scholarly journals IMMU-17. TARGETING GLIOBLASTOMA WITH DNAM-1-BASED CHIMERIC ANTIGEN RECEPTOR (CAR) T CELLS

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi122-vi122
Author(s):  
Tobias Weiss ◽  
Hanna Meister ◽  
Michael Weller ◽  
Charles Sentman ◽  
Patrick Roth
Keyword(s):  
T Cells ◽  
Glioma Cell ◽  
Glioma Cells ◽  
Genetically Engineered ◽  
Human Glioma ◽  
Knock Out ◽  
Car T Cells ◽  
Car T ◽  
Anti Tumor Activity

Abstract BACKGROUND Genetically engineered T cells that express a chimeric antigen receptor (CAR) have shown powerful anti-tumor activity in extracranial malignancies. This concept is now also being explored against glioblastoma. However, many single target antigens used for CAR cell therapy are non-homogeneously expressed. We assessed the therapeutic potential of CAR T cells targeting 2 antigens which are homogeneously expressed by glioma cells which reduces the probability of tumor immune escape due to antigen loss. METHODS We analyzed the expression of CD112 and CD155, which are ligands to the activating immune cell receptor DNAX Accessory Molecule-1 (DNAM-1), in a panel of mouse and human glioma cell lines as well as in human glioblastoma samples and generated glioma cells with a CD112 or CD155 knock-out. To exploit the specific binding properties of DNAM-1, we generated first or second-generation CAR T cells that use DNAM-1 as an antigen-binding domain and investigated their anti-tumor activity in vitro and in vivo using syngeneic orthotopic mouse glioma models. RESULTS CD112 and CD155 are homogeneously expressed in mouse and human glioma cell lines as well as human glioblastoma tissue specimens. CRISPR/Cas9-mediated knock-out of CD112 or CD155 affected the migration of glioma cells, but had no impact on the proliferation or susceptibility to irradiation or temozolomide. DNAM-1-based CAR T cells exerted high cytolytic activity and secretion of various effector cytokines in vitro. Upon intravenous administration, DNAM-1-based CAR T cells did not exert significant toxicity, homed to the tumor site in the brain and prolonged the survival of orthotopic glioma-bearing mice with durable anti-tumor responses in a fraction of mice. CONCLUSION CD112 and CD155 represent attractive targets for glioma immunotherapy using genetically engineered immune cells. Based on the data obtained from our preclincal assessment of DNAM-1-based CAR T cells, this immunotherapeutic strategy might also be explored in human glioma patients.

scholarly journals PL2.1 Exploiting the DNAM-1 system for chimeric antigen receptor (CAR) T cell therapy of glioblastoma

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii2-iii2
Author(s):  
T Weiss ◽  
H Meister ◽  
M Weller ◽  
C Sentman ◽  
P Roth
Keyword(s):  
T Cells ◽  
Cell Lines ◽  
Glioma Cell ◽  
Knock Out ◽  
Car T Cells ◽  
Car T ◽  
Glioma Cell Lines ◽  
Mouse Glioma

Abstract BACKGROUND Cancer immunotherapy with genetically engineered T cells that express a chimeric antigen receptor (CAR) has led to impressive responses in extracranial malignancies and is also explored against glioblastoma. However, CAR T cell strategies that are currently being explored against glioblastoma target single tumor antigens, which are non-homogeneously expressed and are prone to antigen escape. Furthermore, the immunosuppressive brain tumor microenvironment hampers anti-tumor efficacy. METHODS By immunohistochemistry and flow cytometry, we investigated the expression of CD155 and CD112, which are ligands to the activating immune cell receptor DNAX accessory molecule-1 (DNAM-1), in human and mouse glioma cell lines as well as in human glioblastoma samples. To understand their functional role, we generated CD155 or CD112 knock-out glioma cell lines using CRISPR/Cas9 and studied proliferation, sensitivity to irradiation or temozolomide as well as migration. To exploit the promiscuous binding features of DNAM-1, we generated different first or second-generation CAR T cells that use DNAM-1 as a tumor-binding domain. Subsequently, we investigated their anti-tumor activity in vitro in co-culture assays and in vivo in syngeneic orthotopic murine glioma models. RESULTS CD155 and CD112 are homogenously expressed in human and mouse glioma cell lines and human glioblastoma tissues. Knock-out of these ligands affected the migration of tumor cells, but did not affect proliferation or sensitivity to irradition or temozolomide. DNAM-1-based CAR T cells demonstrated high cytolytic activity and effector cytokine secretion in vitro. In vivo, DNAM-1 based CAR T cells reached to the tumor site in the brain upon intravenous administration, prolonged survival of orthotopic glioma-bearing mice and led to a durable anti-tumor response in a fraction of mice. The treatment was tolerated without toxicities. CONCLUSION We elucidated the tumor-intrinisic role of CD155 and CD112 and provide the first systematical preclincal assessment of DNAM-1 CAR T cells against glioma. These findings provide a rationale to test this immunotherapeutic strategy also in human glioma patients.

327 B7-H3 Chimeric Antigen Receptor Modified T Cells Show Potent Anti-Tumor Activity in a Preclinical Model of Glioblastoma

Neurosurgery ◽  
2017 ◽  
Vol 64 (CN_suppl_1) ◽  
pp. 272-272
Author(s):  
Kwong-Hon (Kevin) Chow ◽  
Sabine Heitzeneder ◽  
Peng Xu ◽  
Johanna Theruvath ◽  
Siddhartha S Mitra ◽  
...  
Keyword(s):  
T Cells ◽  
Mouse Model ◽  
Glioma Cell ◽  
Preclinical Model ◽  
Car T Cells ◽  
Car T ◽  
Glioma Cell Lines ◽  
Anti Tumor Activity

Abstract INTRODUCTION While initial phase I data suggest efficacy of local delivery of chimeric antigen receptor (CAR) modified T cells against glioblastoma (GBM), their activity remains limited in part by the intensity of antigen expression. Targeting more robust tumor associated antigens (TAAs) may help to improve anti-tumor responses. B7-H3 (CD276), a transmembrane glycoprotein which is overexpressed on many solid cancers including GBM, is a promising target. Here we generate CAR T cells specific for B7-H3 and characterize their function in a preclinical model of glioblastoma. METHODS B7-H3 CAR T cells were generated by retroviral transduction of healthy donor peripheral blood mononuclear cells (PBMCs) using a vector designed by our lab. The CAR modified T cells were tested in vitro for their ability to produce proinflammatory cytokines and kill B7-H3 positive glioma cell lines. In vivo activity of B7-H3 CAR T cells was tested using an orthotopic GBM xenograft mouse model. RESULTS >B7-H3 CAR T cells produced the proinflammatory cytokines interferon-gamma (IFN-g), interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNF-a) when cocultured with B7-H3 positive glioma cell lines. B7-H3 CAR T cells also killed B7-H3 positive glioma cells in an in vitro cytotoxicity assay. Finally, B7-H3 CAR T cells demonstrated potent anti-tumor activity in vivo, producing tumor regression in our mouse model of GBM and significantly improving survival. CONCLUSION B7-H3 CAR T cells effectively target GBM and demonstrate significant anti-tumor activity in our preclinical studies. Efforts to translate this CAR for clinical use are warranted and will add to the armamentarium for treating patients with GBM and other solid cancers.

High-fidelity genome editing using NextGEN CRISPR (Clo51-dCas9) system for the production of allogeneic CAR-T cells.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 3048-3048
Author(s):  
Xinxin Wang ◽  
Xianghong Li ◽  
Burton Barnett ◽  
Christopher Martin ◽  
David Hermanson ◽  
...  
Keyword(s):  
T Cells ◽  
Genome Editing ◽  
Solid Tumors ◽  
Inhibitory Receptors ◽  
Double Knockout ◽  
Knock Out ◽  
Car T Cells ◽  
Highly Efficient ◽  
Car T

3048 Background: Autologous chimeric antigen receptor (CAR) T therapies are highly efficient at targeting hematological malignancies, but the clinical applications have been limited by individualized manufacturing. Furthermore, there has been little success in treating solid tumors due to immunosuppressive microenvironments. Currently, genome editing technologies are being used to address both issues. However, the CRISPR/Cas9 system has significant safety concerns due to high incidence of off-target mutations and TALEN only works sufficiently in activated cells. A hybrid gene editing system, NextGEN (NG) Clo51-dCas9, can be targeted using gRNA, like CRISPR/Cas9, but exhibits little-to-no off-target cutting like TALEN, thereby overcoming limitations in the genome editing of resting T cells. Methods: We successfully developed a platform for production of allogeneic CAR-T cells with reduced receptivity to inhibitory signaling. Here, T cells were modified by piggyBac-mediated BCMA CAR gene delivery, along with NG reagents to knock out critical genes mediating rejection responses. Gene edited CAR-T cells were assessed by mixed lymphocyte reaction (MLR) and tumor killing. In addition, NG was used to knockout multiple checkpoint inhibitory receptors known to mediate key suppressive signals in T cells. Results: NG demonstrated high gene disruption efficiencies for all targets (84% for TCRα, 91% for TCRβ, 64% for β-2 microglobulin, and 40-60% for the surface inhibitory receptors PD-1, CTLA-4, Tim3, Lag-3, and TGFBRII). In contrast to CRISPR/Cas9, no off-target mutations were detected for multiple targets by deep-sequencing. In MLRs, disruption of TCR eliminated the GvHD response, while disruption of MHCI completely abrogated graft-rejection. Lastly, TCR/MHCI double knockout did not affect the ability to kill BCMA+ multiple myeloma cells in vitro. Conclusions: NG overcomes significant limitations of the CRISPR/Cas9 and TALEN systems with highly efficient genome editing in resting T cells. NG has great potential and flexibility for the manufacture of allogeneic CAR-T cells and for enhancing efficacy against solid tumors.

CD70 as a novel target of CAR-T-cell therapy for gliomas.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 148-148 ◽  
Author(s):  
Linchun Jin ◽  
Haitao Ge ◽  
Changlin Yang ◽  
Yu Long ◽  
Yifan(Emily) Chang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Protein Expression ◽  
Glioma Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Gene And Protein Expression ◽  
Car T ◽  
Human And Mouse

148 Background: Gliomas are the most common primary malignant brain tumor and are uniformly lethal. Cancer immunotherapy has the potential to target gliomas; however, its antitumor effects are restricted by limitations in clinically useful tumor specific targets. Chimeric antigen receptor modified T-cell (CAR-T) therapy is a highly promising option for cancer treatment, due to its combination of precision antibody recognition and T-cell tumor-specific killing. CD70 is an antigen expressed by limited subsets of normal lymphocytes and dendritic cells but is aberrantly overexpressed by glioma cells, which makes it an outstanding glioma-antigen target. Methods: The gene and protein expression of CD70 were evaluated to identify its potential as a glioma target. Human and mouse versions of CD70-specific CAR-T cells were generated, and human primary GBM lines as well as murine lines (GL-261, KR-158B) were used as human and mouse tumor targets, respectively. The antitumor effect of the human and mouse CD70-sepecific CARs were tested in vitro and in orthotopic xenograft and syngeneic murine models. Results: CD70 is only overexpressed by tumor cells in a subset of low-grade gliomas and GBM. The elevated gene and protein expression are associated with increased tumor grade and poor patient survival. Co-culturing CD70-specific CAR-T cells with CD70-positive glioma cells resulted in potent secretion of IFN-gamma and tumor-specific killing in a CD70-dependent manner. Irradiation enhances CD70 expression on glioma cells and thus increases CAR T-cell recognition. Adoptive transfer of the human and mouse CD70 CAR-T cells resulted in tumor regression of immunocompetent and immunodeficient mice, respectively. Conclusions: CD70 can be an excellent tumor target for gliomas, and CD70-specific CAR-T cells have potent antitumor activity against CD70-positive gliomas both in vitro and in vivo. Our study provides crucial preclinical evidence to support the future clinical application of CD70 CAR-T cells to treat gliomas.

scholarly journals 137 Development of anti-Mucin1 CAR-T against solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A146-A146
Author(s):  
Jihyun Lee ◽  
Areum Park ◽  
Jungwon Choi ◽  
Dae Gwan Yi ◽  
Hee Jung Yang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Anti Tumor Activity

BackgroundChimeric antigen receptor (CAR) -T cell therapies have proven to be effective against various liquid tumors. However, the development of CAR-T against solid tumors has been challenging due to insufficient efficacy and potential on-target off-tumor toxicities caused by low expression of tumor antigens on normal tissues. Testing various affinities of CARs has demonstrated that lower affinity CARs maintain its anti-tumor effect while minimizing safety concerns (1). In order to develop a CAR-T against solid tumors expressing Mucin1, we have screened for Mucin1 binding antibodies and tested their anti-tumor effect in vitro and in vivo. The potential of on-target off-tumor toxicity was also measured in vitro.MethodsAnti-Mucin1 human single chain variable fragments (scFv) were obtained via screening against a scFv display library. Anti-Mucin1 scFvs were incorporated into CARs and in vitro, in vivo functions against various tumor cells expressing Mucin1 were tested. For in vivo studies, tumor bearing NOG mice (HCC1954 cells) received anti-Mucin1 CAR-T cells. Therapeutic efficacy was evaluated by measuring tumor volumes. Potential on-target off-tumor toxicity against Mucin1 on normal cells was tested by investigating the killing effect of anti-Mucin1 CAR-T against cancer cell line (HCC70) and non-tumorigenic breast epithelial cell line (MCF-10A) in co-culture systemsResultsIn vitro activity of anti-Mucin1 CAR-T cells that displayed a range of affinities for Mucin1 (27nM to 320nM) showed similar cytokine secretion levels and cytotoxicity against Mucin-1 expressing tumor cell lines (HCC70 and T47D). Robust anti-tumor activity was also demonstrated in vivo against large tumors (400~500 mm3) with relatively small numbers of CAR-T cells (0.5 x 106 CAR-T cells per mouse). In vivo expansion of CAR-T cells were observed in all scFv-CAR-T cases and accompanied by close to complete regression of tumors within 25 days post CAR-T cell injection. Of the 4 scFv CAR-Ts, 2H08 (with a Kd of 94nM) was tested for activity against normal breast epithelial cells. When 2H08-CAR-T was cocultured with a mixture of HCC70 and MCF-10A cells, they preferentially killed only the Mucin1 overexpressing HCC70 cells leaving MCF-10 cells intact.ConclusionsOur study demonstrates anti-tumor activity of a novel scFv-derived CAR-T recognizing Mucin1 and its effectiveness in large pre-established tumors in vivo. We also demonstrate that 2H08-CAR-T can distinguish between target overexpressing cancer cells and normal epithelial cells, which suggests that by toning down the affinity of CAR against antigen one can improve the safety profile of solid tumor antigen targeting CAR-T cell therapies.ReferenceCastellarin M, Sands C, Da T, Scholler J, Graham K, Buza E, Fraietta J, Zhao Y, June C. A rational mouse model to detect on-target, off-tumor CAR T cell toxicity. JCI Insight 2020; 5:e136012Ethics ApprovalAll experiments were done under protocols approved by the Institutional Animal Care and Use Committee (IACUC) (Study#LGME21-011).ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

scholarly journals Locally secreted BiTEs complement CAR T cells by enhancing solid tumor killing

2021 ◽  
Author(s):  
Yibo Yin ◽  
Jesse Rodriguez ◽  
Nannan Li ◽  
Radhika Thokala ◽  
MacLean P Nasrallah ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumor ◽  
Tumor Therapy ◽  
Target Antigen ◽  
Car T Cells ◽  
Car T ◽  
Anti Tumor Activity

Bispecific T-cell engagers (BiTEs) are bispecific antibodies that redirect T cells to target antigen-expressing tumors. BiTEs can be secreted by T cells through genetic engineering and perform anti-tumor activity. We hypothesized that BiTE-secreting T cells could be a valuable T cell-directed therapy in solid tumors, with distinct properties in mono- or multi-valent strategies incorporating chimeric antigen receptor (CAR) T cells. Glioblastomas represent a good model for solid tumor heterogeneity and represent a significant therapeutic challenge. We detected expression of tumor-associated epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), and interleukin-13 receptor alpha 2 (IL13Rα2) on glioma tissues and glioma cancer stem cells. These antigens formed the basis of a multivalent approach, using a conformation-specific tumor-related EGFR targeting antibody (806) and Hu08, an IL13Rα2-targeting antibody, as the scFvs to generate new BiTE molecules. Compared with 806CAR T cells and Hu08CAR T cells, BiTE T cells demonstrated prominent activation, cytokine production, and cytotoxicity in response to target-positive gliomas. Superior response activity was also demonstrated in BiTE secreting bivalent targeting T cells compared with bivalent targeting CAR T cells, which significantly delayed tumor growth in a glioma mouse model. In summary, BiTEs secreted by mono- or multi- valent targeting T cells have potent anti-tumor activity in vitro and in vivo with significant sensitivity and specificity, demonstrating a promising strategy in solid tumor therapy.

Modulation of inhibitory signals in CAR T cells leads to improved activity against glioblastoma.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 3031-3031
Author(s):  
Khaled Sanber ◽  
Zeid Nawas ◽  
Vita Salsman ◽  
Ahmed Gad ◽  
Pretty Matthew ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mouse Model ◽  
Inhibitory Receptors ◽  
Car T Cells ◽  
Ribonucleoprotein Complexes ◽  
Human T Cells ◽  
Car T ◽  
Anti Tumor Activity

3031 Background: Early clinical trials have demonstrated the safety of chimeric antigen receptor (CAR) T cells targeting glioblastoma (GBM), however, their efficacy remains limited by multiple obstacles including the immunosuppressive tumor microenvironment. Adoptively transferred CAR T cells remain susceptible to inhibition via the engagement of co-inhibitory receptors on their surface such as PD1, BTLA, CTLA4 and LAG3. The subsequent recruitment of Src homology region 2 containing protein tyrosine phosphatase 2 (SHP2) by these receptors to the immune synapse may represent a common mechanism of T cell inhibition, as SHP2 can de-phosphorylate key signaling molecules that mediate T cell activation (including CD28 and CD3ζ). We hypothesized that SHP2 deletion will simultaneously offset the effects of multiple co-inhibitory receptors, thereby improving the anti-tumor activity of CAR T cells. Methods: Electroporation of sgRNA/Cas9 ribonucleoprotein complexes into human T cells was used to knockout (KO) SHP2. Retroviral vector transduction was used to express a clinically-utilized second generation CAR (with a CD28 endodomain) targeting HER2. The phenotype of wild-type (WT) and SHP2KO CAR T cells was evaluated with mass cytometry and flow cytometry. Their anti-tumor function was tested in vitro using the xCELLigence assay (an impedance-based cytotoxicity assay), and in vivo, in an orthotopic xenograft mouse model of GBM. Results: Efficient and reproducible depletion of the SHP2 protein in human T cells was verified using western blotting. The Inference of CRISPR Efficiency (ICE) Assay confirmed efficient editing of the PTPN11 gene encoding SHP2. An anti-HER2 CAR was efficiently expressed in the SHP2KO T cells. SHP2 deletion did not significantly affect CAR T cell expansion, proliferation or baseline phenotype. However, following co-culture with HER2+ LN229-GBM cells, the CD8+ central memory (CCR7+ CD45RA-) and effector memory (CCR7- CD45RA-) subsets were enriched to a greater extent in the SHP2KO CAR T cells. The pattern of cytokine co-expression varied between donors in a single-cell analysis comparing SHP2KO to WT CAR T cells after encountering LN229 cells. Functionally, SHP2KO CAR T cells derived from the majority of healthy donor and patient peripheral blood eliminated LN229 cells more rapidly in vitro. In an orthotopic mouse model of GBM, SHP2KO CAR T cells showed better early control of established LN229 xenografts and improved survival in comparison to WT CAR T cells. Conclusions: SHP2 deletion in CD28ζ.CAR T cells improves their anti-tumor activity.

scholarly journals The Application of CAR-T Cells in Haematological Malignancies

2020 ◽  
Vol 68 (6) ◽  
Author(s):  
Katarzyna Skorka ◽  
Katarzyna Ostapinska ◽  
Aneta Malesa ◽  
Krzysztof Giannopoulos
Keyword(s):  
T Cells ◽  
Mechanism Of Action ◽  
B Cell Lymphoma ◽  
Genetically Engineered ◽  
Haematological Malignancies ◽  
Treatment Regimens ◽  
Car T Cells ◽  
Car T

AbstractChimeric antigen receptor (CAR)-T cells (CART) remain one of the most advanced and promising forms of adoptive T-cell immunotherapy. CART represent autologous, genetically engineered T lymphocytes expressing CAR, i.e. fusion proteins that combine components and features of T cells as well as antibodies providing their more effective and direct anti-tumour effect. The technology of CART construction is highly advanced in vitro and every element of their structure influence their mechanism of action in vivo. Patients with haematological malignancies are faced with the possibility of disease relapse after the implementation of conventional chemo-immunotherapy. Since the most preferable result of therapy is a partial or complete remission, cancer treatment regimens are constantly being improved and customized to individual patients. This individualization could be ensured by CART therapy. This paper characterized CART strategy in details in terms of their structure, generations, mechanism of action and published the results of clinical trials in haematological malignancies including acute lymphoblastic leukaemia, diffuse large B-cell lymphoma, chronic lymphocytic leukaemia and multiple myeloma.

scholarly journals 143 Mesothelin (MSLN) targeting allogeneic CAR T cells engineered to overcome tumor immunosuppressive microenvironment

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A151-A151
Author(s):  
Cecile Schiffer-Mannioui ◽  
Sophie Leduc ◽  
Isabelle Chion-Sotinel ◽  
Diane le Clerre ◽  
Valérie Guyot ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Tgfβ Signaling ◽  
Knock Out ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Immunosuppressive Microenvironment

BackgroundChimeric Antigen Receptor (CAR) T cell therapy is emerging as a potential treatment for solid tumors, even if only limited activity has been observed for CAR T therapies to date. Cellular therapies face indeed many hurdles in solid tumors, such as the immunosuppressive microenvironment. TGFβ is an important growth factor of the tumor microenvironment and has been shown to suppress anti-tumor immunity. Gene editing represents a powerful way to enhance properties of CAR T cells and can be used to circumvent the effect of TGFβ signaling. The tumor associated antigen mesothelin (MSLN) is an attractive target for cellular therapy; being expressed at high levels in several tumor types (e.g., pleural mesothelioma and pancreatic cancer) while only modestly expressed in healthy tissues.MethodsUCARTMeso, an allogeneic CAR T cell product targeting MSLN expressing cells is being developed by Cellectis. UCARTMeso bears an anti-MSLN CAR and a triple gene knock-out (KO) for TRAC, CD52 and TGFBR2 genes, all generated using TALEN® gene-editing technology. TRAC KO limits the risk of GvHD, while CD52 KO allows the use of alemtuzumab in the preconditioning regimen. The additional KO of TGFBR2 confers resistance to the immunomodulatory effects of TGFβ within the solid tumor microenvironment.ResultsPreclinical studies showed high specificity of the anti-MSLN CAR, as well as potent anti-tumor activity in vitro against different cell lines expressing MSLN. In addition, this activity was confirmed in mouse studies against pancreatic and pleural mesothelioma tumor models, with comparable activities observed in the latest model upon i.v. or intra-pleural administration of UCARTMeso. Also, we observed that TGFBR2 edited anti-MSLN CAR T cells displayed a blockade in the TGFβ signaling pathway, being able to respond to antigen stimulation in the presence of TGFβ (figure 1).Abstract 143 Figure 1Left panel: TGFβ-induced SMAD2/3 phosphorylation in anti-MSLN CAR T cells. UCARTMeso cells were stained with mesothelin recombinant protein for CAR detection and anti-pSMAD2/3 one hour post exposure to TGFβ. The lack of SMAD2/3 phosphorylation in TGFBR2 KO cells indicates that they are unable to trigger TGFβ signaling. Right panel: Antigen-induced anti-MSLN CAR T cell activation in the presence (blue histogram) or absence (red histogram) of TGFβ. CAR T cells were stained with anti-CD25 antibody and analyzed by flow cytometry 5 days post exposure to antigen ± TGFβ. The data shows that cells not edited at the TGFBR2 locus are unable to be activated upon target exposure in the presence of TGFβ, while edited cells were activated in the presence of TGFβ, triggering CD25 expression at similar levels as those of cells activated in the absence of TGFβ.ConclusionsAltogether, we have demonstrated potent antitumor activity in vitro and in vivo, and that addition of the third knock-out of TGFBR2 gene provide valuable additional properties to UCARTMeso cells, representing a very attractive strategy for their use in the treatment of solid tumors.

scholarly journals 107 Armoring NKG2D CAR T cells with IL-18 improves in vivo anti-tumor activity

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A118-A118
Author(s):  
Eytan Breman ◽  
Ann-Sophie Walravens ◽  
Isabelle Gennart ◽  
Amelie Velghe ◽  
Thuy Nguyen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Car T Cells ◽  
Tumor Bearing ◽  
Car T Cell ◽  
Car T ◽  
High Doses ◽  
Anti Tumor Activity

BackgroundWhilst delivering impressive clinical efficacy in certain hematological malignancies, Chimeric Antigen Receptor (CAR) T cell therapy has yet to deliver significant clinical impact across a broader array of cancer indications. Armoring CAR T through the co-expression of immune modifying cytokines is an approach that may aid anti-cancer activity but is currently at an embryonic stage of development. In this study, the potential benefit of expressing IL-18 alongside a NKG2D CAR was assessed.MethodsA series of retroviral vectors encoding the NKG2D CAR (a fusion of NKG2D with CD3z), a cell surface tag to facilitate cell selection and tracking (truncated CD19) either with or without full length IL-18 were compared. In certain vectors, a single shRNA targeting CD3z was included to generate allogeneic CAR T versions. All transgenes were delivered as a single vector expressed under the control of the retroviral promoter with individual 2A elements ensuring equimolar levels of protein expression. T cells transduced with the individual vectors were challenged in vitro and in vivo to determine the impact of IL-18 upon NKG2D CAR directed function.ResultsArmored NKG2D CAR T cells that included the IL-18 transgene showed high levels of IL-18 secretion in culture and increased levels of interferon gamma secretion upon antigen challenge as compared to non-armored NKG2D CAR T cells. Armored NKG2D CAR T cells also showed prolonged sequential target cell killing as compared to non-armored CAR T versions. Importantly, in an in vivo stress test where the dose of non-armored NKG2D T cells was reduced to a level where minimal anti-tumor activity and survival above control was seen using an established THP-1 model, armored CAR T cells showed enhanced anti-tumor activity (as determined by bioluminescence) and overall survival. Interestingly, at high doses of armored CAR T cells, toxicity was seen in some tumor bearing models. This toxicity was abrogated by systemic infusion of human IL-18 binding protein (IL-18BP).ConclusionsArmoring NKG2D CAR T cells with IL-18 resulting in increased in vitro and in vivo target-dependent anti-tumor activity. The transient toxicity observed with high doses of the armored CAR T in tumor bearing models was eliminated by IL-18BP. Together, these observations imply that armoring NKG2D CAR T cells with IL-18 is likely to drive improved anti-tumor activity of the CAR T cell in line with previous publications1 2 while the presence of systemic IL-18BP3 should negate possible toxicities arising from high level constitutive expression of the cytokine.ReferencesChmielewski M, Abken H. Cell Reports 2017;21(11): 3205–32192.Hu B, Ren J, Luo Y, Keith B, Young R, Scholler J, Zhao Y, June C. Cell Reports 2017; 20(13): 3025–30333.Dinarello C, Novick D, Kim S, Kaplamski G. Frontiers in Immunology 2013;4;289

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