Car Genes Research Articles

Bibliometric analysis of chimeric antigen receptor macrophage therapy

Chimeric antigen receptor macrophage (CAR-M) therapy refers to the implantation of specific edited CAR genes into macrophages to equip macrophages to bind to the surface of tumor cells through specific antigens and subsequently activate the activity of macrophages to achieve tumor-killing function. This bibliometric analysis aims to decipher the emerging trends in CAR-M therapy and provide insights for future research. In this review, all relevant literature on CAR-M therapy in the Web of Science has been analyzed for its research trends in this field by using VOS viewer, Pajek, Microsoft Excel, and Endnote software. According to the research findings, it is the most productive in the United States. The institution with the highest number of publications is the University of Pennsylvania. Frontiers in Immunology is one of the most productive journals. Kenderian Saad Sirop publishes the largest number of articles. Keyword cluster analysis shows that the current research trend is more focused on tumor-associated macrophages as well as immunotherapy, exploring the mechanisms and modalities of CAR-M therapy. This study provides a comprehensive summary and analysis of global research trends in CAR-M therapy. In the past few decades, the number of high-quality papers in this field has increased significantly, and CAR-M therapy has provided hope for tumor treatment, truly bringing health gospel to the majority of tumor patients, patients with major diseases, and sub-healthy people.

The Art of Nano-CAR T Cells in Ovarian Cancer Management

The combination of nanotechnology and CAR T-cell therapeutics heralds a paradigm shift in the management of ovarian cancer, a disease characterized by substantial obstacles such as antigenic heterogeneity, an immunosuppressive tumor microenvironment, and physical barriers to treatment efficacy. This paper examines these obstacles critically and proposes innovative nano-engineered solutions, such as the development of nano-CAR T cells and nanoparticle-mediated delivery of CAR genes and immunomodulatory agents, as strategic approaches to improve CAR T-cell delivery, survival, and function within the hostile tumor microenvironment. The convergence of these two disciplines represents a promising strategy for overcoming these obstacles and optimizing the therapeutic potential of CAR T cells in solid malignancies. To translate this promising approach into a viable clinical reality in the fight against ovarian cancer, however, additional scientific research and rigorous clinical trials are required to ensure safety, optimize the design, and establish standardized production and application protocols.

Building a novel TRUCK by harnessing the endogenous IFN-gamma promoter for cytokine expression

Despite the remarkable success of CAR T therapy in hematological malignancies, its efficacy in solid tumors remains limited. Cytokine-engineered CAR T cells offer a promising avenue, yet their clinical translation is hindered by the risks associated with constitutive cytokine expression. In this proof-of-concept study, we leverage the endogenous IFN-γ promoter for transgenic IL-15 expression. We demonstrate that IFN-γ expression is tightly regulated by TCR signaling. By introducing IRES-IL15 into the 3’-UTR of the IFN-γ gene via HDR-mediated knock-in, we confirm that IL-15 expression can co-express with IFN-γ in an antigen-stimulation-dependent manner. Importantly, the insertion of transgenes does not compromise endogenous IFN-γ expression. In vitro and in vivo data demonstrate that IL-15 driven by the IFN-γ promoter dramatically improves CAR T cells' antitumor activity, suggesting the effectiveness of IL-15 expression. Lastly, as part of our efforts toward clinical translation, we have developed an innovative two-gene knock-in approach. This approach enables the simultaneous integration of CAR and IL-15 genes into TRAC and IFN-γ gene loci using a single AAV vector. CAR T cells engineered to express IL-15 using this approach demonstrate enhanced antitumor efficacy. Overall, our study underscores the feasibility of utilizing endogenous promoters for transgenic cytokines expression in CAR T cells.

Use of IL-3RB to mediate antitumor effect of CAR-T cells through the JAK-STAT signaling pathway.

2541 Background: The application of CAR-T in solid tumors has not yet demonstrated significant clinical benefits. The continuous proliferation and decreased terminal differentiation of CAR-T cells in vivo are considered potential strategies that could alter this predicament. Cytokines, as the 3rd signal for T-cell activation, play a crucial role in the lifecycle and fate of T cells. In this study, we innovatively incorporated the IL-3 receptor β chain (IL-3RB) into the CAR structure to enhance signal transduction, potentially activating the anti-tumor function of CAR-T and maintaining the persistent proliferative activity. Methods: Transcriptional profiling analysis performed on HER2 or VEGFR1 CAR-T cells, with a specific focus on the gp140 cytokine family (IL-3/IL-5/CSF-2) and the receptor expression, validated by ELISA and Western blot. By cloning CAR genes containing different structural domains of the IL-3RB into the lentiviral vector pWPXLd, CAR expression, activation, depletion, proliferation, cytotoxicity, pSTAT3 and pSTAT5, and cytokine release of CAR-T cells were assessed using flow cytometry (FCM), Western blot (WB), RTCA, CCK-8, and ELISA. Subcutaneous/abdominal injections of SKOV3 cells or SKOV3-Luc cells into NSG mice, followed treated by CAR-T cells. Tumor burden was regularly monitored, and FCM and Q-PCR were utilized to assess the quantity and phenotype of CAR-T cells in vivo. Results: RNA-sequence, ELISA and WB revealed that upon activation of CAR-T cells, the expression of genes such as IL-3/IL-5/CSF-2 rapidly increased. However, their receptors were hardly expressed. IL-3RB modification did not affect the phenotype of resting CAR-T cells. But, co-cultured with target cells, IL-3RB significantly enhanced the anti-tumor effects of CAR-T cells. This was manifested by more rapid activation, promotion of persistent proliferation, delayed exhaustion phenotype, secretion of higher levels of cytokines (IL-2, IFN-γ, TNF-α) and efficient cytotoxicity. IL-3RB-expressing CAR-T cells exhibited activation of the JAK kinase and STAT3, STAT5 transcription factor signaling pathways in vitro. We established solid tumor models of subcutaneous (SKOV3) and intraperitoneal metastasis, where HER2/VEGFR1 CAR-T cells partially inhibited tumor cell growth in vivo. In contrast, IL-3RB-enhanced CAR-T cells completely eradicated tumor cells, and even achieving a state of complete remission. FCM and Q-PCR also confirmed that IL-3RB significantly improved the proliferation and survival of CAR-T cells in vivo. Conclusions: IL-3RB is scarcely expressed in T cells and not regulated by T cell activation. IL-3RB-modified CAR-T cells exhibit superior persistence and significant anti-tumor advantages in a solid tumor through the JAK-STAT signaling pathway. Our innovative CAR structure design has the potential to demonstrate promising anti-tumor effects in clinical translational applications.

CAR-T Therapy in Treating Solid Tumor: Application and Limitations

CAR-T therapy has emerged as a revolutionary approach in the battle against cancer. By leveraging the body's own immune system, this technique harnesses the power of T cells, which are genetically engineered to recognize specific antigens found on cancer cells. The procedure involves isolating lymphocytes from patients, followed by laboratory-based genetic modifications to infuse them with distinct CAR genes. Once these enhanced CAR-T cells are reintroduced into the patient, they actively target and neutralize cancer cells. However, the journey of CAR-T therapy is not without challenges. The selection of the right antigen is paramount, as an incorrect choice can lead to unintended consequences. Moreover, the application of this therapy in solid tumors presents its own set of complexities. This review aims to shed light on the clinical potential of CAR-T therapy, the obstacles faced in the realm of solid tumor treatment, and the ongoing research endeavors to refine and improve this promising therapeutic strategy.

Impact of Glycine-Serine Linker on Target Antigen Binding and Subsequent CD37CAR-T Performance

Introduction/Objectives: Gene-modified therapy expressing CD19-chimeric antigen receptor (CD19CAR) has shown promising outcomes in B-cell malignancies. However, some patients experience relapses due to poor CAR-T persistence and antigen escape. Various approaches have been studied to address these issues, including a novel signaling domain; CD28/CD40 to potentiate CAR-T effectiveness, linker modification to improve antigen-binding affinity, and new target identification such as CD37 in mature T-/B-cell malignancies. Consequently, we developed the third-generation CD37CAR incorporating the dual T- and B-cell costimulatory molecules; CD28/CD40 with two different single-chain variable fragment (scFv) linkers to improve CAR-T efficacy against CD37 + malignancies. Methods: The two different scFv linkers, GSTSGSGKPGSGEGSTKG (18aaL) and GGGGSGGGGSGGGGSGGGGS (GS4L), were fused into the CD37CAR; V L-linker-V H-H-CD28TM-CD28/CD40-CD3z-tEGFR backbone. CAR genes were subcloned into retroviral packaging cells to produce viral particles and transduced into primary CD3 + cells. The CAR +-Tcells were enriched using biotinylated anti-EGFR mAbs and further expanded with anti-CD3/CD28 microbeads. The cells were then used for subsequent experiments compared with untransduced T-cells. The in-silico studyinvestigated the structural behavior and binding characteristics of scFv at the atomistic level. Results: All CD37CAR genes were successfully transduced into primary CD3 + cells with significantly higher transduction efficiency in the glysine-serine linker (CD37.GS4L) CAR than CD37.18aaL. We initially evaluated the influence of linkers on CAR-T proliferation by stimulating once with gamma-irradiated Raji cells and found the robust CD37.GS4L CAR expansion regardless of IL-2. Short- and long-term co-culture assays against various CD37 + malignancies were then performed to examine tumoricidal activity. The overnight specific cytolysis demonstrated greater tumor eradication in CD37.GS4L CAR-T cells against Ramos (burkitt's lymphoma), Jeko-1 (mantle cell lymphoma; MCL), MM1S, KMS12BM (multiple myeloma), Hut78 (cutaneous T-lymphocyte), SNU449 (hepatocellular carcinoma), NCI-H522 (lung adenocarcinoma), primary-MCL and primary-chronic lymphocytic leukemia at various E:T ratios. In addition, we observed significant tumor suppression against Raji cells (burkitt's lymphoma) in CD37.GS4L CAR throughout the 12-day long-term cytotoxicity assay. Concerning the possibility of fratricide, CTV-labeled CD3 + were co-cultured with CARs and found that CD37.18aaL CAR revealed significant autologous T-cell killing compared to others. In terms of cytokine secretion, similar levels of IL-2, IFN-g, perforin, and granzyme B positive cells were noted after being stimulated with target cells. To replicate the effects of prolonged antigen exposure in humans, three consecutive weeklytarget cell stimulations revealed substantial expansion with significant preservation of central memory T-cells and less expressed exhaustion phenotypes in CD37.GS4L CAR compared to CD37.18aaL. Furthermore, the potential anti-tumor efficiency in vivo was studied using Raji-bearing NSG mice model. CD37.GS4L CAR-T effectively suppressed tumor cell growth and prolonged overall survival with persistent T CM positive cells in mice's spleen, liver, and bone marrow on day 60 compared to tEGFR-T and CD37.18aaL CAR-T (Figure 1). Additionally, the preliminary results of MM1S- and HUT78-inoculated mice models illustrated greater in vivo cytotoxicity in CD37.GS4L CAR-T than tEGFR-T. Regarding the in-silico study, molecular dynamic simulation and docking were evaluated. The scFv-specific CD37.GS4L CAR displayed more flexibility with higher binding affinity compared to CD37.18aaL CAR with ∆G = -11.1 kcal/mol, K d = 1.4 x 10 -8 molar, and ∆G = -9.7 kcal/mol, K d of 1.5 x 10 -7 molar, respectively. Conclusions: The considerable flexibility and affinity of CD37.GS4L CAR augment the CAR-T function in terms of proliferation, persistence, and tumoricidal activity. The glysine-serine linker could facilitate scFv activity and maximize CAR-T effectiveness.

HmbC, a Protein of the HMG Family, Participates in the Regulation of Carotenoid Biosynthesis in Fusarium fujikuroi.

In the fungus Fusarium fujikuroi, carotenoid production is up-regulated by light and down-regulated by the CarS RING finger protein, which modulates the mRNA levels of carotenoid pathway genes (car genes). To identify new potential regulators of car genes, we used a biotin-mediated pull-down procedure to detect proteins capable of binding to their promoters. We focused our attention on one of the proteins found in the screening, belonging to the High-Mobility Group (HMG) family that was named HmbC. The deletion of the hmbC gene resulted in increased carotenoid production due to higher mRNA levels of car biosynthetic genes. In addition, the deletion resulted in reduced carS mRNA levels, which could also explain the partial deregulation of the carotenoid pathway. The mutants exhibited other phenotypic traits, such as alterations in development under certain stress conditions, or reduced sensitivity to cell wall degrading enzymes, revealed by less efficient protoplast formation, indicating that HmbC is also involved in other cellular processes. In conclusion, we identified a protein of the HMG family that participates in the regulation of carotenoid biosynthesis. This is probably achieved through an epigenetic mechanism related to chromatin structure, as is frequent in this class of proteins.

Surficial nano-deposition locoregionally yielding bactericidal super CAR-macrophages expedites periprosthetic osseointegration.

Tracking and eradicating Staphylococcus aureus in the periprosthetic microenvironment are critical for preventing periprosthetic joint infection (PJI), yet effective strategies remain elusive. Here, we report an implant nanoparticle coating that locoregionally yields bactericidal super chimeric antigen receptor macrophages (CAR-MΦs) to prevent PJI. We demonstrate that the plasmid-laden nanoparticle from the coating can introduce S. aureus-targeted CAR genes and caspase-11 short hairpin RNA (CASP11 shRNA) into macrophage nuclei to generate super CAR-MΦs in mouse models. CASP11 shRNA allowed mitochondria to be recruited around phagosomes containing phagocytosed bacteria to deliver mitochondria-generated bactericidal reactive oxygen species. These super CAR-MΦs targeted and eradicated S. aureus and conferred robust bactericidal immunologic activity at the bone-implant interface. Furthermore, the coating biodegradability precisely matched the bone regeneration process, achieving satisfactory osteogenesis. Overall, our work establishes a locoregional treatment strategy for priming macrophage-specific bactericidal immunity with broad application in patients suffering from multidrug-resistant bacterial infection.

Association of CAR-T cell secreting CD47 blocking protein with an enhanced antitumor activity in small cell lung cancer.

e20512 Background: Notch ligand family DLL3 has been considered a therapeutic target for small cell lung cancer. CD47/SIRPa is an important immunosuppressive factor in the tumor microenvironment and is considered to be a "checkpoint" of innate immune cells. Here, we described an DLL3 targeting CAR-T cells secreting CD47/SIRPa signal blocking protein. We hypothesized that the armored CAR-T cells, by blocking the CD47/SIRPa signal with specific antibodies may promote macrophages to engulf tumor cells and induce anti-tumor immune response, thus addressing a clinical need of cell therapy in SCLC. Methods: Bioinformatics analysis of immune cell composition in the microenvironment of small cell lung cancer, focusing on macrophage content and phenotypic characteristics. The CAR genes were cloned into the lentiviral vector pWPXLd, the expression, phenotype, activation, proliferation, killing and cytokine release of CAR-T cells were detected by flow cytometry, Western blot, RTCA and ELISA; NSG mice were injected subcutaneously with H446-DLL3 cells or H446-DLL3(+/-) combined with CAR-T cells or armored CAR-T cells. Tumor size was measured every 3rd day. The number and phenotype of macrophages in tumors were detected by FCM. Results: Bioinformatics analysis showed that tumors with high expression of DLL3 were often accompanied by downregulation of antigen presentation-related genes; there is a large amount of macrophage infiltration in SCLC, and the later the stage, the more the infiltration. CAR-T cells co-cultured with target cells could effectively activate T cells, promote CAR-T cell proliferation, secrete IL-2, IFN-γ and TNF-α cytokines, and specifically kill DLL3 positive tumor cells. We have established a solid tumor model with heterogeneous antigen expression. CAR-T cells can effectively inhibit the growth of tumors with positive DLL3 expression in vivo, but the inhibition on tumors with heterogeneous antigens is not obvious. Blocking CD47 can promote phagocytosis of tumor cells by macrophages. Western blot analysis revealed the presence of CD47 blocking protein CV1-Fc in the supernatant of secreted CAR-T cells, which can effectively promote the phagocytic effect of macrophages; Armored CAR-T cells have a better anti-tumor effect in antigen heterogeneous tumor model in vivo, delays tumor progression, and promotes the aggregation of M1 type macrophages. Conclusions: CAR-T cells that specifically target DLL3 can effectively kill antigen-positive tumor cells in vivo and in vitro. Blocking CD47 can promote the non-specific anti-tumor effect of macrophages. CAR-T cells secreting CD47 blocking protein have dual anti-tumor effects, not only killing antigen-positive tumor cells, but also recruit and activate macrophages to exert phagocytosis, and have better anti-tumor effects in antigenic heterogeneous solid tumors.

Melatonin ameliorates trimethyltin chloride-induced cardiotoxicity: The role of nuclear xenobiotic metabolism and Keap1-Nrf2/ARE axis-mediated pyroptosis.

Trimethyltin chloride (TMT) is a stabilizer for polyvinyl chloride plastics that causes serious health hazards in nontarget organisms. Melatonin (MT) exhibits powerful protective effects in cardiac diseases. As a new environmental pollutant, TMT-induced cardiotoxicity and the protective effects of MT remain unclear. To explore this, the mice were treated with TMT (2.8mg/kg) and/or MT (10mg/kg) for 7 days. Firstly, the histopathological and ultrastructural evaluation showed that TMT induced cardiac damage, tumescent rupture and nuclear pyknosis. Moreover, TMT elevated the expressions of pyroptosis genes NLRP3, ASC and Cas1 and inflammation factors IL-6, IL-17 and TNFα. Secondly, TMT reduced antioxidant enzymes (GSH, CAT and T-AOC) via decreasing the expression of genes associated with the Keap1-Nrf2/ARE pathway to increase oxidative stress. Thirdly, TMT decreased the expression of genes associated with the ARE-driven drug metabolizing enzymes (DMEs), including Akr7a3, Akr1b8, and Akr1b10. Besides, TMT upregulated the mRNA expression of nuclear Xenobiotic metabolism on cytochrome P450s enzymes via increasing the expression of CAR, PXP, and AHR genes. Furthermore, MT treatment mitigated the aforementioned adverse changes induced by TMT. Overall, these results demonstrated that TMT caused pyroptosis and inflammation to aggravate cardiac damage via inducing excessive oxidative stress, imbalance of DMEs homeostasis, and nuclear Xenobiotic metabolism disorder, which could be alleviated by MT.

Microfluidic and Nanofluidic Intracellular Delivery.

Innate cell function can be artificially engineered and reprogrammed by introducing biomolecules, such as DNAs, RNAs, plasmid DNAs, proteins, or nanomaterials, into the cytosol or nucleus. This process of delivering exogenous cargos into living cells is referred to as intracellular delivery. For instance, clustered regularly interspaced short palindromic repeats (CRISPR)‐Cas9 gene editing begins with internalizing Cas9 protein and guide RNA into cells, and chimeric antigen receptor‐T (CAR‐T) cells are prepared by delivering CAR genes into T lymphocytes for cancer immunotherapies. To deliver external biomolecules into cells, tools, including viral vectors, and electroporation have been traditionally used; however, they are suboptimal for achieving high levels of intracellular delivery while preserving cell viability, phenotype, and function. Notably, as emerging solutions, microfluidic and nanofluidic approaches have shown remarkable potential for addressing this open challenge. This review provides an overview of recent advances in microfluidic and nanofluidic intracellular delivery strategies and discusses new opportunities and challenges for clinical applications. Furthermore, key considerations for future efforts to develop microfluidics‐ and nanofluidics‐enabled next‐generation intracellular delivery platforms are outlined.

Human CAR NK Cells: A New Non-viral Method Allowing High Efficient Transfection and Strong Tumor Cell Killing.

CAR-NK cells may represent a valuable tool, complementary to CAR-T cells, in adoptive immunotherapy of leukemia and solid tumors. However, gene transfer to human NK cells is a challenging task, particularly with non-virus-based techniques. Here, we describe a new procedure allowing efficient electroporation-based transfection of plasmid DNA, including CAR and CCR7 genes, in resting or cytokine-expanded human NK cell populations and NK-92 cell line. This procedure may offer a suitable platform for a safe and effective use of CAR-NK cells in adoptive immunotherapy of cancer.

Triclocarban Disrupts the Epigenetic Status of Neuronal Cells and Induces AHR/CAR-Mediated Apoptosis

Triclocarban is a phenyl ether that has recently been classified as a contaminant of emerging concern. Evidence shows that triclocarban is present in human tissues, but little is known about the impact of triclocarban on the nervous system, particularly at early developmental stages. This study demonstrated that triclocarban that was used at environmentally relevant concentrations induced apoptosis in mouse embryonic neurons, inhibited sumoylation, and changed the epigenetic status, as evidenced by impaired activities of HDAC, sirtuins, and DNMT, global DNA hypomethylation, and alterations of methylation levels of bax, bcl2, Ahr, and Car genes. The use of selective antagonists and specific siRNAs, which was followed by the co-localization of aryl hydrocarbon receptor (AHR) and constitutive androstane receptor (CAR) in mouse neurons, points to the involvement of AHR and CAR in triclocarban-induced neurotoxicity. A 24-h treatment with triclocarban enhanced protein levels of the receptors which was paralleled by Car hypomethylation and Ahr hypermethylation. Car hypomethylation is in line with global DNA hypomethylation and explains the increased mRNA and protein levels of CAR in response to triclocarban. Ahr hypermethylation could reflect reduced Ahr mRNA expression and corresponds to lowered protein levels after 3- and 6-h exposures to triclocarban that is likely related to proteasomal degradation of activated AHR. We hypothesize that the triclocarban-induced apoptosis in mouse neurons and the disruption of epigenetic status involve both AHR- and CAR-mediated effects, which may substantiate a fetal basis of the adult onset of neurological diseases; however, the expression of the receptors is regulated in different ways.

CAR gene cluster and transcript levels of carotenogenic genes in Rhodotorula mucilaginosa.

A molecular approach was applied to the study of the carotenoid biosynthetic pathway of Rhodotorula mucilaginosa. At first, functional annotation of the genome of R. mucilaginosa C2.5t1 was carried out and gene ontology categories were assigned to 4033 predicted proteins. Then, a set of genes involved in different steps of carotenogenesis was identified and those coding for phytoene desaturase, phytoene synthase/lycopene cyclase and carotenoid dioxygenase (CAR genes) proved to be clustered within a region of ~10 kb. Quantitative PCR of the genes involved in carotenoid biosynthesis showed that genes coding for 3-hydroxy-3-methylglutharyl-CoA reductase and mevalonate kinase are induced during exponential phase while no clear trend of induction was observed for phytoene synthase/lycopene cyclase and phytoene dehydrogenase encoding genes. Thus, in R. mucilaginosa the induction of genes involved in the early steps of carotenoid biosynthesis is transient and accompanies the onset of carotenoid production, while that of CAR genes does not correlate with the amount of carotenoids produced. The transcript levels of genes coding for carotenoid dioxygenase, superoxide dismutase and catalase A increased during the accumulation of carotenoids, thus suggesting the activation of a mechanism aimed at the protection of cell structures from oxidative stress during carotenoid biosynthesis. The data presented herein, besides being suitable for the elucidation of the mechanisms that underlie carotenoid biosynthesis, will contribute to boosting the biotechnological potential of this yeast by improving the outcome of further research efforts aimed at also exploring other features of interest.

Immunoengineering: Valet parking for CAR genes

T-cell-binding nanoparticles bypass the complexities of in vitro gene transfer to deliver genes to T cells within the host.

Abstract PR09: A novel chimeric antigen receptor containing JAK-STAT signaling domains mediates superior antitumor effects

Abstract Adoptive transfer of anti-CD19 CAR-engineered T cells have shown impressive and often durable clinical responses in patients with refractory and relapsed pre-B-cell acute lymphoblastic leukemia (ALL). However, clinical efficacy of anti-CD19 CAR-T cell therapy for lymphoma and chronic lymphocytic leukemia as well as CAR-T cell therapies that target solid tumors is not yet satisfactory. Optimal T cell activation and proliferation requires multiple signals including T cell receptor engagement (signal 1), costimulation (signal 2), and cytokine engagement (signal 3). To the best of our knowledge, CAR genes currently tested in the clinic contain a CD3z domain (signal 1) and costimulatory domain(s) (signal 2), but not a domain that can transmit signal 3. Although forced expression of cytokine genes in CAR-T cells can improve their persistence and antitumor effects, constitutive cytokine expression poses a risk for autonomous T cell growth causing cancers. In this study, we have developed a novel CAR design, 28-IL2RB-z (YXXQ), which can activate the JAK-STAT pathway in an antigen-dependent manner, recapitulating cytokine exposure upon TCR engagement. We incorporated a minimal cytoplasmic domain of the IL-2 receptor β (IL2RB) between CD28 and CD3z, which contains a box1 motif for the binding of JAK family protein tyrosine kinases and the tyrosine residue at position 510, an essential site for STAT5 association. To promote STAT3 recruitment, we introduced a YXXQ motif to the C-terminus end of CD3z (28-IL2RB-z (YXXQ)). T cells transduced with the novel CD19 CAR gene induced phosphorylation of both STAT3 and STAT5 proteins only upon CD19 encounter, and exhibited increased expression of JAK-STAT target genes. Moreover, upon stimulation with CD19+ target cells, these T cells, compared to 2nd and 3rd generation CAR-T cells, demonstrated superior proliferative capacity with increased cellular division and reduced activation-induced cell death. Interestingly, CD8+ T cells engineered with this novel CAR maintained a stem cell-like memory (TSCM) phenotype and polyfunctional cytokine secretion capacity even after repeated antigenic stimulation. Treatment of 28-IL2RB-z (YXXQ) CAR-T cells with a STAT3 inhibitor largely abrogated their proliferative advantage and decreased the frequency of TSCM cells, suggesting that STAT3 signaling played a predominant role in the maintenance of the TSCM phenotype. All the 2nd generation, 3rd generation, and our new generation CAR-T cells similarly demonstrated potent in vitro cytotoxicity and IFN-γ secretion upon initial exposure to CD19+ cells. However, our novel CAR-T cells were superior to the other CAR-T cells in both cytolytic activity and cytokine secretion following multiple antigen exposures. NSG mice were intravenously injected with the human CD19+ leukemia cell line, NALM6, and treated with adoptive transfer of each of the 2nd, 3rd, or new generation CAR-transduced T cells. The 28-IL2RB-z (YXXQ) new generation CAR-T cells demonstrated superior persistence compared to other generation CAR-T cells, which resulted in significantly better overall survival. Long-term persisting 28-IL2RB-z (YXXQ) CAR-T cells from treated mice secreted more IFN-γ when restimulated ex vivo, suggesting the maintenance of a functional and less-exhausted phenotype in vivo. Furthermore, the 28-IL2RB-z (YXXQ) CAR-T cells also demonstrated better in vivo persistence and therapeutic effects than the 28-z and 28-BB-z CAR-T cells in NSG mice inoculated with primary human CD19+ pre-B-ALL cells. Importantly, the 28-IL2RB-z (YXXQ) CD19 CAR-T cells did not exhibit any proliferative responses when adoptively transferred into leukemia-free mice, affirming that JAK-STAT signals are activated only when exposed to the antigen. Taken together, these results suggest that our new generation CAR has the potential to demonstrate superior antitumor effects with minimal toxicities in the clinic. Clinical translation of this novel CAR is warranted. This abstract is also being presented as Poster A73. Citation Format: Yuki Kagoya, Shinya Tanaka, Marcus O. Butler, Mark Minden, Naoto Hirano. A novel chimeric antigen receptor containing JAK-STAT signaling domains mediates superior antitumor effects. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr PR09.

Bioinformatic analysis of miRNAs targeting the key nuclear receptors regulating CYP3A4 gene expression: The challenge of the CYP3A4 “missing heritability” enigma

The expression of the main cytochrome P450 enzyme CYP3A4 displays enormous interindividual variability. Studies addressing the genetic variability of either the CYP3A4 gene itself or its key transcription factors have not found any crucial polymorphism contributing to this variability in expression, a phenomenon is referred to as the “missing heritability of CYP3A4 variability.” Several reports have recently described microRNAs (miRNAs) targeting the CYP3A4 gene and/or its major transcription factors.A comprehensive bioinformatic analysis was performed using the miRDB, PITA, miRanda and TargetScan programs in the search for hypothetical miRNAs targeting 3′-untranslated regions of PXR, CAR, VDR, HNF4α, RXRα, SHP and GRα genes controlling CYP3A4 expression.We propose several novel miRNAs identified parallelly by at least three algorithms to the target analyzed genes. In particular, we found novel promising miRNAs which may be involved in the indirect PXR-mediated CYP3A4 gene expression such as miR-18a and miR-18b, miR-449a, miR-449b and miR-34a. We also hypothesize that some miRNAs may play the role of a master regulator of NRs since they are predicted to target more than three genes.The identification of miRNAs determining CYP3A4 interindividual variability might be an important step toward progress in pharmacogenetics and personalized medicine.

The flavoproteins CryD and VvdA cooperate with the white collar protein WcoA in the control of photocarotenogenesis in Fusarium fujikuroi.

Light stimulates carotenoid biosynthesis in the ascomycete fungus Fusarium fujikuroi through transcriptional activation of the structural genes of the pathway carRA, carB, and cart, but the molecular basis of this photoresponse is unknown. The F. fujikuroi genome contains genes for different predicted photoreceptors, including the WC protein WcoA, the DASH cryptochrome CryD and the Vivid-like flavoprotein VvdA. We formerly found that null mutants of wcoA, cryD or vvdA exhibit carotenoid photoinduction under continuous illumination. Here we show that the wild type exhibits a biphasic response in light induction kinetics experiments, with a rapid increase in carotenoid content in the first hours, a transient arrest and a subsequent slower increase. The mutants of the three photoreceptors show different kinetic responses: the wcoA mutants are defective in the rapid response, the cryD mutants are affected in the slower response, while the fast and slow responses were respectively enhanced and attenuated in the vvdA mutants. Transcriptional analyses of the car genes revealed a strong reduction of dark and light-induced transcript levels in the wcoA mutants, while minor or no reductions were found in the cryD mutants. Formerly, we found no change on carRA and carB photoinduction in vvdA mutants. Taken together, our data suggest a cooperative participation of WcoA and CryD in early and late stages of photoinduction of carotenoid biosynthesis in F. fujikuroi, and a possible modulation of WcoA activity by VvdA. An unexpected transcriptional induction by red light of vvdA, cryD and carRA genes suggest the participation of an additional red light-absorbing photoreceptor.

Impact of Drug Transporters ABCB1 and ABCG2 and Regulators of Xenobiotic Transport and Metabolism Pxr and CAR Gene Polymorphisms on Clinical Efficacy of Imatinib in Chronic Myeloid Leukemia (CML)

Background: Inherited background in mechanisms regulating cellular uptake, elimination and metabolism of imatinib and other BCR-ABL tyrosine kinase inhibitors may influence treatment outcome in chronic myeloid leukemia (CML). Despite good efficacy of imatinib in CML therapy, a proportion of patients show suboptimal responses or even primary resistance. Imatinib is a well-established substrate of ABC-superfamily xenobiotic transporters ABCB1 (MDR1, Pg-P) and ABCG2 (BCRP). However, the results of published studies regarding impact of ABCB1 and ABCG2 single nucleotide polymorphisms (SNPs) on imatinib treatment outcome have shown discrepant results. Moreover, little is known on the potential role of polymorphisms of regulators of xenobiotic transport and metabolism, pregnane X receptor (PXR, NR1|2) and constitutive androstane receptor (CAR, NR1|3) in imatinib efficacy.Aim: In this study we verified whether 61 tagging SNPs in drug transporters genes (ABCB1 and ABCG2) and regulators of xenobiotic transport and metabolism gene (PXR and CAR) genes affect imatinib therapy outcomes in CML.Methods: Genotyping was performed in 256 patients of Polish Caucasian origin (141 men and 117 women) treated with imatinib as first line treatment (229; 89.5%) or after second-line after interferon therapy (27; 10.5%). Tagging SNP approach was used to select the genetic variants and resulted in a selection of 61 tagging SNPs including 26 SNPs for ABCB1, 17 SNPs for ABCG2, 11 SNPs for PXR and 7 SNPs for CAR gene. Complete clinical data on imatinib treatment outcome were assessable for analyses of associations with tested SNPs. The influence of SNPs on imatinib outcomes including achievement of complete cytogenetic response (CCyR) at 12 months, achievement of major molecular response (MMR) at 18 months, progression-free survival (PFS), time to treatment failure (TTF) and overall survival (OS) were tested in regard to established clinical and laboratory predictive factors using logistic regression and proportional-hazards regression models. Three inheritance models (co-dominant, dominant and recessive) were tested for all allelic variants.Results: In this study we found that carriers of minor allele of CAR rs4073054A>C SNP significantly associated with shorter TTF on imatinib therapy (OR=2.42, 95%CI 1.41 - 4.17; p=0.0014; Bonferroni corrected p=0.048). In contrast, no impact of other tested allelic variants in ABCB1, ABCG2, PXR and CAR genes on proportion of patients who achieved CCyR at 12 months, MMR at 18 months as well as on probability of PFS, TTF and OS was observed.Conclusion: Our data suggest that inherited variants in the regulator of xenobiotic transport and metabolism CAR gene may modify results of imatinib therapy in CML patients. In contrast, the results of this relatively large study indicate that polymorphic variants in ABCB1, ABCG2 and PXR genes do not seem to play major role as determinants of response to imatinib in CML. DisclosuresRobak:MorphoSys AG: Research Funding.

Light-mediated participation of the VIVID-like protein of Fusarium fujikuroi VvdA in pigmentation and development

The phytopathogen Fusarium fujikuroi is a model fungus for the production of different secondary metabolites. These include the acidic xanthophyll neurosporaxanthin, produced through the activity of the enzymes encoded by the car genes, of which carRA and carB play a major role. Expression of these genes is induced by light but, in contrast to other fungi, the induction is not impaired under continuous illumination in null mutants of the F. fujikuroi wc-1-like gene, wcoA. Therefore, we investigate the role of other blue-light photoreceptors. Here we describe the identification, regulation and targeted mutation of the F. fujikuroi gene vvdA, homologous of the Neurospora crassa vivid (vvd) gene. As found for vvd in N. crassa, expression of vvdA in F. fujikuroi is strongly stimulated by light, an activation that is severely reduced in the wcoA mutants. Deletion of vvdA in F. fujikuroi results in a paler pigmentation under constant light, explained by a reduced carotenoid production, a regulatory effect opposite to the enhanced carotenoid accumulation characteristic of the vvd mutants of N. crassa. No major changes were appreciated in the transcriptional regulation of the car genes following exposure to light, but a noticeable reduction was found upon long-term incubation under constant illumination. Additionally, the vvdA mutants produce less conidia and their colonies exhibit morphological alterations under constant light, such as a more compact development of aerial mycelia and a more solid attachment to the agar surface. The results indicate that VvdA participates in the regulation by light of mycelial development and affects the accumulation of carotenoids but it is not responsible of the transcriptional photoadaptation of the car genes.