scholarly journals Selectively targeting myeloid-derived suppressor cells through TRAIL receptor 2 to enhance the efficacy of CAR T cell therapy for treatment of breast cancer

2021 ◽  
Vol 9 (11) ◽  
pp. e003237
Author(s):  
Saisha A Nalawade ◽  
Paul Shafer ◽  
Pradip Bajgain ◽  
Mary K McKenna ◽  
Arushana Ali ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nuclear Translocation ◽  
Suppressor Cells ◽  
T Cell Proliferation ◽  
Myeloid Derived Suppressor Cells ◽  
Tumor Site ◽  
Costimulatory Receptor ◽  
Car T Cells ◽  
Car T

BackgroundSuccessful targeting of solid tumors such as breast cancer (BC) using chimeric antigen receptor (CAR) T cells has proven challenging, largely attributed to the immunosuppressive tumor microenvironment (TME). Myeloid-derived suppressor cells (MDSCs) inhibit CAR T cell function and persistence within the breast TME. To overcome this challenge, we have developed CAR T cells targeting tumor-associated mucin 1 (MUC1) with a novel chimeric costimulatory receptor that targets tumor necrosis factor–related apoptosis-inducing ligand receptor 2 (TR2) expressed on MDSCs.MethodsThe function of the TR2.41BB costimulatory receptor was assessed by exposing non-transduced (NT) and TR2.41BB transduced T cells to recombinant TR2, after which nuclear translocation of NFκB was measured by ELISA and western blot. The cytolytic activity of CAR.MUC1/TR2.41BB T cells was measured in a 5-hour cytotoxicity assay using MUC1+ tumor cells as targets in the presence or absence of MDSCs. In vivo antitumor activity was assessed using MDSC-enriched tumor-bearing mice treated with CAR T cells with or without TR2.41BB.ResultsNuclear translocation of NFκB in response to recombinant TR2 was detected only in TR2.41BB T cells. The presence of MDSCs diminished the cytotoxic potential of CAR.MUC1 T cells against MUC1+ BC cell lines by 25%. However, TR2.41BB expression on CAR.MUC1 T cells induced MDSC apoptosis, thereby restoring the cytotoxic activity of CAR.MUC1 T cells against MUC1+ BC lines. The presence of MDSCs resulted in an approximately twofold increase in tumor growth due to enhanced angiogenesis and fibroblast accumulation compared with mice with tumor alone. Treatment of these MDSC-enriched tumors with CAR.MUC1.TR2.41BB T cells led to superior tumor cell killing and significant reduction in tumor growth (24.54±8.55 mm3) compared with CAR.MUC1 (469.79±81.46 mm3) or TR2.41BB (434.86±64.25 mm3) T cells alone. CAR.MUC1.TR2.41BB T cells also demonstrated improved T cell proliferation and persistence at the tumor site, thereby preventing metastases. We observed similar results using CAR.HER2.TR2.41BB T cells in a HER2+ BC model.ConclusionsOur findings demonstrate that CAR T cells that coexpress the TR2.4-1BB receptor exhibit superior antitumor potential against breast tumors containing immunosuppressive and tumor promoting MDSCs, resulting in TME remodeling and improved T cell proliferation at the tumor site.

Overcoming the breast tumor microenvironment by targeting MDSCs through CAR-T cell therapy.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 1032-1032
Author(s):  
Saisha Abhay Nalawade ◽  
Paul Shafer ◽  
Pradip Bajgain ◽  
Katie McKenna ◽  
Arushana Ali ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Nuclear Translocation ◽  
T Cell Proliferation ◽  
Tumor Site ◽  
Car T Cells ◽  
Car T

1032 Background: Successful targeting of solid tumors such as breast cancer (BC) using CAR T cells (CARTs) has proven challenging, largely due to the immune suppressive tumor microenvironment (TME). Myeloid derived suppressor cells (MDSCs) inhibit CART’s function and persistence within the breast TME. We generated CAR T cells targeting tumor-expressed mucin 1 (MUC1) (Bajgain P et al, 2018) for BC. To potentiate expansion and persistence of MUC1 CARTs and modulate the suppressive TME, we developed a novel chimeric co-stimulatory receptor, TR2.4-1BB, encoding a ScFv derived from a TNF-related apoptosis-inducing ligand receptor 2 (TR2) mAb followed by a 4-1BB endodomain. We hypothesize that engagement with TR2 expressed on TME-resident MDSCs, will lead to both MDSC apoptosis and CART co-stimulation, promoting T cell persistence and expansion at tumor site. Methods: Function of the novel TR2.4-1BB receptor, was assessed by exposing non-transduced (NT) and TR2.4-1BB transduced T cells to recombinant TR2 and nuclear translocation of NFκB was measured by ELISA. Functionality of in vitro generated MDSCs was determined by the suppression assay. In vitro CART/costimulatory receptor T cell function was measured by cytotoxicity assays using MUC1+ tumor targets in presence or absence of MDSCs. In vivo anti-tumor activity was assessed using MDSC enriched tumor-bearing mice using calipers to assess tumor volume and bioluminescence imaging to track T cells. Results: Nuclear translocation of NFκB was detected only in TR2.4-1BB T cells. MDSCs significantly attenuated T cell proliferation by 50±5% and IFNγ production by half compared with T cells cultured alone. Additionally, presence of MDSCs, diminished cytotoxic potential of MUC1 CARTs against MUC1+ BC cell lines by 25%. However, TR2.4-1BB expression on CAR.MUC1 T cells induced MDSC apoptosis thereby restoring the cytotoxic activity of CAR.MUC1 against MUC1+ BC lines in presence of TR2.4-1BB (67±8.5%). There was an approximate two-fold increase in tumor growth due enhanced angiogenesis and fibroblast accumulation in mice receiving tumors + MDSCs compared to tumors alone. Treatment of these MDSC-enriched tumors with MUC1.TR2.4-1BB CARTs led to superior tumor cell killing and significant reduction in tumor growth (24.54±8.55 mm3) compared to CAR.MUC1 (469.79.9±81.46mm3) or TR2.4-1BB (434.86±64.25 mm3) T cells alone (Day 28 after T cell injection). The treatment also improved T cell proliferation and persistence at the tumor site. Thereby, leading to negligible metastasis demonstrating ability of CARTs to eliminate tumor and prevent dissemination. We observed similar results using HER2.TR2.4-1BB CARTs in a HER2+ BC model. Conclusions: Our findings demonstrate that CARTs co-expressing our novel TR2.4-1BB receptor have higher anti-tumor potential against BC tumors and infiltrating MDSCs, resulting in TME remodeling and improved T cell proliferation at the tumor site.

scholarly journals Polymorphonuclear myeloid-derived suppressor cells impair the anti-tumor efficacy of GD2.CAR T-cells in patients with neuroblastoma

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Nicola Tumino ◽  
Gerrit Weber ◽  
Francesca Besi ◽  
Francesca Del Bufalo ◽  
Valentina Bertaina ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clinical Trial ◽  
T Cells ◽  
T Cell ◽  
High Risk ◽  
Suppressor Cells ◽  
Myeloid Derived Suppressor Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

AbstractThe outcome of patients affected by high-risk or metastatic neuroblastoma (NB) remains grim, with ≥ 50% of the children experiencing relapse or progression of the disease despite multimodal, intensive treatment. In order to identify new strategies to improve the overall survival and the quality of life of these children, we recently developed and optimized a third-generation GD2-specific chimeric antigen receptor (CAR) construct, which is currently under evaluation in our Institution in a phase I/II clinical trial (NCT03373097) enrolling patients with relapsed/refractory NB. We observed that our CAR T-cells are able to induce marked tumor reduction and even achieve complete remission with a higher efficiency than that of other CAR T-cells reported in previous studies. However, often responses are not sustained and relapses occur. Here, we demonstrate for the first time a mechanism of resistance to GD2.CAR T-cell treatment, showing how polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) increase in the peripheral blood (PB) of NB patients after GD2.CAR T-cell treatment in case of relapse and loss of response. In vitro, isolated PMN-MDSC demonstrate to inhibit the anti-tumor cytotoxicity of different generations of GD2.CAR T-cells. Gene-expression profiling of GD2.CAR T-cells “conditioned” with PMN-MDSC shows downregulation of genes involved in cell activation, signal transduction, inflammation and cytokine/chemokine secretion. Analysis of NB gene-expression dataset confirms a correlation between expression of these genes and patient outcome. Moreover, in patients treated with GD2.CAR T-cells, the frequency of circulating PMN-MDSC inversely correlates with the levels of GD2.CAR T-cells, resulting more elevated in patients who did not respond or lost response to the treatment. The presence and the frequency of PMN-MDSC in PB of high-risk and metastatic NB represents a useful prognostic marker to predict the response to GD2.CAR T-cells and other adoptive immunotherapy. This study underlines the importance of further optimization of both CAR T-cells and clinical trial in order to target elements of the tumor microenvironment.

scholarly journals Expansion of Functional Myeloid-Derived Suppressor Cells in Controlled Human Malaria Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Carlos Lamsfus Calle ◽  
Rolf Fendel ◽  
Anurag Singh ◽  
Thomas L. Richie ◽  
Stephen L. Hoffman ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Malaria Infection ◽  
Malaria Vaccine ◽  
Suppressor Cells ◽  
T Cell Proliferation ◽  
Myeloid Derived Suppressor Cells ◽  
Controlled Human Malaria Infection

Malaria can cause life-threatening complications which are often associated with inflammatory reactions. More subtle, but also contributing to the burden of disease are chronic, often subclinical infections, which result in conditions like anemia and immunologic hyporesponsiveness. Although very frequent, such infections are difficult to study in endemic regions because of interaction with concurrent infections and immune responses. In particular, knowledge about mechanisms of malaria-induced immunosuppression is scarce. We measured circulating immune cells by cytometry in healthy, malaria-naïve, adult volunteers undergoing controlled human malaria infection (CHMI) with a focus on potentially immunosuppressive cells. Infectious Plasmodium falciparum (Pf) sporozoites (SPZ) (PfSPZ Challenge) were inoculated during two independent studies to assess malaria vaccine efficacy. Volunteers were followed daily until parasites were detected in the circulation by RT-qPCR. This allowed us to analyze immune responses during pre-patency and at very low parasite densities in malaria-naïve healthy adults. We observed a consistent increase in circulating polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) in volunteers who developed P. falciparum blood stage parasitemia. The increase was independent of preceding vaccination with a pre-erythrocytic malaria vaccine. PMN-MDSC were functional, they suppressed CD4+ and CD8+ T cell proliferation as shown by ex-vivo co-cultivation with stimulated T cells. PMN-MDSC reduced T cell proliferation upon stimulation by about 50%. Interestingly, high circulating PMN-MDSC numbers were associated with lymphocytopenia. The number of circulating regulatory T cells (Treg) and monocytic MDSC (M-MDSC) showed no significant parasitemia-dependent variation. These results highlight PMN-MDSC in the peripheral circulation as an early indicator of infection during malaria. They suppress CD4+ and CD8+ T cell proliferation in vitro. Their contribution to immunosuppression in vivo in subclinical and uncomplicated malaria will be the subject of further research. Pre-emptive antimalarial pre-treatment of vaccinees to reverse malaria-associated PMN-MDSC immunosuppression could improve vaccine response in exposed individuals.

scholarly journals P06.07 CXCR6 expression enhances accumulation of anti-mesothelin CAR T cells at the tumor site and their therapeutic efficacy in pancreatic cancer xenografts

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A44.2-A44
Author(s):  
S Stoiber ◽  
S Lesch ◽  
J Ogonek ◽  
B Cadilha ◽  
M Benmebarek ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Tumor Site ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

BackgroundChimeric antigen receptor (CAR) T cell therapy is currently approved for the treatment of some hematological malignancies. However, CAR T cells have so far lacked efficacy in the treatment of solid tumors. A major hurdle of CAR T cell therapy is the limited infiltration of CAR T cells into tumor tissue. Chemokine receptors enable immune cells to migrate along a chemokine gradient. Here, we show that overexpression of the C-X-C chemokine receptor 6 (CXCR6) enhances CAR T cell accumulation in C-X-C motif ligand 16 (CXCL16)-positive xenograft pancreatic cancer models, resulting in increased anti-tumor potency of anti-mesothelin CAR T cells.Materials and MethodsHuman T cells were retrovirally transduced with an anti-mesothelin CAR and CXCR6. NSG mice were injected subcutaneously with mesothelin-CXCL16-overexpressing tumor cells. Mice were treated once with CAR-, CAR-CXCR6- or mock-transduced T cells when tumors were palpable and tumor size was monitored with a caliper. In a separate tracking experiment, subcutaneous tumors were established as described above and the presence of T cells at the tumor site was determined by FACS analysis within one week after adoptive T cell transfer. For orthotopic xenograft experiments mesothelin-CXCL16-overexpressing tumor cells were directly injected into the pancreas of NSG mice and one-time treatment with CAR-, CAR-CXCR6- or mock T cells was performed 5 days post tumor injection.ResultsIn a subcutaneous xenograft model of pancreatic cancer CXCR6-expressing CAR T cells displayed improved anti-tumoral potency compared to CAR T cells without CXCR6, resulting in prolonged survival of mice and tumor clearance in 9 out of 10 CAR-CXCR6-treated mice. A tracking experiment confirmed the increased accumulation of CAR-CXCR6 T cells compared to CAR T cells at the subcutaneous tumor site, suggesting increased migratory capacity of CAR-CXCR6-transduced T cells towards CXCL16-expressing tumors as the mode of action. Treatment of orthotopic pancreatic cancer xenografts similarly revealed prolonged survival of CAR-CXCR6-treated animals in comparison to CAR-treated animals, suggesting improved anti-tumor efficacy of CAR-CXCR6-transduced T cells.ConclusionsForced expression of CXCR6 in anti-mesothelin CAR T cells increased the accumulation of CAR T cells at the CXCL16-positive tumor site, resulting in improved survival of treated mice and in complete tumor rejection in the majority of cases. This data reveals the potential of CXCR6 to direct CAR T cells to the tumor site and this approach may therefore be an attractive strategy to target a major pitfall in the translation of CAR T cell therapy to solid tumors.Disclosure InformationS. Stoiber: None. S. Lesch: None. J. Ogonek: None. B. Cadilha: None. M. Benmebarek: None. A. Gottschlich: None. P. Metzger: None. C. Hörth: None. A. Nottebrock: None. S. Endres: None. S. Kobold: None.

A killer sidekick for antitumor T cells

2019 ◽  
Vol 11 (479) ◽  
pp. eaaw5325
Author(s):  
Christian S. Hinrichs
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Suppressor Cells ◽  
Myeloid Derived Suppressor Cells ◽  
Car T Cell ◽  
Car T

Engineered NK cells kill myeloid-derived suppressor cells to aid CAR-T cell antitumor responses.

scholarly journals Preinfusion polyfunctional anti-CD19 chimeric antigen receptor T cells are associated with clinical outcomes in NHL

Blood ◽  
2018 ◽  
Vol 132 (8) ◽  
pp. 804-814 ◽  
Author(s):  
John Rossi ◽  
Patrick Paczkowski ◽  
Yueh-Wei Shen ◽  
Kevin Morse ◽  
Brianna Flynn ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Clinical Response ◽  
Clinical Outcomes ◽  
Chimeric Antigen Receptor ◽  
T Cell Proliferation ◽  
Car T Cells ◽  
Key Points ◽  
Car T

Key Points The PSI of manufactured CAR T cells was associated with clinical response and toxicities. Monitoring CAR T-cell polyfunctionality as a key product attribute may complement other characteristics including T-cell proliferation.

scholarly journals Recent Advances in Allogeneic CAR-T Cells

Biomolecules ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 263 ◽  
Author(s):  
Dong Wook Kim ◽  
Je-Yoel Cho
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Cell Proliferation ◽  
Downstream Signaling ◽  
Car T Cells ◽  
Cell Based Therapy ◽  
Car T ◽  
Genes Encoding ◽  
Signaling Domains

In recent decades, great advances have been made in the field of tumor treatment. Especially, cell-based therapy targeting tumor associated antigen (TAA) has developed tremendously. T cells were engineered to have the ability to attack tumor cells by generating CAR constructs consisting of genes encoding scFv, a co-stimulatory domain (CD28 or TNFRSF9), and CD247 signaling domains for T cell proliferation and activation. Principally, CAR-T cells are activated by recognizing TAA by scFv on the T cell surface, and then signaling domains inside cells connected by scFv are subsequently activated to induce downstream signaling pathways involving T cell proliferation, activation, and production of cytokines. Many efforts have been made to increase the efficacy and persistence and also to decrease T cell exhaustion. Overall, allogeneic and universal CAR-T generation has attracted much attention because of their wide and prompt usage for patients. In this review, we summarized the current techniques for generation of allogeneic and universal CAR-T cells along with their disadvantages and limitations that still need to be overcome.

scholarly journals Myeloid-derived suppressor cells are bound and inhibited by anti-thymocyte globulin

2019 ◽  
Vol 25 (1) ◽  
pp. 46-59 ◽  
Author(s):  
Young Suk Lee ◽  
Eduardo Davila ◽  
Tianshu Zhang ◽  
Hugh P Milmoe ◽  
Stefanie N Vogel ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Suppressor Cells ◽  
Arginase Activity ◽  
T Cell Proliferation ◽  
Myeloid Derived Suppressor Cells ◽  
Tumor Bearing ◽  
And Function

Myeloid-derived suppressor cells (MDSCs) inhibit T cell responses and are relevant to cancer, autoimmunity and transplant biology. Anti-thymocyte globulin (ATG) is a commonly used T cell depletion agent, yet the effect of ATG on MDSCs has not been investigated. MDSCs were generated in Lewis Lung Carcinoma 1 tumor-bearing mice. MDSC development and function were assessed in vivo and in vitro with and without ATG administration. T cell suppression assays, RT-PCR, flow cytometry and arginase activity assays were used to assess MDSC phenotype and function. MDSCs increased dramatically in tumor-bearing mice and the majority of splenic MDSCs were of the polymorphonuclear subset. MDSCs potently suppressed T cell proliferation. ATG-treated mice developed 50% fewer MDSCs and these MDSCs were significantly less suppressive of T cell proliferation. In vitro, ATG directly bound 99.6% of MDSCs. CCR7, L-selectin and LFA-1 were expressed by both T cells and MDSCs, and binding of LFA-1 was inhibited by ATG pre-treatment. Arg-1 and PD-L1 transcript expression were reduced 30–40% and arginase activity decreased in ATG-pretreated MDSCs. MDSCs were bound and functionally inhibited by ATG. T cells and MDSCs expressed common Ags which were also targets of ATG. ATG may be helpful in tumor models seeking to suppress MDSCs. Alternatively, ATG may inadvertently inhibit important T cell regulatory events in autoimmunity and transplantation.

scholarly journals AMV564 Depletes Myeloid-Derived Suppressor Cells and Expands T Cells: Potential to Address Key Limitations of Cellular Therapies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1702-1702
Author(s):  
Sterling Eckard ◽  
Bianca Rojo ◽  
Victoria Smith ◽  
Patrick Chun
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Ex Vivo ◽  
Suppressor Cells ◽  
Target Cells ◽  
Myeloid Derived Suppressor Cells ◽  
Car T

Abstract Background Myeloid-derived suppressor cells (MDSC) contribute to an immunosuppressive tumor environment and are a barrier to immune therapeutic approaches, including cell-based therapies such as chimeric antigen receptor T cells (CAR T). Despite good overall response rates with certain subsets of B cell leukemias and lymphomas, a significant percentage of patients treated with CAR T therapy do not respond or subsequently relapse. Poor CAR T expansion, poor persistence of infused cells, and clinical treatment failure are associated with tumor and systemic immune dysregulation including high blood levels of peripheral blood monocytic MDSC (M-MDSCs) and interleukin-6, both of which are associated with lack of durable responses 1. In addition, CAR T therapy has been limited by the occurrence of severe cytokine release syndrome (CRS), which is associated with high IL-6 production 2 by myeloid cells such as MDSC. AMV564 is a potent T cell engager that selectively depletes MDSC while promoting T cell activation and proliferation without significant IL-6 induction 3. In phase 1 studies in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and solid tumors, AMV564 has been demonstrated to be clinically safe and active with some patients achieving complete remissions. Methods Cell lines, primary human cells, and patient samples were analyzed using flow cytometry with appropriate marker panels. T cell activation and cytotoxicity assays were conducted using primary human T cells from healthy donors and target cells (3:1 ratio) for 72 hours. T cell activation using ImmunoCult Human CD3/CD28 served as an assay reference. Results Analysis of patients treated with AMV564 demonstrated statistically significant selective depletion of M-MDSC by cycle 2 (Fig. 1A). While on AMV564 therapy, median IL-6 levels remained below 100 pg/mL despite robust T cell activation and expansion. Granzyme B production by CD8 T cells increased significantly between Cycle 1 and Cycle 2 in patients on therapy, and effector CD8 T cells expand over the course of treatment (Fig. 1B-C). These data collectively support the finding that AMV564 both removes a key source of immune suppression and is a potent agonist of T cell function and differentiation in patients. AMV564 potently activates and expands primary T cells ex vivo. Across donors, peak proliferation was significantly higher with AMV564 than with the CD3/CD28 reference (Fig. 2A). Importantly, T cell viability remained significantly higher with AMV564 when compared to reference control (CD3/CD28), and there was no evidence of activation-induced cell death (AICD) in AMV564-treated samples (Fig. 2B). Conclusions AMV564 depletes MDSC and stimulates expansion and longevity of T cells without significant IL-6 induction, suggesting a possible strategy for improvement in efficacy of cell-based therapy such as CAR T approaches. As circulating M-MDSC both at baseline and after CAR T infusion correlate with poor clinical efficacy 4, AMV564 may have beneficial effects during the conditioning phase of cell therapy, after re-infusion of CAR T products into patients, or both. Ex vivo studies using donor T cells and ongoing in vitro studies using CAR T molecules suggest that AMV564 may provide dual benefit with respect to both depletion of MDSC and T cell agonism. References 1. Jain, et al; Blood 2021; 137 (19): 2621-2633. doi: https://doi.org/10.1182/blood.2020007445 2. Li et al., Sci. Transl. Med. 11, eaax8861 (2019) 3. Eckard et al; Cancer Res 2021; (81) (13 Supplement) 528; DOI: 10.1158/1538-7445.AM2021-528 4. Jain, et al; Blood 2019; 134 (Supplement_1): 2885. doi: https://doi.org/10.1182/blood-2019-131041 Figure 1 Figure 1. Disclosures Eckard: Amphivena Therapeutics: Current Employment. Rojo: Amphivena Therapeutics: Current Employment. Smith: Amphivena Therapeutics: Current Employment. Chun: Amphivena Therapeutics: Current Employment.

A Novel Strategy of Switching on/Off CD19CAR Expression Under Tetracycline-Based System

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4424-4424
Author(s):  
Reona Sakemura ◽  
Seitaro Terakura ◽  
Keisuke Watanabe ◽  
Kotaro Miyao ◽  
Daisuke Koyama ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Research Funding ◽  
T Cell Proliferation ◽  
Target Cells ◽  
Car T Cells ◽  
Car T ◽  
Release Assay

Abstract Introduction: Genetic modification of T cells with chimeric antigen receptor (CAR) has emerged with astonishing treatment outcomes for B cell malignancies. Clinical trials of CAR-T therapy demonstrated toxicities such as hypogammaglobulinemia due to B cell aplasia or hemophagocytic syndrome after overactivation of CAR-T cells. These toxicities are considered as major drawbacks for broader application of CAR-T therapy. To overcome these serious adverse events, further modification of CAR-T technology to control CAR expression arbitrary is needed. Therefore we aimed to develop inducible CAR expressing T cells based on tetracycline-regulation system. Methods: We developed a novel inducible CD19CAR system by infusing anti-CD19-CD3z-CD28-tEGFR into pRetroX-TetOne vector (Tet-19CAR). By using Tet-19CAR transduced SUPT1 (T cell line), expression and disappearance kinetics of CAR were determined. We also retrovirally transduced Tet-19CAR into human CD8+ T cells, and achieved more than 90% purity of CAR positive T cells after a selection with anti-EGFR mAb. These CAR-T cells were again expanded with anti-CD3/28 beads and used in 51 Cr release assay, coculture assay, cytokine release assay and T cell proliferation assay. Regarding coculture assay, CD19 transduced K562-CD19 (K562-CD19) was labeled with 0.1 nM CFSE and plated with CAR-T cells at a ratio of 1:1 without IL-2 supplementation and incubated for 96 hours. Finally we examined this system in NOG mice. We injected 0.5 x 106 Raji-ffluc (fire-fly luciferase) followed by 5.0 x 106 CAR-T cells from the tail vein, then we evaluated the tumor flux by in vivo imaging system on days 7, 14, 21, and 30. Results: With more than 100 ng/mL of Doxycycline (Dox), CD19CAR was successfully expressed on both of SUPT1 and CD8+ T cells. For maximum and minimum expression, 24 and 72 hours were needed after addition and discontinuation of Dox, respectively. To determine the cytotoxicity of Tet-19CAR-T cells according to presence or absence of Dox, we performed 51 Cr release assay and coculture assay against K562-CD19. In the presence of Dox, Tet-19CAR showed an equivalent lytic activity to conventional CD19CAR-T cells (c19CAR). In contrast, Tet-19CAR without Dox exhibited significantly lower cytotoxicity against CD19+ target cells. (Dox (-) Tet-19CAR, Dox (+) Tet-19CAR and c19CAR: 14.0±4.0%, 38.0±4.0% and 37.0±2.0% at an E:T ratio = 10:1, respectively). In the coculture assay, Tet-19CAR with Dox eradiated K562-CD19, while they failed to suppress the target cells without Dox. In the intracellular IFN-g assay against K562-CD19, a similar proportion of responder was IFN-g + in Tet-19CAR with Dox and c19CAR. On the other hand, a significantly low proportion of IFN-g + cells were observed in Tet-19CAR without Dox. (Dox (-) Tet-19CAR, 1.0%±0%, Dox (+) Tet-19CAR, 19.1%±6.0% and c19CAR 21.5%±4.0%, respectively) Similar to intracellular IFN-g assay, ELISA revealed that Tet-19CAR with Dox and c19CAR produced IL-2 and IFN-g equally well. However, Tet-19CAR without Dox hardly did. [IL-2 (ng/ml): Dox (-) Tet-19CAR, 1.00±0.060, Dox (+) Tet-19CAR, 9.25±0.30 and c19CAR 8.75±0.68; IFN-g (ng/ml): 2.32±1.24, 57.96±6.95 and 62.42±5.95] (Fig). We next analyzed CAR-T cell proliferation upon stimulation with K562-CD19 over 96 hours. Tet-19CAR with Dox showed 6-7 fold expansion, whereas Tet-19CAR without Dox failed to proliferate. Regarding in vivo model, the mice treated with c19CAR or Tet-19CAR with Dox showed significantly low tumor flux but the mice treated with Tet-19CAR without Dox showed higher tumor burden at day 21 of CAR-T cell infusion [Photons/sec: Dox (-) Tet-19CAR, 2.5 x 1010, Dox (+) Tet-19CAR, 6.4 x 108 and c19CAR, 8.4 x 108 ]. Conclusions: We generated tetracycline-inducible CAR-T cells and successfully controlled the CAR expression with Dox administration. Tet-19CAR without Dox still demonstrated some CD19CAR expression and subsequent cytotoxicity against CD19 positive cells. Nonetheless the CAR expression level of Tet-19CAR without Dox was lower than the threshold for exhibiting positive responses in the function assays such as cytokine production and proliferation. This phenomenon was also confirmed in the xenograft model. To regulate CAR expression more precisely and pursue clinical translations in combinations with other CARs, further efforts are needed to reduce any leaky CAR expression by modification of the system. Figure 1. Figure 1. Disclosures Kiyoi: Pfizer Inc.: Research Funding; Eisai Co., Ltd.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Alexion Pharmaceuticals: Research Funding; MSD K.K.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Teijin Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Japan Blood Products Organization: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding.

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