scholarly journals Epigenetic Priming of Bladder Cancer Cells With Decitabine Increases Cytotoxicity of Human EGFR and CD44v6 CAR Engineered T-Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Camilla M. Grunewald ◽  
Corinna Haist ◽  
Carolin König ◽  
Patrick Petzsch ◽  
Arthur Bister ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Antigen Expression ◽  
Cell Cytotoxicity ◽  
Malignant Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
T Cell Cytotoxicity

BackgroundTreatment of B-cell malignancies with CD19-directed chimeric antigen receptor (CAR) T-cells marked a new era in immunotherapy, which yet has to be successfully adopted to solid cancers. Epigenetic inhibitors of DNA methyltransferases (DNMTi) and histone deacetylases (HDACi) can induce broad changes in gene expression of malignant cells, thus making these inhibitors interesting combination partners for immunotherapeutic approaches.MethodsUrothelial carcinoma cell lines (UCC) and benign uroepithelial HBLAK cells pretreated with the DNMTi decitabine or the HDACi romidepsin were co-incubated with CAR T-cells directed against EGFR or CD44v6, and subsequent cytotoxicity assays were performed. Effects on T-cell cytotoxicity and surface antigen expression on UCC were determined by flow cytometry. We also performed next-generation mRNA sequencing of inhibitor-treated UCC and siRNA-mediated knockdown of potential regulators of CAR T-cell killing.ResultsExposure to decitabine but not romidepsin enhanced CAR T-cell cytotoxicity towards all UCC lines, but not towards the benign HBLAK cells. Increased killing could neither be attributed to enhanced target antigen expression (EGFR and CD44v6) nor fully explained by changes in the T-cell ligands PD-L1, PD-L2, ICAM-1, or CD95. Instead, gene expression analysis suggested that regulators of cell survival and apoptosis were differentially induced by the treatment. Decitabine altered the balance between survival and apoptosis factors towards an apoptosis-sensitive state associated with increased CAR T-cell killing, while romidepsin, at least partially, tilted this balance in the opposite direction. Knockdown experiments with siRNA in UCC confirmed BID and BCL2L1/BCLX as two key factors for the altered susceptibility of the UCC.ConclusionOur data suggest that the combination of decitabine with CAR T-cell therapy is an attractive novel therapeutic approach to enhance tumor-specific killing of bladder cancer. Since BID and BCL2L1 are essential determinants for the susceptibility of a wide variety of malignant cells, their targeting might be additionally suitable for combination with immunotherapies, e.g., CAR T-cells or checkpoint inhibitors in other malignancies.

Effect of dendritic cells (DC) transduced with chimeric antigen receptor (CAR) on CAR T-cell cytotoxicity.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 144-144 ◽  
Author(s):  
Hyung Chan Suh ◽  
Katherine Pohl ◽  
Anna Patricia L. Javier ◽  
Dennis J. Slamon ◽  
John P Chute
Keyword(s):  
T Cells ◽  
T Cell ◽  
Critical Role ◽  
Cell Cytotoxicity ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Mock Control ◽  
T Cell Cytotoxicity

144 Background: T cells interacting DC could be superior in T cell cytotoxicity. CD141+/Cleg9a+ intra-tumoral DC play a critical role in tumor cytotoxicity. Therefore, combining intra-tumoral DC in CAR T cell would safely increase localized CAR T cell cytotoxicity. We hypothesized that bioengineered DC compartment could be an excellent source for enhanced CAR T cell cytotoxicity. Methods: DC precursors and T cells of PBMC were transduced with a CAR (pCCL-anti-CD33-4-1BB-CD3z-T2A-GFP; CAR-DC or CAR T). For comparison, additional DC were transduced with 4-1BB cDNA (pCCL-4-1BB-T2A-GFP; 4-1BB-DC) or mock control (pCCL-eGFP). In addition to lentivirus transduction, differentiation of DC in vitro employed Flt3L/GM-CSF/IL-4. Transduced CAR T and CAR-DC were sorted by GFP expression at day 5. After further 10 days of culture, cells were harvested and analyzed for phenotype. An acute myeloid leukemia (AML) cell line (Kasumi-1) was treated with CAR T +/- CAR-DC, 4-1BB-DC, or mock control for functional assays. Results: Frequencies of cells expressing CD141+/Cleg9a+ were higher in 4-1BB-DC vs. control DC (33% vs. 1.5%). After mixing CAR T and CAR-DC (5X105) with Kasumi-1 (1X105) for 6 hours, CAR T/CAR-DC showed 100% Kasumi-1 cell cytotoxicity compared to 70% of CAR T by trypan blue. CAR T/CAR-DC also demonstrated higher Annexin V positive Kasumi-1 cells compared with CAR-T (91% vs. 52%). CAR T with or without CAR-DC were also assessed with multiplex immunoassays. CAR T cells mixed with CAR-DC induced higher level of IFN-gamma (10,316 vs. 6,186 pg/ml), IL-2 (68,840 vs. 64,708 pg/ml), and TNFalpha (1,361 vs. 905 pg/ml) (Kasumi-1 cells mixed with CAR-T cells of 10 E/T ratio) than CAR T cells. CAR-DC produced significantly higher IL-12 cytokine production (1,352 vs. 161 pg/ml) than CAR T cells in response to CD33 but independent to T cells, confirmed by comparing IL-12 production with CAR T/4-1BB-DC. Conclusions: These data show that in vitro differentiation of DC bearing 4-1BB increases CD141+/Cleg9a+ DC population and that interaction with CAR-DC to CAR T cells enhances anti-AML cytotoxicity. Our finding may implicate the development of CAR-DC therapy combined to CAR T cells to increase the efficiency of cancer immunotherapy.

scholarly journals Efficient Human Acute Myeloid Leukemia Targeting By Universal Chimeric Antigen Receptor T-Cells Via Combinatorial Use of Linkers

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2781-2781
Author(s):  
Laura Volta ◽  
Renier Myburgh ◽  
Christian Edoardo Pellegrino ◽  
Jan Müller ◽  
C. Matthias Wilk ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Half Life ◽  
Antigen Expression ◽  
Car T Cells ◽  
Car T Cell ◽  
Short Half Life ◽  
Car T ◽  
Single Antigen

Abstract Chimeric antigen receptor (CAR) T-cells are genetically engineered T-cells with potent biocidal activity against respective target-expressing cells. Recently, CAR T-cells have been successfully used clinically to eradicate B cell-derived malignancies by targeting B-cell lineage specific surface antigens (e.g. CD19, BCMA). However, several limitations of current single-antigen targeting CAR T-cell therapies are becoming evident: a) low target tumor-antigen expression might lead to low CAR T-cell targeting efficacy; b) single antigen-targeting might lead to rapid selection of tumor cells with low or loss of antigen expression; c) single antigen-targeting does frequently not generate a high tumor-selectivity as tumor-antigens are frequently also expressed on healthy tissues; d) production of single-antigen CAR T-cells is time- and resource-consuming but results in effectors that target only one antigen; e) on-target off-tumor as well as off-target side-effects are difficult to control without terminally eliminating CAR T-cells in the recipient. While a-c) might be overcome by combinatorial tumor-antigen targeting, d-e) might be addressed by production of Universal CAR T-cells that recognize a specific tag on selective bridging molecules with short half-life. To address some of these limitations in principle, we have here developed universal CAR T-cells targeting fluorescein, as well as fluorescein-labeled antibody-constructs directed against several cell surface antigens, that would serve as versatile, combinatorial selective linkers and, upon target binding, also CAR T-cells activators. We then tested the system on human acute myeloid leukemia (AML) cell lines and primary patient AML blasts. Specifically, we engineered CAR T-cells, termed FluA-CAR T-cells, to display the anti-fluorescein engineered lipocalin FluA, which mediated recognition of randomly or site-specifically fluorescein-labeled antibodies in IgG or short half-life diabody (Db) format, directed against the frequently AML expressed antigens CD33, CD117 and CD371. Site-specific chemical modification methods and cysteine-tagged Db mediated the strongest AML killing results in vitro over a broad range of antibody concentrations. We then hypothesized that FluA-CAR T-cells, targeting AML cells via combinatorial use of linker molecules adapted to specific AML antigen-expression profiles, would allow to most efficiently eliminate AML cells in a dose- and timely-regulated fashion. To this end, we tested single and combinatorial use of fluoresceinated anti-CD117 Db and anti-CD371 Db on MOLM13 AML cell lines, engineered to be either CD117 highCD371 neg, CD117 negCD371 high, or CD117 highCD371 high. Indeed, combination of anti-CD117 and anti-CD371 Db-linkers, resulted in significantly improved MOLM13 cell lysis compared to equimolar concentrations of single agents in vitro (example figure). We then tested the same approach, targeting CD117 +CD371 + primary AML cells from two different patients, using either patient-derived or allogeneic FluA CAR T-cells. Again, the combinatorial use of linkers generated a significantly higher AML cell lysis than the use of single Db linkers. We thus here provide proof-of-concept for the generation of highly potent universal targeting FluA CAR T-cells from healthy donors and AML patients. By choosing suitable CAR-adaptors with respect to their conjugation chemistry and size, it is possible to tightly regulate CAR-T cell activity against CD33, CD117 and CD371 expressing AML cells and likely any tumor cell expressed antigen. Short half-life small molecule linkers will allow to control FluA CAR T-cell on-off activity and combinatorial use of linkers will allow to maximize anti-tumor activity and to minimize on-target off-tumor toxicity. Figure 1 Figure 1. Disclosures Myburgh: University of Zurich: Patents & Royalties: CD117xCD3 TEA. Neri: Philogen S.p.A.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Multiple patents on vascular targeting; ETH Zurich: Patents & Royalties: CD117xCD3 TEA. Manz: CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company; University of Zurich: Patents & Royalties: CD117xCD3 TEA.

scholarly journals Overcoming Heterogeneity of Antigen Expression for Effective CAR T Cell Targeting of Cancers

Cancers ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 1075 ◽  
Author(s):  
Sareetha Kailayangiri ◽  
Bianca Altvater ◽  
Malena Wiebel ◽  
Silke Jamitzky ◽  
Claudia Rossig
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Antigen Expression ◽  
T Cell Therapy ◽  
Major Mechanism ◽  
Car T Cells ◽  
Car T Cell ◽  
Combination Strategies ◽  
Car T

Chimeric antigen receptor (CAR) gene-modified T cells (CAR T cells) can eradicate B cell malignancies via recognition of surface-expressed B lineage antigens. Antigen escape remains a major mechanism of relapse and is a key barrier for expanding the use of CAR T cells towards solid cancers with their more diverse surface antigen repertoires. In this review we discuss strategies by which cancers become amenable to effective CAR T cell therapy despite heterogeneous phenotypes. Pharmaceutical approaches have been reported that selectively upregulate individual target antigens on the cancer cell surface to sensitize antigen-negative subclones for recognition by CARs. In addition, advanced T cell engineering strategies now enable CAR T cells to interact with more than a single antigen simultaneously. Still, the choice of adequate targets reliably and selectively expressed on the cell surface of tumor cells but not normal cells, ideally by driving tumor growth, is limited, and even dual or triple antigen targeting is unlikely to cure most solid tumors. Innovative receptor designs and combination strategies now aim to recruit bystander cells and alternative cytolytic mechanisms that broaden the activity of CAR-engineered T cells beyond CAR antigen-dependent tumor cell recognition.

scholarly journals Repurposing Bi-Specific Chimeric Antigen Receptor (CAR) Approach to Enhance CAR T Cell Activity Against Low Antigen Density Tumors

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-30
Author(s):  
Sherly Mardiana ◽  
Olga Shestova ◽  
Marco Ruella ◽  
Saar Gill
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Research Funding ◽  
Cell Activity ◽  
Antigen Expression ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Lentivirus Transduction

BACKGROUND Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of relapsed/refractory B-cell malignancies, as highlighted by high complete remission rates and FDA approval of CD19-specific CAR T cell products. However, depth and duration of remission are limited by antigen loss/downregulation on tumors, as observed in clinical trials using CAR T cells targeting the CD19, CD22, or B cell maturation antigen (BCMA). Antigen density is an important factor modulating CAR T cell responses, and therefore antigen expression below a certain threshold may fail to induce optimal CAR-T cell response. This presents a significant problem as antigen expression varies highly in many cancers including lymphoma, leukemia, multiple myeloma, and solid tumors, both between and within patients. Given that signal strength induced upon antigen encounter determines CAR T cell activity, we hypothesized that simultaneous targeting of two antigens will result in enhanced CAR T cell signaling, allowing them to attack low antigen density (LAD) tumors that would otherwise escape CAR T cells. METHODS Lentivirus transduction was performed to generate CAR T cells from healthy human T cells, using second generation 4-1BBz CARs specific for either human CD19 (FMC63), CD22 (CD22-12), or both, herein referred to as CAR19, CAR22, or CAR19/22, respectively. Tumor cells expressing variegated antigen densities were generated using lentivirus transduction or mRNA electroporation. For in vitro functional characterization, co-culture assay of T cells and tumor cells was performed, and CAR T cell anti-tumor potency was determined by assessing T cell functional parameters including T cell cytotoxicity in real-time using the Incucyte® systems for live-cell imaging and analysis. In addition, cytokine production and CFSE-based proliferation were also assessed using flow cytometry. RESULTS Using CAR19/22, we found LAD tumors are indeed more susceptible to bi-specific CAR T cells compared to either CAR19 or CAR22 T cells, as demonstrated by significantly higher amounts of cytokines produced by bi-specific CAR T cells such as IL-2 (p<0.0001) and IFNg (p<0.0001) compared to CAR19 or CAR22 T cells. Results from cytotoxicity assay revealed increased killing of LAD tumors by CAR19/22 T cells compared to CAR19 (p<0.0001) or CAR22 (p=0.0002) T cells. Further, we found bi-specific CAR19/22 T cells have increased proliferation capacity (p<0.0001) compared to either of the mono-specific CAR T cells. Using NanoString-based RNA analysis, we confirmed that graded antigen density on tumor cells modulates CAR T cell gene expression, with cytotoxicity-associated genes being more susceptible to modulation by antigen expression than cytokine-associated genes. Upcoming experiments aim to test and compare therapeutic efficacy of bi-specific and mono-specific CAR T cells in vivo against LAD tumors. Further characterization of the immune synapse using confocal microscopy as well as analyses of early and late events in downstream CAR signaling will further illuminate the mechanism by which bi-specific CARs can overcome the challenge of low tumor-associated antigen density. CONCLUSIONS Here we showed that antigen density on tumors modulates T cell transcriptional profiles. Our results demonstrated that bi-specific CAR19/22 T cells are superior than mono-specific CAR19 or CAR22 T cells against LAD tumors, as demonstrated by their enhanced cytokine-producing function, cytotoxic capacity and proliferation. Results from this study will provide a novel rationale for repurposing multi-specific CAR T cells as a strategy to improve efficacy against LAD tumors, in addition to the recognized benefit of reducing the risk of antigen-negative escape. Disclosures Ruella: Abclon, BMS, NanoString: Consultancy; Abclon: Consultancy, Research Funding; UPenn/Novartis: Patents & Royalties. Gill:Tmunity Therapeutics: Research Funding; Sensei: Consultancy; Aileron: Consultancy; Fate: Consultancy; Carisma Therapeutics: Patents & Royalties, Research Funding; Novartis: Research Funding.

scholarly journals Aged CAR T Cells Exhibit Enhanced Cytotoxicity and Effector Function but Shorter Persistence and Less Memory-like Phenotypes

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2047-2047 ◽  
Author(s):  
Hiroshi Kotani ◽  
Gongbo Li ◽  
Jiqiang Yao ◽  
Tania E. Mesa ◽  
Jon Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Aged Mice ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Signaling Domain ◽  
The Difference ◽  
Signaling Domains

Abstract [Introduction] CD19 chimeric antigen receptor (CAR) T cell therapies have been approved by the FDA for children and young adults with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL) and adults with r/r large B-cell lymphoma. Recent reports about long-term follow-up of CD19 CAR T cell therapy in B-ALL (Maude et. al. NEJM 2018, Park et. al. NEJM 2018) suggest that the median event-free survival of children and young adult patients is longer than that of adult patients (Over 11 months versus 6.1 months). The reason for the difference between survival of pediatric and adult patient is unclear, but we hypothesize it is due to age-related changes in the T cells collected from patients. Therefore, we compared the function of CAR T cells derived from young or aged mice. [Methods] Young C57BL/6J (B6) mice (6-12 weeks) and aged B6 mice (³ 72 weeks) were used as donors for CAR T cell preparation. Four types of mouse specific CD19 CAR encoded GFP fusion proteins were evaluated with all having the same anti-CD19 scFv and CD8 hinge and transmembrane domains but differing in their intracellular domain (m19Δz: lacks the CD3ξ signaling domain, m19z: CD3ξ signaling domain only, m1928z: CD28 and CD3ξ signaling domains, m19-humBBz: 4-1BB and CD3ξ signaling domains). [Results] T cells isolated from the spleen of aged B6 mice were significantly fewer than those of young B6 mice. However, CAR transduction efficiency, viability and yield were similar between young and aged CAR T cells for each CAR group. All groups of aged CAR T cells predominate with CD8+ and effector-like phenotypes at the expense of CD4+ and memory-like phenotypes after CD19+ artificial antigen presenting cell (aAPC) stimulation (Fig. 1A-1B). Furthermore, compared to CAR T cells derived from young mice, aged CAR T cells (m19z, m1928z and m19BBz) exhibited superior cytotoxicity in a real-time cell analysis for CD19+ aAPC killing (Fig. 1C). Using our immune competent in vivo murine model, aged CAR T cells were short-lived and expanded poorly despite their superior in vitro cytotoxicity. To evaluate for potential mechanisms involving preferential production of effector-like CAR T cells from aged mice we performed gene-expression, as well as single cell secretory polyfunctional analyses. While the polyfunctional strength index (PSI) of CD8+ aged CAR T cells was higher for aged CAR T cells, the increased score was due mostly to abundant secretion of a chemokine (Fig. 1D). Furthermore, the RNA-DESeq analysis demonstrated increased expression of chemokines and perturbation of the EOMES/TBET transcription factor axis. RNA-DESeq also suggested that young CAR T cells were highly active in cell proliferation and cell differentiation whereas aged CAR T cells upregulated gene expression pathways that regulated responses to stimulus and exocytosis. [Conclusions] CAR T cells derived from aged mice exhibited enhanced cytotoxicity but shorter persistence and less memory-like phenotypes. Our results suggest that the difference of clinical outcome between younger patients and older patients may be due to an age-dependent CAR T cell phenotype that is reflected by its unique gene expression pattern, secretory profile, and/or transcription factor balance. In our future directions we are extending these observations to human CAR T cells and identifying potential methods to improve the function of aged CAR T cells. Disclosures Davila: Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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.

Chimeric Antigen Receptor-Engineered T-Cells - A New Way and Era for Lymphoma Treatment

2020 ◽  
Vol 14 (4) ◽  
pp. 312-323
Author(s):  
Romeo G. Mihăilă
Keyword(s):  
T Cells ◽  
T Cell ◽  
Response Rate ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Car T Cells ◽  
Future Developments ◽  
Car T Cell ◽  
Large B Cell Lymphoma ◽  
Car T

Background: Patients with refractory or relapsed diffuse large B-cell lymphoma have a poor prognosis with the current standard of care. Objective: Chimeric Antigen Receptor T-cells (CAR T-cells) are functionally reprogrammed lymphocytes, which are able to recognize and kill tumor cells. The aim of this study is to make progress in this area. Method: A mini-review was achieved using the articles published in Web of Science and PubMed in the last year and the new patents were made in this field. Results: The responses to CAR T-cell products axicabtagene ciloleucel and tisagenlecleucel are promising; the objective response rate can reach up to 83%, and the complete response rate ranges between 40 and 58%. About half of the patients may have serious side effects, such as cytokine release syndrome and neurotoxicity. Current and future developments include the improvement of CAR T-cell expansion and polyfunctionality, the combined use of CAR T-cells with a fusion protein between interferon and an anti-CD20 monoclonal antibody, with checkpoint inhibitors or small molecule sensitizers that have apoptotic-regulatory effects. Furthermore, the use of IL-12-expressing CAR T-cells, an improved technology for the production of CAR T-cells based on targeted nucleases, the widespread use of allogeneic CAR T-cells or universal CAR T-cells obtained from genetically engineered healthy donor T-cells are future developments actively considered. Conclusion: CAR T-cell therapy significantly improved the outcome of patients with relapsed or refractory diffuse large B-cell lymphoma. The advances in CAR T-cells production technology will improve the results and enable the expansion of this new immunotherapy.

scholarly journals Structure of the Signal Transduction Domain in Second-Generation CAR Regulates the Input Efficiency of CAR Signals

2021 ◽  
Vol 22 (5) ◽  
pp. 2476
Author(s):  
Kento Fujiwara ◽  
Masaki Kitaura ◽  
Ayaka Tsunei ◽  
Hotaka Kusabuka ◽  
Erika Ogaki ◽  
...  
Keyword(s):  
Signal Transduction ◽  
T Cells ◽  
T Cell ◽  
Second Generation ◽  
Independent Manner ◽  
Cell Functions ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
The Impact

T cells that are genetically engineered to express chimeric antigen receptor (CAR) have a strong potential to eliminate tumor cells, yet the CAR-T cells may also induce severe side effects due to an excessive immune response. Although optimization of the CAR structure is expected to improve the efficacy and toxicity of CAR-T cells, the relationship between CAR structure and CAR-T cell functions remains unclear. Here, we constructed second-generation CARs incorporating a signal transduction domain (STD) derived from CD3ζ and a 2nd STD derived from CD28, CD278, CD27, CD134, or CD137, and investigated the impact of the STD structure and signaling on CAR-T cell functions. Cytokine secretion of CAR-T cells was enhanced by 2nd STD signaling. T cells expressing CAR with CD278-STD or CD137-STD proliferated in an antigen-independent manner by their STD tonic signaling. CAR-T cells incorporating CD28-STD or CD278-STD between TMD and CD3ζ-STD showed higher cytotoxicity than first-generation CAR or second-generation CARs with other 2nd STDs. The potent cytotoxicity of these CAR-T cells was not affected by inhibiting the 2nd STD signals, but was eliminated by placing the STDs after the CD3ζ-STD. Our data highlighted that CAR activity was affected by STD structure as well as by 2nd STD signaling.

scholarly journals Anti-Mesothelin CAR T cell therapy for malignant mesothelioma

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Laura Castelletti ◽  
Dannel Yeo ◽  
Nico van Zandwijk ◽  
John E. J. Rasko
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Malignant Mesothelioma ◽  
Oncolytic Viruses ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

AbstractMalignant mesothelioma (MM) is a treatment-resistant tumor originating in the mesothelial lining of the pleura or the abdominal cavity with very limited treatment options. More effective therapeutic approaches are urgently needed to improve the poor prognosis of MM patients. Chimeric Antigen Receptor (CAR) T cell therapy has emerged as a novel potential treatment for this incurable solid tumor. The tumor-associated antigen mesothelin (MSLN) is an attractive target for cell therapy in MM, as this antigen is expressed at high levels in the diseased pleura or peritoneum in the majority of MM patients and not (or very modestly) present in healthy tissues. Clinical trials using anti-MSLN CAR T cells in MM have shown that this potential therapeutic is relatively safe. However, efficacy remains modest, likely due to the MM tumor microenvironment (TME), which creates strong immunosuppressive conditions and thus reduces anti-MSLN CAR T cell tumor infiltration, efficacy and persistence. Various approaches to overcome these challenges are reviewed here. They include local (intratumoral) delivery of anti-MSLN CAR T cells, improved CAR design and co-stimulation, and measures to avoid T cell exhaustion. Combination therapies with checkpoint inhibitors as well as oncolytic viruses are also discussed. Preclinical studies have confirmed that increased efficacy of anti-MSLN CAR T cells is within reach and offer hope that this form of cellular immunotherapy may soon improve the prognosis of MM patients.

scholarly journals CARTmath—A Mathematical Model of CAR-T Immunotherapy in Preclinical Studies of Hematological Cancers

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2941
Author(s):  
Luciana R. C. Barros ◽  
Emanuelle A. Paixão ◽  
Andrea M. P. Valli ◽  
Gustavo T. Naozuka ◽  
Artur C. Fassoni ◽  
...  
Keyword(s):  
Mathematical Model ◽  
T Cells ◽  
T Cell ◽  
Mouse Models ◽  
In Silico ◽  
Car T Cells ◽  
Car T Cell ◽  
Cell Immunotherapy ◽  
Car T ◽  
T Cell Immunotherapy

Immunotherapy has gained great momentum with chimeric antigen receptor T cell (CAR-T) therapy, in which patient’s T lymphocytes are genetically manipulated to recognize tumor-specific antigens, increasing tumor elimination efficiency. In recent years, CAR-T cell immunotherapy for hematological malignancies achieved a great response rate in patients and is a very promising therapy for several other malignancies. Each new CAR design requires a preclinical proof-of-concept experiment using immunodeficient mouse models. The absence of a functional immune system in these mice makes them simple and suitable for use as mathematical models. In this work, we develop a three-population mathematical model to describe tumor response to CAR-T cell immunotherapy in immunodeficient mouse models, encompassing interactions between a non-solid tumor and CAR-T cells (effector and long-term memory). We account for several phenomena, such as tumor-induced immunosuppression, memory pool formation, and conversion of memory into effector CAR-T cells in the presence of new tumor cells. Individual donor and tumor specificities are considered uncertainties in the model parameters. Our model is able to reproduce several CAR-T cell immunotherapy scenarios, with different CAR receptors and tumor targets reported in the literature. We found that therapy effectiveness mostly depends on specific parameters such as the differentiation of effector to memory CAR-T cells, CAR-T cytotoxic capacity, tumor growth rate, and tumor-induced immunosuppression. In summary, our model can contribute to reducing and optimizing the number of in vivo experiments with in silico tests to select specific scenarios that could be tested in experimental research. Such an in silico laboratory is an easy-to-run open-source simulator, built on a Shiny R-based platform called CARTmath. It contains the results of this manuscript as examples and documentation. The developed model together with the CARTmath platform have potential use in assessing different CAR-T cell immunotherapy protocols and its associated efficacy, becoming an accessory for in silico trials.

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