Wu, A. Birt, C. Ambrose, R. Lobb","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A040","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A040","url":null,"abstract":"Introduction: CAR T-cells targeting CD19 (CAR19s) can eradicate B cell leukemias and lymphomas. The effectiveness of CAR19s is linked to their robust expansion properties but also their long-term persistence. Persistence is maintained by normal CD19+ B cells: a non-tumor dependent, self-renewing source of antigen. In this manner, CAR19s are quite unique. We have re-engineered CAR19s to secrete a wide variety of retargeting fusion proteins (FPs) by encoding expression cassettes downstream of the CAR sequence in lentiviral vectors. By hijacking CAR19s, we utilize their inherent persistence properties. By designing multispecific FP, we directly counter the clinically critical issues of tumor heterogeneity and antigen loss. Experimental Procedures: A lentiviral vector with an MCSV promoter was used to express the CAR19 construct and FPs. FPs and multispecific-FPs were designed to encode the extracellular domain of the CD19 protein, followed by one or two scFv sequences, separated from the CAR sequence by a P2A cleavage site, a design termed IMPACTtm (Integrated Modular Proteins for Adoptive Cell Therapy). The FPs therefore consist of the CD19 extracellular domain linked to one or more scFvs. The FPs redirect CAR19 T-cell cytotoxic activity to any tumor antigen of interest by coating that antigen with CD19 via the scFv. Further, multiple antigens can be coated with CD19 by encoding multiple scFv in the FP. Hijacked CAR19s therefore serve as a platform for targeting diverse antigens. Results: Here we describe one example in detail, focusing on Her2+ solid tumors. The CD19/anti-Her2 FP was highly potent in cytotoxicity assays targeting Her2+/CD19- solid tumor cell lines. The concentration of fusion protein required to reduce tumor cell number by 50% was 10 pM (0.7 ng/ml). Primary donor T-cells transduced with the CAR19 - CD19/anti-Her2 FP lentiviral vector secreted > 20 ng/ml of FP in cell culture. Cytotoxic activity of this redirected CAR19 against Her2+ SKOV3 tumor cells was demonstrated in vitro and in vivo. CD19-mediated persistence was demonstrated in serial restimulation assays. A bispecific FP containing CD19 linked to anti-Her2 and anti-EGFR scFv had specific activity against both antigens with a potency of 0.75 pM. For each antigen, the potent cytotoxicity was specifically mediated by the secreted fusion protein. Additional program examples of multispecific targeting for diverse hematologic and solid tumor types will be shown. Conclusions: The IMPACT platform addresses critical issues in cell therapy including CAR persistence, antigen escape and antigen heterogeneity, and provides important solutions for treating both hematologic and solid tumors. The potency of redirected cytotoxicity supports clinical development of CAR19/IMPACT programs, four of which are now ready for IND enabling studies. Citation Format: Paul Rennert, Fay Dufort, Lihe Su, Lan Wu, Alyssa Birt, Christine Ambrose, Roy Lobb. Hijacking CAR19 T-cells for use in targeting divers","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"53 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":"127271288","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 A029: Unexpected antagonism between oncolytic virus derived type I interferon and EGFRvIII CAR T-cells","authors":"Laura Evgin, Amanda L. Huff, Phonphimon Wongthida, Jill Thompson, T. Kottke, J. Sampson, L. Perez, R. Vile","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A029","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A029","url":null,"abstract":"Although the chimeric antigen receptor (CAR) T-cell platform has experienced clinical success in patients with hematologic malignancies, CAR T-cells specific to solid tumor targets have met with more limited efficacy. The highly inflammatory nature of oncolytic viruses and their ability to remodel the tumor microenvironment suggested to us that they would provide a complementary mechanism of action to both recruit and potentiate the functionality of CAR T-cells. VSVmIFNβ injection into B16EGFRvIII tumors increased the expression of chemokines such as CXCL10 and CCL5, which we would expect to recruit CXCR3+and CCR5+ EGFRvIII specific murine CAR T-cells. However, we did not observe an increase in overall survival or tumor control using the combination strategy compared to monotherapy with CAR T-cells. We recovered fewer viable CD8+ CAR T-cells from tumors injected with VSVmIFNβ early after adoptive transfer, and observed a similar reduction in the number of CD8+ CAR T-cells which persisted long term in the blood. We have shown that type I interferon increases the expression of the CAR from the retroviral LTR and in turn sensitizes these cells to tonic signaling mediated exhaustion and apoptosis. Correspondingly, CAR T-cells prepared from IFNAR KO T-cells were protected from the deleterious effect of type I IFN in vivo. We are currently investigating strategies to overcome the interference between these two modalities and to uncouple the regulation of the expression of the CAR from type I IFN signaling. Citation Format: Laura Evgin, Amanda L. Huff, Phonphimon Wongthida, Jill Thompson, Timothy Kottke, John Sampson, Luis Sanchez Perez, Richard Vile. Unexpected antagonism between oncolytic virus derived type I interferon and EGFRvIII 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 A029.","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"1 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":"116030753","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 A042: A novel nonviral, nonintegrative DNA vector system for T-cell engineering","authors":"P. Schmidt, Matthias Bozza, D. Jaeger, R. Harbottle","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-A042","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-A042","url":null,"abstract":"Adoptive immunotherapy is one of the most encouraging therapeutic strategies for the treatment of a range of cancers. A particularly promising avenue of research is the functional introduction of chimeric antigen receptors (CARs) into naive human T-cells for autologous immunotherapy. Currently, the genetic engineering of these cells is achieved through the use of integrating vector systems such as lentiviruses or the sleeping beauty transposon system, which present a potential risk of genotoxicity associated with their random genomic integration. We have invented a novel DNA vector platform for the safe and efficient generation of genetically engineered T-cells for human immunotherapy. This DNA vector system contains no viral components and comprises only clinically approved sequences; it does not integrate into the target-cell’s genome but it can replicate autonomously and extrachromosomally in the nuclei of dividing human primary cells. These DNA vectors offer several advantages over currently used vector systems; they are not subject to commercial licenses, they are cheaper and easier to produce, and they can more quickly genetically modify human cells without the inherent risk of integrative mutagenesis. In preclinical experiments we have successfully generated genetically engineered human T-cells that sustain the expression of a reporter gene for over a month at persistently high levels without decline. We have also successfully modified these cells with a range of transgenic CAR receptors against several known cancer cell epitopes and we have demonstrated their viability and capability in the targeted killing of these human cancer cells. We showed that CAR-T-cells generated with our technology killed more efficiently target cells when compared to T-cells engineered with current state-of-the-art integrative lentivirus. The expression of functional CARs was detected over a period of two weeks of administration in culture and the anticancer activity of our DNA-CAR-T-cells was evaluated in vivo using xenotransplanted cell lines in immunodeficient mice. We are currently performing analyses in order to determine the molecular behavior of the vector in the cells and its impact on cellular viability. Furthermore, we are developing a protocol for large scale electroporation in order to manufacture a clinical grade CAR-T DNA product. We believe that this novel DNA vector system provides a unique and innovative approach to this exciting therapeutic strategy for cancer therapy. We estimate that this novel methodology will provide a simpler method of CAR T-cell manufacturing, resulting in a 10-fold reduction in the cost of the CAR-T product. Citation Format: Patrick Schmidt, Matthias Bozza, Dirk Jaeger, Richard Harbottle. A novel nonviral, nonintegrative DNA vector system for T-cell engineering [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 201","PeriodicalId":254712,"journal":{"name":"Genetically Engineered T-cells","volume":"86 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":"124738532","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}