31 results on '"Labanieh L"'
Search Results
2. Engineered CD47 protects T cells for enhanced antitumour immunity.
- Author
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Yamada-Hunter SA, Theruvath J, McIntosh BJ, Freitas KA, Lin F, Radosevich MT, Leruste A, Dhingra S, Martinez-Velez N, Xu P, Huang J, Delaidelli A, Desai MH, Good Z, Polak R, May A, Labanieh L, Bjelajac J, Murty T, Ehlinger Z, Mount CW, Chen Y, Heitzeneder S, Marjon KD, Banuelos A, Khan O, Wasserman SL, Spiegel JY, Fernandez-Pol S, Kuo CJ, Sorensen PH, Monje M, Majzner RG, Weissman IL, Sahaf B, Sotillo E, Cochran JR, and Mackall CL
- Subjects
- Animals, Female, Humans, Male, Mice, Antigens, Differentiation immunology, Antigens, Differentiation metabolism, Cell Line, Tumor, Macrophages cytology, Macrophages immunology, Receptors, Antigen, T-Cell genetics, Receptors, Antigen, T-Cell immunology, Receptors, Antigen, T-Cell metabolism, Receptors, Chimeric Antigen genetics, Receptors, Chimeric Antigen immunology, Receptors, Chimeric Antigen metabolism, Receptors, Immunologic immunology, Receptors, Immunologic metabolism, Tumor Microenvironment immunology, Antibodies immunology, Antibodies therapeutic use, Macrophage Activation, CD47 Antigen genetics, CD47 Antigen immunology, CD47 Antigen metabolism, Immunotherapy, Adoptive methods, Neoplasms immunology, Neoplasms metabolism, Neoplasms therapy, T-Lymphocytes immunology, T-Lymphocytes metabolism, T-Lymphocytes transplantation
- Abstract
Adoptively transferred T cells and agents designed to block the CD47-SIRPα axis are promising cancer therapeutics that activate distinct arms of the immune system
1,2 . Here we administered anti-CD47 antibodies in combination with adoptively transferred T cells with the goal of enhancing antitumour efficacy but observed abrogated therapeutic benefit due to rapid macrophage-mediated clearance of T cells expressing chimeric antigen receptors (CARs) or engineered T cell receptors. Anti-CD47-antibody-mediated CAR T cell clearance was potent and rapid enough to serve as an effective safety switch. To overcome this challenge, we engineered the CD47 variant CD47(Q31P) (47E ), which engages SIRPα and provides a 'don't eat me' signal that is not blocked by anti-CD47 antibodies. TCR or CAR T cells expressing 47E are resistant to clearance by macrophages after treatment with anti-CD47 antibodies, and mediate substantial, sustained macrophage recruitment to the tumour microenvironment. Although many of the recruited macrophages manifested an M2-like profile3 , the combined therapy synergistically enhanced antitumour efficacy. Our study identifies macrophages as major regulators of T cell persistence and illustrates the fundamental challenge of combining T-cell-directed therapeutics with those designed to activate macrophages. It delivers a therapeutic approach that is capable of simultaneously harnessing the antitumour effects of T cells and macrophages, offering enhanced potency against solid tumours., (© 2024. The Author(s).)- Published
- 2024
- Full Text
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3. Directed evolution of genetically encoded LYTACs for cell-mediated delivery.
- Author
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Yang JL, Yamada-Hunter SA, Labanieh L, Sotillo E, Cheah JS, Roberts DS, Mackall CL, Bertozzi CR, and Ting AY
- Subjects
- Humans, HEK293 Cells, Proteolysis, Lysosomes
- Abstract
Lysosome-targeting chimeras (LYTACs) are a promising therapeutic modality to drive the degradation of extracellular proteins. However, early versions of LYTAC contain synthetic glycopeptides that cannot be genetically encoded. Here, we present our designs for a fully genetically encodable LYTAC (GELYTAC), making our tool compatible with integration into therapeutic cells for targeted delivery at diseased sites. To achieve this, we replaced the glycopeptide portion of LYTACs with the protein insulin-like growth factor 2 (IGF2). After showing initial efficacy with wild-type IGF2, we increased the potency of GELYTAC using directed evolution. Subsequently, we demonstrated that our engineered GELYTAC construct not only secretes from HEK293T cells but also from human primary T-cells to drive the uptake of various targets into receiver cells. Immune cells engineered to secrete GELYTAC thus represent a promising avenue for spatially selective targeted protein degradation., Competing Interests: Competing interests statement:C.R.B. is a co-founder and scientific advisory board member of Lycia Therapeutics, Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent), OliLux Bio, InterVenn Bio, GanNA Bio, Firefly Bio, Neuravid, and Valora Therapeutics. S.A.Y.-H. is a consultant for Quince Therapeutics. L.L. is a cofounder of, consults for, and holds equity in CARGO Therapeutics. E.S. is a consultant for Lepton Pharmaceuticals and Galaria, and holds equity in Lyell Immunopharma. C.L.M. is a cofounder of Lyell Immunopharma, CARGO Therapeutics, and Link Cell Therapies, which are developing CAR-based therapies, and consults for CARGO, Link Immatics, Ensoma and Red Tree Capital. A.Y.T. is a scientific advisor to Third Rock Ventures and Nereid Therapeutics. The remaining authors declare no competing interest. C.R.B. owns founders shares of Lycia Therapeutics, which is developing medicines based on targeted extracellular protein degradation.
- Published
- 2024
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4. Inosine induces stemness features in CAR-T cells and enhances potency.
- Author
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Klysz DD, Fowler C, Malipatlolla M, Stuani L, Freitas KA, Chen Y, Meier S, Daniel B, Sandor K, Xu P, Huang J, Labanieh L, Keerthi V, Leruste A, Bashti M, Mata-Alcazar J, Gkitsas N, Guerrero JA, Fisher C, Patel S, Asano K, Patel S, Davis KL, Satpathy AT, Feldman SA, Sotillo E, and Mackall CL
- Subjects
- Humans, T-Lymphocytes metabolism, Inosine
- Abstract
Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8
+ CAR-T cells express CD39 and CD73, which mediate proximal steps in Ado generation. Here, we sought to enhance CAR-T cell potency by knocking out CD39, CD73, or adenosine receptor 2a (A2aR) but observed only modest effects. In contrast, overexpression of Ado deaminase (ADA-OE), which metabolizes Ado to inosine (INO), induced stemness and enhanced CAR-T functionality. Similarly, CAR-T cell exposure to INO augmented function and induced features of stemness. INO induced profound metabolic reprogramming, diminishing glycolysis, increasing mitochondrial and glycolytic capacity, glutaminolysis and polyamine synthesis, and reprogrammed the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR-T cell products meeting criteria for clinical dosing. These results identify INO as a potent modulator of CAR-T cell metabolism and epigenetic stemness programming and deliver an enhanced potency platform for cell manufacturing., Competing Interests: Declaration of interests D.D.K, S.A.F., and C.L.M. are co-inventors on a pending patent application for inosine media supplementation during cell manufacturing. D.D.K and C.L.M. are inventors on a patent application for the use of T cells overexpressing ADA1/2 for cancer immunotherapy. C.L.M. holds equity in and receives research funding from Lyell Immunopharma, holds equity in and consults for Link Cell Therapies and C.L.M., and L.L. hold equity and consult for CARGO Therapeutics. L.L. and E.S. hold equity in Lyell Immunopharma. E.S consults for Lepton Pharmaceuticals and Galaria. S.A.F. serves on the Scientific Advisory Boards for Alaunos Therapeutics and Fresh Wind Biotech and has equity interest in both; S.A.F. receives research funding from CARGO and Tune Therapeutics. S.P. is a current employee of and holds equity in CARGO. C.L.M. consults for Immatics, Mammoth, and Ensoma. A.T.S. is a cofounder of Immunai and Cartography Biosciences. A.T.S. receives research funding from Allogene Therapeutics and Merck Research Laboratories., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2024
- Full Text
- View/download PDF
5. Directed Evolution of Genetically Encoded LYTACs for Cell-Mediated Delivery.
- Author
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Yang JL, Yamada-Hunter SA, Labanieh L, Sotillo E, Cheah JS, Roberts DS, Mackall CL, Ting AY, and Bertozzi CR
- Abstract
Lysosome-targeting chimeras (LYTACs) are a promising therapeutic modality to drive the degradation of extracellular proteins. However, early versions of LYTAC contain synthetic glycopeptides that cannot be genetically encoded. Here we present our designs for a fully genetically encodable LYTAC (GELYTAC), making our tool compatible with integration into therapeutic cells for targeted delivery at diseased sites. To achieve this, we replaced the glycopeptide portion of LYTACs with the protein insulin like growth factor 2 (IGF2). After showing initial efficacy with wild type IGF2, we increased the potency of GELYTAC using directed evolution. Subsequently, we demonstrated that our engineered GELYTAC construct not only secretes from HEK293T cells but also from human primary T-cells to drive the uptake of various targets into receiver cells. Immune cells engineered to secrete GELYTAC thus represent a promising avenue for spatially-selective targeted protein degradation., Competing Interests: Competing Interest Statement: C.R.B. is a co-founder and scientific advisory board member of Lycia Therapeutics, Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent), OliLux Bio, InterVenn Bio, GanNA Bio, Firefly Bio, Neuravid and Valora Therapeutics. S.A.Y.-H. is a consultant for Quince Therapeutics. L.L. is a cofounder of, consults for, and holds equity in CARGO Therapeutics. E.S. is a consultant for Lepton Pharmaceuticals and Galaria, and holds equity in Lyell Immunopharma. C.L.M. is a cofounder of Lyell Immunopharma, CARGO Therapeutics, and Link Cell Therapies, which are developing CAR-based therapies, and consults for CARGO, Link Immatics, Ensoma and Red Tree Capital. A.Y.T. is a scientific advisor to Third Rock Ventures and Nereid Therapeutics. The remaining authors declare no competing interests.
- Published
- 2023
- Full Text
- View/download PDF
6. Author Correction: CAR immune cells: design principles, resistance and the next generation.
- Author
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Labanieh L and Mackall CL
- Published
- 2023
- Full Text
- View/download PDF
7. Inosine Induces Stemness Features in CAR T cells and Enhances Potency.
- Author
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Klysz DD, Fowler C, Malipatlolla M, Stuani L, Freitas KA, Meier S, Daniel B, Sandor K, Xu P, Huang J, Labanieh L, Leruste A, Bashti M, Keerthi V, Mata-Alcazar J, Gkitsas N, Guerrero JA, Fisher C, Patel S, Asano K, Patel S, Davis KL, Satpathy AT, Feldman SA, Sotillo E, and Mackall CL
- Abstract
Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8
+ CAR T cells mediate Ado-induced immunosuppression through CD39/73-dependent Ado production. Knockout of CD39, CD73 or A2aR had modest effects on exhausted CAR T cells, whereas overexpression of Ado deaminase (ADA), which metabolizes Ado to inosine (INO), induced stemness features and potently enhanced functionality. Similarly, and to a greater extent, exposure of CAR T cells to INO augmented CAR T cell function and induced hallmark features of T cell stemness. INO induced a profound metabolic reprogramming, diminishing glycolysis and increasing oxidative phosphorylation, glutaminolysis and polyamine synthesis, and modulated the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR T cell products meeting criteria for clinical dosing. These data identify INO as a potent modulator of T cell metabolism and epigenetic stemness programming and deliver a new enhanced potency platform for immune cell manufacturing., Competing Interests: Conflict of interests D.D.K, S.A.F., and C.L.M. are co-inventors on a pending patent application number 63/358,996 for inosine media supplementation during cell manufacturing. D.D.K and C.L.M. are inventors on a patent application number PCT/US2022/075584 that covers the use of T cells overexpressing ADA1/2 for cancer immunotherapy. C.L.M. is a cofounder of and holds equity in Lyell Immunopharma and Link Cell Therapies. C.L.M., and L.L. are co-founders of and hold equity in CARGO Therapeutics. L.L. consults for and holds equity in Lyell Immunopharma. E.S holds equity in Lyell Immunopharma and consults for Lepton Pharmaceuticals and Galaria. S.A.F. serves on the Scientific Advisory Boards for Alaunos Therapeutics and Fresh Wind Biotech and has equity interest in both; S.A.F. receives research funding from CARGO and Tune Therapeutics. S.P. is a current employee of and holds equity in CARGO. C.L.M. consults for Lyell, CARGO, Link, Apricity, Nektar, Immatics, Mammoth, and Ensoma. A.T.S. is a cofounder of Immunai and Cartography Biosciences. A.T.S. receives research funding from Allogene Therapeutics and Merck Research Laboratories.- Published
- 2023
- Full Text
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8. Co-opting signalling molecules enables logic-gated control of CAR T cells.
- Author
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Tousley AM, Rotiroti MC, Labanieh L, Rysavy LW, Kim WJ, Lareau C, Sotillo E, Weber EW, Rietberg SP, Dalton GN, Yin Y, Klysz D, Xu P, de la Serna EL, Dunn AR, Satpathy AT, Mackall CL, and Majzner RG
- Subjects
- Humans, Leukemia, B-Cell, Lymphoma, B-Cell, Cell Engineering methods, Immunotherapy, Adoptive adverse effects, Logic, Neoplasms immunology, Neoplasms metabolism, Neoplasms therapy, Receptors, Antigen, T-Cell immunology, Receptors, Antigen, T-Cell metabolism, Receptors, Chimeric Antigen immunology, Receptors, Chimeric Antigen metabolism, Signal Transduction, T-Lymphocytes immunology, T-Lymphocytes metabolism
- Abstract
Although chimeric antigen receptor (CAR) T cells have altered the treatment landscape for B cell malignancies, the risk of on-target, off-tumour toxicity has hampered their development for solid tumours because most target antigens are shared with normal cells
1,2 . Researchers have attempted to apply Boolean-logic gating to CAR T cells to prevent toxicity3-5 ; however, a truly safe and effective logic-gated CAR has remained elusive6 . Here we describe an approach to CAR engineering in which we replace traditional CD3ζ domains with intracellular proximal T cell signalling molecules. We show that certain proximal signalling CARs, such as a ZAP-70 CAR, can activate T cells and eradicate tumours in vivo while bypassing upstream signalling proteins, including CD3ζ. The primary role of ZAP-70 is to phosphorylate LAT and SLP-76, which form a scaffold for signal propagation. We exploited the cooperative role of LAT and SLP-76 to engineer logic-gated intracellular network (LINK) CAR, a rapid and reversible Boolean-logic AND-gated CAR T cell platform that outperforms other systems in both efficacy and prevention of on-target, off-tumour toxicity. LINK CAR will expand the range of molecules that can be targeted with CAR T cells, and will enable these powerful therapeutic agents to be used for solid tumours and diverse diseases such as autoimmunity7 and fibrosis8 . In addition, this work shows that the internal signalling machinery of cells can be repurposed into surface receptors, which could open new avenues for cellular engineering., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)- Published
- 2023
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9. CAR immune cells: design principles, resistance and the next generation.
- Author
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Labanieh L and Mackall CL
- Subjects
- Humans, Cell- and Tissue-Based Therapy methods, Cell- and Tissue-Based Therapy trends, Genetic Therapy methods, Genetic Therapy trends, Neoplasms immunology, Neoplasms pathology, Neoplasms therapy, Tumor Microenvironment, B-Lymphocytes pathology, Immunotherapy, Adoptive adverse effects, Immunotherapy, Adoptive methods, Immunotherapy, Adoptive standards, Immunotherapy, Adoptive trends, T-Lymphocytes immunology, Receptors, Chimeric Antigen
- Abstract
The remarkable clinical activity of chimeric antigen receptor (CAR) therapies in B cell and plasma cell malignancies has validated the use of this therapeutic class for liquid cancers, but resistance and limited access remain as barriers to broader application. Here we review the immunobiology and design principles of current prototype CARs and present emerging platforms that are anticipated to drive future clinical advances. The field is witnessing a rapid expansion of next-generation CAR immune cell technologies designed to enhance efficacy, safety and access. Substantial progress has been made in augmenting immune cell fitness, activating endogenous immunity, arming cells to resist suppression via the tumour microenvironment and developing approaches to modulate antigen density thresholds. Increasingly sophisticated multispecific, logic-gated and regulatable CARs display the potential to overcome resistance and increase safety. Early signs of progress with stealth, virus-free and in vivo gene delivery platforms provide potential paths for reduced costs and increased access of cell therapies in the future. The continuing clinical success of CAR T cells in liquid cancers is driving the development of increasingly sophisticated immune cell therapies that are poised to translate to treatments for solid cancers and non-malignant diseases in the coming years., (© 2023. Springer Nature Limited.)
- Published
- 2023
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10. Enhanced safety and efficacy of protease-regulated CAR-T cell receptors.
- Author
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Labanieh L, Majzner RG, Klysz D, Sotillo E, Fisher CJ, Vilches-Moure JG, Pacheco KZB, Malipatlolla M, Xu P, Hui JH, Murty T, Theruvath J, Mehta N, Yamada-Hunter SA, Weber EW, Heitzeneder S, Parker KR, Satpathy AT, Chang HY, Lin MZ, Cochran JR, and Mackall CL
- Subjects
- Humans, Immunotherapy, Adoptive methods, Peptide Hydrolases, Receptors, Antigen, T-Cell, T-Lymphocytes pathology, Neoplasms drug therapy, Neoplasms pathology, Receptors, Chimeric Antigen
- Abstract
Regulatable CAR platforms could circumvent toxicities associated with CAR-T therapy, but existing systems have shortcomings including leakiness and attenuated activity. Here, we present SNIP CARs, a protease-based platform for regulating CAR activity using an FDA-approved small molecule. Design iterations yielded CAR-T cells that manifest full functional capacity with drug and no leaky activity in the absence of drug. In numerous models, SNIP CAR-T cells were more potent than constitutive CAR-T cells and showed diminished T cell exhaustion and greater stemness. In a ROR1-based CAR lethality model, drug cessation following toxicity onset reversed toxicity, thereby credentialing the platform as a safety switch. In the same model, reduced drug dosing opened a therapeutic window that resulted in tumor eradication in the absence of toxicity. SNIP CARs enable remote tuning of CAR activity, which provides solutions to safety and efficacy barriers that are currently limiting progress in using CAR-T cells to treat solid tumors., Competing Interests: Declaration of interests L.L., R.G.M., M.Z.L., and C.L.M. are coinventors on a patent related to this work. C.L.M. is a cofounder of Lyell Immunopharma, Syncopation Life Sciences, and Link Cell Therapies, which are developing CAR-based therapies, and consults for Lyell, NeoImmune Tech, Apricity, Nektar, Immatics, Ensoma, Mammoth, Glaxo Smith Kline, and Bristol Myers Squibb. L.L., R.G.M., E.S., and E.W.W. are consultants for and hold equity in Lyell Immunopharma. L.L. is a cofounder of, consults for, and holds equity in Syncopation Life Sciences. R.G.M. is a cofounder of, consults for, and holds equity in Syncopation Life Sciences and Link Cell Therapies. R.G.M. is a consultant for Illumina Radiopharmaceuticals, NKarta, ImmunAI, Arovella Therapeutics, Zai Lab, and Aptorum Group. R.G.M. serves on the Data and Safety Monitoring Board for Fate Therapeutics. J.T. is a consultant for Dorian Therapeutics. E.W.W. consults for and holds equity in VISTAN Health. A.T.S. is a founder of Immunai and Cartography Biosciences and receives research funding from Arsenal Biosciences, Allogene Therapeutics, and 10x Genomics. K.R.P. is a cofounder and employee of Cartography Biosciences. H.Y.C. is a cofounder of Accent Therapeutics, Boundless Bio, and Cartography Biosciences and is an advisor to 10x Genomics, Arsenal Biosciences, and Spring Discovery. J.R.C. is a cofounder and equity holder of Trapeze Therapeutics, Combangio, and Virsti Therapeutics; he has financial interests in Aravive, Xyence Therapeutics, and Syncopation Life Sciences; and he is a member of the Board of Directors of Ligand Pharmaceuticals and Revel Pharmaceuticals. S.A.Y.-H. is a consultant for Trapeze Therapeutics and Xyence Therapeutics., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
- Full Text
- View/download PDF
11. Delivery of CAR-T cells in a transient injectable stimulatory hydrogel niche improves treatment of solid tumors.
- Author
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Grosskopf AK, Labanieh L, Klysz DD, Roth GA, Xu P, Adebowale O, Gale EC, Jons CK, Klich JH, Yan J, Maikawa CL, Correa S, Ou BS, d'Aquino AI, Cochran JR, Chaudhuri O, Mackall CL, and Appel EA
- Subjects
- Cytokines, Humans, Hydrogels, Immunotherapy, Adoptive methods, T-Lymphocytes pathology, Neoplasms pathology, Neoplasms therapy, Receptors, Chimeric Antigen genetics
- Abstract
Adoptive cell therapy (ACT) has proven to be highly effective in treating blood cancers, but traditional approaches to ACT are poorly effective in treating solid tumors observed clinically. Novel delivery methods for therapeutic cells have shown promise for treatment of solid tumors when compared with standard intravenous administration methods, but the few reported approaches leverage biomaterials that are complex to manufacture and have primarily demonstrated applicability following tumor resection or in immune-privileged tissues. Here, we engineer simple-to-implement injectable hydrogels for the controlled co-delivery of CAR-T cells and stimulatory cytokines that improve treatment of solid tumors. The unique architecture of this material simultaneously inhibits passive diffusion of entrapped cytokines and permits active motility of entrapped cells to enable long-term retention, viability, and activation of CAR-T cells. The generation of a transient inflammatory niche following administration affords sustained exposure of CAR-T cells, induces a tumor-reactive CAR-T phenotype, and improves efficacy of treatment.
- Published
- 2022
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12. An engineered ligand trap inhibits leukemia inhibitory factor as pancreatic cancer treatment strategy.
- Author
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Hunter SA, McIntosh BJ, Shi Y, Sperberg RAP, Funatogawa C, Labanieh L, Soon E, Wastyk HC, Mehta N, Carter C, Hunter T, and Cochran JR
- Subjects
- Humans, Leukemia Inhibitory Factor Receptor alpha Subunit metabolism, Ligands, Protein Engineering, Leukemia Inhibitory Factor antagonists & inhibitors, Leukemia Inhibitory Factor Receptor alpha Subunit genetics, Pancreatic Neoplasms therapy
- Abstract
Leukemia inhibitory factor (LIF), a cytokine secreted by stromal myofibroblasts and tumor cells, has recently been highlighted to promote tumor progression in pancreatic and other cancers through KRAS-driven cell signaling. We engineered a high affinity soluble human LIF receptor (LIFR) decoy that sequesters human LIF and inhibits its signaling as a therapeutic strategy. This engineered 'ligand trap', fused to an antibody Fc-domain, has ~50-fold increased affinity (~20 pM) and improved LIF inhibition compared to wild-type LIFR-Fc, potently blocks LIF-mediated effects in pancreatic cancer cells, and slows the growth of pancreatic cancer xenograft tumors. These results, and the lack of apparent toxicity observed in animal models, further highlights ligand traps as a promising therapeutic strategy for cancer treatment.
- Published
- 2021
- Full Text
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13. Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling.
- Author
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Weber EW, Parker KR, Sotillo E, Lynn RC, Anbunathan H, Lattin J, Good Z, Belk JA, Daniel B, Klysz D, Malipatlolla M, Xu P, Bashti M, Heitzeneder S, Labanieh L, Vandris P, Majzner RG, Qi Y, Sandor K, Chen LC, Prabhu S, Gentles AJ, Wandless TJ, Satpathy AT, Chang HY, and Mackall CL
- Subjects
- Animals, Cell Line, Tumor, Cytotoxicity, Immunologic, Down-Regulation, Enhancer of Zeste Homolog 2 Protein metabolism, Epigenome, Female, Hepatocyte Nuclear Factor 1-alpha metabolism, High Mobility Group Proteins metabolism, Humans, Immunologic Memory, Lymphocyte Activation, Lymphoid Enhancer-Binding Factor 1 metabolism, Male, Mice, Neoplasms, Experimental therapy, Protein Domains, Protein Stability, Receptors, Chimeric Antigen chemistry, Receptors, Chimeric Antigen immunology, Signal Transduction, T-Lymphocytes metabolism, Transcription, Genetic, Xenograft Model Antitumor Assays, Dasatinib pharmacology, Epigenesis, Genetic, Immunotherapy, Adoptive, Receptors, Chimeric Antigen metabolism, T-Lymphocytes immunology
- Abstract
T cell exhaustion limits immune responses against cancer and is a major cause of resistance to chimeric antigen receptor (CAR)-T cell therapeutics. Using murine xenograft models and an in vitro model wherein tonic CAR signaling induces hallmark features of exhaustion, we tested the effect of transient cessation of receptor signaling, or rest, on the development and maintenance of exhaustion. Induction of rest through enforced down-regulation of the CAR protein using a drug-regulatable system or treatment with the multikinase inhibitor dasatinib resulted in the acquisition of a memory-like phenotype, global transcriptional and epigenetic reprogramming, and restored antitumor functionality in exhausted CAR-T cells. This work demonstrates that rest can enhance CAR-T cell efficacy by preventing or reversing exhaustion, and it challenges the notion that exhaustion is an epigenetically fixed state., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)
- Published
- 2021
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14. Global analysis of shared T cell specificities in human non-small cell lung cancer enables HLA inference and antigen discovery.
- Author
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Chiou SH, Tseng D, Reuben A, Mallajosyula V, Molina IS, Conley S, Wilhelmy J, McSween AM, Yang X, Nishimiya D, Sinha R, Nabet BY, Wang C, Shrager JB, Berry MF, Backhus L, Lui NS, Wakelee HA, Neal JW, Padda SK, Berry GJ, Delaidelli A, Sorensen PH, Sotillo E, Tran P, Benson JA, Richards R, Labanieh L, Klysz DD, Louis DM, Feldman SA, Diehn M, Weissman IL, Zhang J, Wistuba II, Futreal PA, Heymach JV, Garcia KC, Mackall CL, and Davis MM
- Subjects
- Algorithms, Antigen Presentation, Antigens, Neoplasm metabolism, Cells, Cultured, Cross Reactions, Epitopes, T-Lymphocyte metabolism, HLA-A2 Antigen metabolism, Humans, Protein Binding, T-Cell Antigen Receptor Specificity, Carcinoma, Non-Small-Cell Lung immunology, Epitope Mapping methods, Epitopes, T-Lymphocyte genetics, Lung Neoplasms immunology, Receptors, Antigen, T-Cell, alpha-beta genetics, T-Lymphocytes immunology
- Abstract
To identify disease-relevant T cell receptors (TCRs) with shared antigen specificity, we analyzed 778,938 TCRβ chain sequences from 178 non-small cell lung cancer patients using the GLIPH2 (grouping of lymphocyte interactions with paratope hotspots 2) algorithm. We identified over 66,000 shared specificity groups, of which 435 were clonally expanded and enriched in tumors compared to adjacent lung. The antigenic epitopes of one such tumor-enriched specificity group were identified using a yeast peptide-HLA A
∗ 02:01 display library. These included a peptide from the epithelial protein TMEM161A, which is overexpressed in tumors and cross-reactive epitopes from Epstein-Barr virus and E. coli. Our findings suggest that this cross-reactivity may underlie the presence of virus-specific T cells in tumor infiltrates and that pathogen cross-reactivity may be a feature of multiple cancers. The approach and analytical pipelines generated in this work, as well as the specificity groups defined here, present a resource for understanding the T cell response in cancer., Competing Interests: Declaration of interests C.L.M. is a founder of, holds equity in, and receives consulting fees from Lyell Immunopharma and receives consulting fees from NeoImmuneTech, Nektar, Apricity, and Roche. J.W.N. reports research support from Genentech/Roche, Merck, Novartis, Boehringer Ingelheim, Exelixis, Takeda Pharmaceuticals, Nektar Therapeutics, Adaptimmune, and GSK and has served in a consulting or advisory role for AstraZeneca, Genentech/Roche, Exelixis Inc., Jounce Therapeutics, Takeda Pharmaceuticals, Eli Lilly and Company, Calithera Biosciences, Amgen, Regeneron Pharmaceuticals, Natera, and Iovance Biotherapeutics. H.A.W. has received research support from Celgene, Clovis Oncology, Genentech/Roche, Arrys Therapeutics, Novartis, Merck, BMS, Exelixis, Lilly, Pfizer, and has participated on the advisory boards of Helsinn, Mirati, Cellworks, Genentech/Roche, Merck, and ITMIG. N.S.L. has received research funding from Intuitive Foundation and Auspex Diagnostics. E.S. is a consultant for Lyell Immunopharma. L.L. is a consultant for Lyell Immunopharma. S.A.F. is consulting for Lonza PerMed and Samsara BioCapital. M.D. reports research funding from Varian Medical Systems and Illumina; ownership interest in CiberMed and Foresight Diagnostics; patent filings related to cancer biomarkers; paid consultancy from Roche, AstraZeneca, BioNTech, Genentech, Novartis, and Gritstone Oncology; and travel/honoraria from Reflexion. K.C.G. is founder of 3T therapeutics. I.I.W. has received honoraria from Genentech/Roche, Bayer, Bristol-Myers Squibb, AstraZeneca/Medimmune, Pfizer, HTG Molecular, Asuragen, Merck, GlaxoSmithKline, Guardant Health, Oncocyte, and MSD. I.I.W. is also supported by Genentech, Oncoplex, HTG Molecular, DepArray, Merck, Bristol-Myers Squibb, Medimmune, Adaptive, Adaptimmune, EMD Serono, Pfizer, Takeda, Amgen, Karus, Johnson & Johnson, Bayer, Iovance, 4D, Novartis, and Akoya. J.Z. reports grants from Merck and Johnson & Johnson, as well as adversary/consulting/Hornoraria fees from Bristol Myers Squibb, AstraZeneca, GenePlus, Innovent, OrigMed, and Roche outside the submitted work. This study was supported in part by a Cancer Prevention Research Institute of Texas Multi-Investigator Research Award (grant number RP160668) and the University of Texas Lung Specialized Programs of Research Excellence grant (grant number P50CA70907). S.-H.C., D.T., C.L.M., and M.M.D have a patent related to this work., (Copyright © 2021. Published by Elsevier Inc.)- Published
- 2021
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15. PET Reporter Gene Imaging and Ganciclovir-Mediated Ablation of Chimeric Antigen Receptor T Cells in Solid Tumors.
- Author
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Murty S, Labanieh L, Murty T, Gowrishankar G, Haywood T, Alam IS, Beinat C, Robinson E, Aalipour A, Klysz DD, Cochran JR, Majzner RG, Mackall CL, and Gambhir SS
- Subjects
- Animals, Antiviral Agents pharmacology, B7 Antigens immunology, Cell Line, Tumor, Cell Movement immunology, Ganciclovir pharmacology, Genes, Transgenic, Suicide, Herpesvirus 1, Human, Humans, Mice, Receptors, Chimeric Antigen immunology, Viral Proteins analysis, Xenograft Model Antitumor Assays, Genes, Reporter, Immunotherapy, Adoptive methods, Osteosarcoma, Positron Emission Tomography Computed Tomography methods, Thymidine Kinase analysis
- Abstract
Imaging strategies to monitor chimeric antigen receptor (CAR) T-cell biodistribution and proliferation harbor the potential to facilitate clinical translation for the treatment of both liquid and solid tumors. In addition, the potential adverse effects of CAR T cells highlight the need for mechanisms to modulate CAR T-cell activity. The herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene has previously been translated as a PET reporter gene for imaging of T-cell trafficking in patients with brain tumor. The HSV1-TK enzyme can act as a suicide gene of transduced cells through treatment with the prodrug ganciclovir. Here we report the molecular engineering, imaging, and ganciclovir-mediated destruction of B7H3 CAR T cells incorporating a mutated version of the HSV1-tk gene (sr39tk) with improved enzymatic activity for ganciclovir. The sr39tk gene did not affect B7H3 CAR T-cell functionality and in vitro and in vivo studies in osteosarcoma models showed no significant effect on B7H3 CAR T-cell antitumor activity. PET/CT imaging with 9-(4-[
18 F]-fluoro-3-[hydroxymethyl]butyl)guanine ([18 F]FHBG) of B7H3-sr39tk CAR T cells in an orthotopic model of osteosarcoma revealed tumor homing and systemic immune expansion. Bioluminescence and PET imaging of B7H3-sr39tk CAR T cells confirmed complete tumor ablation with intraperitoneal ganciclovir administration. This imaging and suicide ablation system can provide insight into CAR T-cell migration and proliferation during clinical trials while serving as a suicide switch to limit potential toxicities. SIGNIFICANCE: This study showcases the only genetically engineered system capable of serving the dual role both as an effective PET imaging reporter and as a suicide switch for CAR T cells., (©2020 American Association for Cancer Research.)- Published
- 2020
- Full Text
- View/download PDF
16. An engineered antibody binds a distinct epitope and is a potent inhibitor of murine and human VISTA.
- Author
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Mehta N, Maddineni S, Kelly RL, Lee RB, Hunter SA, Silberstein JL, Parra Sperberg RA, Miller CL, Rabe A, Labanieh L, and Cochran JR
- Subjects
- Animals, Antigen-Antibody Reactions, B7 Antigens genetics, B7 Antigens immunology, Binding Sites, Cell Adhesion Molecules metabolism, Cell Surface Display Techniques, Cross Reactions, Epitopes genetics, Female, Humans, Immunoglobulins metabolism, Macaca fascicularis, Melanoma, Experimental immunology, Melanoma, Experimental therapy, Membrane Glycoproteins metabolism, Membrane Proteins genetics, Membrane Proteins immunology, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Models, Molecular, Protein Binding, Protein Engineering, Antibodies metabolism, B7 Antigens antagonists & inhibitors, Epitopes metabolism, Membrane Proteins antagonists & inhibitors
- Abstract
V-domain immunoglobulin (Ig) suppressor of T cell activation (VISTA) is an immune checkpoint that maintains peripheral T cell quiescence and inhibits anti-tumor immune responses. VISTA functions by dampening the interaction between myeloid cells and T cells, orthogonal to PD-1 and other checkpoints of the tumor-T cell signaling axis. Here, we report the use of yeast surface display to engineer an anti-VISTA antibody that binds with high affinity to mouse, human, and cynomolgus monkey VISTA. Our anti-VISTA antibody (SG7) inhibits VISTA function and blocks purported interactions with both PSGL-1 and VSIG3 proteins. SG7 binds a unique epitope on the surface of VISTA, which partially overlaps with other clinically relevant antibodies. As a monotherapy, and to a greater extent as a combination with anti-PD1, SG7 slows tumor growth in multiple syngeneic mouse models. SG7 is a promising clinical candidate that can be tested in fully immunocompetent mouse models and its binding epitope can be used for future campaigns to develop species cross-reactive inhibitors of VISTA.
- Published
- 2020
- Full Text
- View/download PDF
17. Rapid Detection of β-Lactamase-Producing Bacteria Using the Integrated Comprehensive Droplet Digital Detection (IC 3D) System.
- Author
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Li Y, Cherukury H, Labanieh L, Zhao W, and Kang DK
- Subjects
- Biosensing Techniques, Humans, Microfluidics, Polymerase Chain Reaction, Bacteria, Sepsis, beta-Lactamases
- Abstract
Antibiotic-resistant bacteria have emerged as an imminent global threat. The lack of rapid and sensitive diagnostic techniques leaves health care providers with inadequate resources for guiding therapy and risks the lives of patients. The traditional plate culturing methods for identifying antibiotic-resistant bacteria is laborious and time-consuming. Bulk PCR (Polymerase Chain Reaction) and qPCR are limited by poor detection sensitivity, which is critical for the early-stage detection of bloodstream infections. In this study, we introduce a technique for detecting β-lactamase-producing bacteria at single-cell sensitivity based on a commercial β-lactamase sensor (Fluorocillin), droplet microfluidics, and a custom 3D particle counter. Bacteria-containing samples were encapsulated within picoliter-sized droplets at the single-cell level and cultured within water-in-oil droplets containing antibiotics and the Fluorocillin sensor. Then, fluorescent droplets were digitally quantified with the 3D particle counter, which is capable of analyzing milliliter-scale volumes of collected droplets within 10 min. The fluorescence signal from single-colony droplets was detectable in less than 5 h, and the 3D scanning was performed in less than 10 min, which was significantly faster than conventional culture-based methods. In this approach, the limit of detection achieved was about 10 bacterial cells per mL of sample, and the turnaround time from sample to result was less than 6 h. This study demonstrates a promising strategy for the detection of β-lactamase-producing bacteria using the recently developed IC 3D system.
- Published
- 2020
- Full Text
- View/download PDF
18. Novel NanoLuc substrates enable bright two-population bioluminescence imaging in animals.
- Author
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Su Y, Walker JR, Park Y, Smith TP, Liu LX, Hall MP, Labanieh L, Hurst R, Wang DC, Encell LP, Kim N, Zhang F, Kay MA, Casey KM, Majzner RG, Cochran JR, Mackall CL, Kirkland TA, and Lin MZ
- Subjects
- Animals, Enzyme Assays methods, Substrate Specificity, Furans chemistry, Luciferases chemistry, Luminescent Measurements methods, Luminescent Proteins chemistry
- Abstract
Sensitive detection of two biological events in vivo has long been a goal in bioluminescence imaging. Antares, a fusion of the luciferase NanoLuc to the orange fluorescent protein CyOFP, has emerged as a bright bioluminescent reporter with orthogonal substrate specificity to firefly luciferase (FLuc) and its derivatives such as AkaLuc. However, the brightness of Antares in mice is limited by the poor solubility and bioavailability of the NanoLuc substrate furimazine. Here, we report a new substrate, hydrofurimazine, whose enhanced aqueous solubility allows delivery of higher doses to mice. In the liver, Antares with hydrofurimazine exhibited similar brightness to AkaLuc with its substrate AkaLumine. Further chemical exploration generated a second substrate, fluorofurimazine, with even higher brightness in vivo. We used Antares with fluorofurimazine to track tumor size and AkaLuc with AkaLumine to visualize CAR-T cells within the same mice, demonstrating the ability to perform two-population imaging with these two luciferase systems.
- Published
- 2020
- Full Text
- View/download PDF
19. Tuning the Antigen Density Requirement for CAR T-cell Activity.
- Author
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Majzner RG, Rietberg SP, Sotillo E, Dong R, Vachharajani VT, Labanieh L, Myklebust JH, Kadapakkam M, Weber EW, Tousley AM, Richards RM, Heitzeneder S, Nguyen SM, Wiebking V, Theruvath J, Lynn RC, Xu P, Dunn AR, Vale RD, and Mackall CL
- Subjects
- Animals, Humans, Mice, Signal Transduction, Receptors, Chimeric Antigen metabolism
- Abstract
Insufficient reactivity against cells with low antigen density has emerged as an important cause of chimeric antigen receptor (CAR) T-cell resistance. Little is known about factors that modulate the threshold for antigen recognition. We demonstrate that CD19 CAR activity is dependent upon antigen density and that the CAR construct in axicabtagene ciloleucel (CD19-CD28ζ) outperforms that in tisagenlecleucel (CD19-4-1BBζ) against antigen-low tumors. Enhancing signal strength by including additional immunoreceptor tyrosine-based activation motifs (ITAM) in the CAR enables recognition of low-antigen-density cells, whereas ITAM deletions blunt signal and increase the antigen density threshold. Furthermore, replacement of the CD8 hinge-transmembrane (H/T) region of a 4-1BBζ CAR with a CD28-H/T lowers the threshold for CAR reactivity despite identical signaling molecules. CARs incorporating a CD28-H/T demonstrate a more stable and efficient immunologic synapse. Precise design of CARs can tune the threshold for antigen recognition and endow 4-1BBζ-CARs with enhanced capacity to recognize antigen-low targets while retaining a superior capacity for persistence. SIGNIFICANCE: Optimal CAR T-cell activity is dependent on antigen density, which is variable in many cancers, including lymphoma and solid tumors. CD28ζ-CARs outperform 4-1BBζ-CARs when antigen density is low. However, 4-1BBζ-CARs can be reengineered to enhance activity against low-antigen-density tumors while maintaining their unique capacity for persistence. This article is highlighted in the In This Issue feature, p. 627 ., (©2020 American Association for Cancer Research.)
- Published
- 2020
- Full Text
- View/download PDF
20. Locoregionally administered B7-H3-targeted CAR T cells for treatment of atypical teratoid/rhabdoid tumors.
- Author
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Theruvath J, Sotillo E, Mount CW, Graef CM, Delaidelli A, Heitzeneder S, Labanieh L, Dhingra S, Leruste A, Majzner RG, Xu P, Mueller S, Yecies DW, Finetti MA, Williamson D, Johann PD, Kool M, Pfister S, Hasselblatt M, Frühwald MC, Delattre O, Surdez D, Bourdeaut F, Puget S, Zaidi S, Mitra SS, Cheshier S, Sorensen PH, Monje M, and Mackall CL
- Subjects
- Adult, Animals, Brain drug effects, Brain immunology, Brain pathology, Brain Neoplasms immunology, Brain Neoplasms pathology, Cells, Cultured, Child, Preschool, Female, Fetus pathology, Humans, Infant, Injections, Intraventricular, Mice, Mice, Inbred NOD, Mice, SCID, Receptors, Chimeric Antigen administration & dosage, Receptors, Chimeric Antigen genetics, Receptors, Chimeric Antigen immunology, Rhabdoid Tumor immunology, Rhabdoid Tumor pathology, T-Lymphocytes immunology, T-Lymphocytes metabolism, T-Lymphocytes transplantation, Teratoma immunology, Teratoma pathology, Xenograft Model Antitumor Assays, B7 Antigens immunology, Brain Neoplasms therapy, Cancer Vaccines administration & dosage, Immunotherapy, Adoptive methods, Rhabdoid Tumor therapy, Teratoma therapy
- Abstract
Atypical teratoid/rhabdoid tumors (ATRTs) typically arise in the central nervous system (CNS) of children under 3 years of age. Despite intensive multimodal therapy (surgery, chemotherapy and, if age permits, radiotherapy), median survival is 17 months
1,2 . We show that ATRTs robustly express B7-H3/CD276 that does not result from the inactivating mutations in SMARCB1 (refs.3,4 ), which drive oncogenesis in ATRT, but requires residual SWItch/Sucrose Non-Fermentable (SWI/SNF) activity mediated by BRG1/SMARCA4. Consistent with the embryonic origin of ATRT5,6 , B7-H3 is highly expressed on the prenatal, but not postnatal, brain. B7-H3.BB.z-chimeric antigen receptor (CAR) T cells administered intracerebroventricularly or intratumorally mediate potent antitumor effects against cerebral ATRT xenografts in mice, with faster kinetics, greater potency and reduced systemic levels of inflammatory cytokines compared to CAR T cells administered intravenously. CAR T cells administered ICV also traffic from the CNS into the periphery; following clearance of ATRT xenografts, B7-H3.BB.z-CAR T cells administered intracerebroventricularly or intravenously mediate antigen-specific protection from tumor rechallenge, both in the brain and periphery. These results identify B7-H3 as a compelling therapeutic target for this largely incurable pediatric tumor and demonstrate important advantages of locoregional compared to systemic delivery of CAR T cells for the treatment of CNS malignancies.- Published
- 2020
- Full Text
- View/download PDF
21. Rapid bacterial detection and antibiotic susceptibility testing in whole blood using one-step, high throughput blood digital PCR.
- Author
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Abram TJ, Cherukury H, Ou CY, Vu T, Toledano M, Li Y, Grunwald JT, Toosky MN, Tifrea DF, Slepenkin A, Chong J, Kong L, Del Pozo DV, La KT, Labanieh L, Zimak J, Shen B, Huang SS, Gratton E, Peterson EM, and Zhao W
- Subjects
- Enterobacteriaceae isolation & purification, Humans, Lab-On-A-Chip Devices, Methicillin-Resistant Staphylococcus aureus isolation & purification, Microfluidic Analytical Techniques instrumentation, Particle Size, Surface Properties, Vancomycin-Resistant Enterococci isolation & purification, Anti-Bacterial Agents pharmacology, Enterobacteriaceae drug effects, Methicillin-Resistant Staphylococcus aureus drug effects, Polymerase Chain Reaction, Vancomycin-Resistant Enterococci drug effects
- Abstract
Sepsis due to antimicrobial resistant pathogens is a major health problem worldwide. The inability to rapidly detect and thus treat bacteria with appropriate agents in the early stages of infections leads to excess morbidity, mortality, and healthcare costs. Here we report a rapid diagnostic platform that integrates a novel one-step blood droplet digital PCR assay and a high throughput 3D particle counter system with potential to perform bacterial identification and antibiotic susceptibility profiling directly from whole blood specimens, without requiring culture and sample processing steps. Using CTX-M-9 family ESBLs as a model system, we demonstrated that our technology can simultaneously achieve unprecedented high sensitivity (10 CFU per ml) and rapid sample-to-answer assay time (one hour). In head-to-head studies, by contrast, real time PCR and BioRad ddPCR only exhibited a limit of detection of 1000 CFU per ml and 50-100 CFU per ml, respectively. In a blinded test inoculating clinical isolates into whole blood, we demonstrated 100% sensitivity and specificity in identifying pathogens carrying a particular resistance gene. We further demonstrated that our technology can be broadly applicable for targeted detection of a wide range of antibiotic resistant genes found in both Gram-positive (vanA, nuc, and mecA) and Gram-negative bacteria, including ESBLs (bla
CTX-M-1 and blaCTX-M-2 families) and CREs (blaOXA-48 and blaKPC ), as well as bacterial speciation (E. coli and Klebsiella spp.) and pan-bacterial detection, without requiring blood culture or sample processing. Our rapid diagnostic technology holds great potential in directing early, appropriate therapy and improved antibiotic stewardship in combating bloodstream infections and antibiotic resistance.- Published
- 2020
- Full Text
- View/download PDF
22. Live imaging of Aiptasia larvae, a model system for coral and anemone bleaching, using a simple microfluidic device.
- Author
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Van Treuren W, Brower KK, Labanieh L, Hunt D, Lensch S, Cruz B, Cartwright HN, Tran C, and Fordyce PM
- Subjects
- Animals, Anthozoa parasitology, Climate Change, Ecosystem, Larva parasitology, Molecular Imaging, Sea Anemones parasitology, Anthozoa physiology, Lab-On-A-Chip Devices, Larva physiology, Models, Biological, Photosynthesis, Sea Anemones physiology, Symbiosis
- Abstract
Coral reefs, and their associated diverse ecosystems, are of enormous ecological importance. In recent years, coral health has been severely impacted by environmental stressors brought on by human activity and climate change, threatening the extinction of several major reef ecosystems. Reef damage is mediated by a process called 'coral bleaching' where corals, sea anemones, and other cnidarians lose their photosynthetic algal symbionts (family Symbiodiniaceae) upon stress induction, resulting in drastically decreased host energy harvest and, ultimately, coral death. The mechanism by which this critical cnidarian-algal symbiosis is lost remains poorly understood. The larvae of the sea anemone, Exaiptasia pallida (commonly referred to as 'Aiptasia') are an attractive model organism to study this process, but they are large (∼100 mm in length, ∼75 mm in diameter), deformable, and highly motile, complicating long-term imaging and limiting study of this critical endosymbiotic relationship in live organisms. Here, we report 'Traptasia', a simple microfluidic device with multiple traps designed to isolate and image individual, live larvae of Aiptasia and their algal symbionts over extended time courses. Using a trap design parameterized via fluid flow simulations and polymer bead loading tests, we trapped Aiptasia larvae containing algal symbionts and demonstrated stable imaging for >10 hours. We visualized algae within Aiptasia larvae and observed algal expulsion under an environmental stressor. To our knowledge, this device is the first to enable time-lapsed, high-throughput live imaging of cnidarian larvae and their algal symbionts and, in further implementation, could provide important insights into the cellular mechanisms of cnidarian bleaching under different environmental stressors. The 'Traptasia' device is simple to use, requires minimal external equipment and no specialized training to operate, and can easily be adapted using the trap optimization data presented here to study a variety of large, motile organisms.
- Published
- 2019
- Full Text
- View/download PDF
23. CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors.
- Author
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Majzner RG, Theruvath JL, Nellan A, Heitzeneder S, Cui Y, Mount CW, Rietberg SP, Linde MH, Xu P, Rota C, Sotillo E, Labanieh L, Lee DW, Orentas RJ, Dimitrov DS, Zhu Z, Croix BS, Delaidelli A, Sekunova A, Bonvini E, Mitra SS, Quezado MM, Majeti R, Monje M, Sorensen PHB, Maris JM, and Mackall CL
- Subjects
- Animals, B7 Antigens antagonists & inhibitors, Brain Neoplasms pathology, Brain Neoplasms therapy, Cell Line, Tumor, Disease Models, Animal, Humans, Immunohistochemistry, Mice, Receptors, Antigen, T-Cell genetics, Receptors, Antigen, T-Cell metabolism, Treatment Outcome, Xenograft Model Antitumor Assays, Antigens, Neoplasm immunology, B7 Antigens immunology, Brain Neoplasms etiology, Brain Neoplasms metabolism, Immunotherapy, Adoptive methods, Receptors, Chimeric Antigen metabolism, T-Lymphocytes immunology, T-Lymphocytes metabolism
- Abstract
Purpose: Patients with relapsed pediatric solid tumors and CNS malignancies have few therapeutic options and frequently die of their disease. Chimeric antigen receptor (CAR) T cells have shown tremendous success in treating relapsed pediatric acute lymphoblastic leukemia, but this has not yet translated to treating solid tumors. This is partially due to a paucity of differentially expressed cell surface molecules on solid tumors that can be safely targeted. Here, we present B7-H3 (CD276) as a putative target for CAR T-cell therapy of pediatric solid tumors, including those arising in the central nervous system., Experimental Design: We developed a novel B7-H3 CAR whose binder is derived from a mAb that has been shown to preferentially bind tumor tissues and has been safely used in humans in early-phase clinical trials. We tested B7-H3 CAR T cells in a variety of pediatric cancer models., Results: B7-H3 CAR T cells mediate significant antitumor activity in vivo , causing regression of established solid tumors in xenograft models including osteosarcoma, medulloblastoma, and Ewing sarcoma. We demonstrate that B7-H3 CAR T-cell efficacy is largely dependent upon high surface target antigen density on tumor tissues and that activity is greatly diminished against target cells that express low levels of antigen, thus providing a possible therapeutic window despite low-level normal tissue expression of B7-H3., Conclusions: B7-H3 CAR T cells could represent an exciting therapeutic option for patients with certain lethal relapsed or refractory pediatric malignancies, and should be tested in carefully designed clinical trials., (©2019 American Association for Cancer Research.)
- Published
- 2019
- Full Text
- View/download PDF
24. Programming CAR-T cells to kill cancer.
- Author
-
Labanieh L, Majzner RG, and Mackall CL
- Subjects
- Apoptosis, Humans, Models, Biological, Protein Binding, Cell Engineering, Immunotherapy, Adoptive, Neoplasms therapy, T-Lymphocytes cytology, T-Lymphocytes immunology, T-Lymphocytes physiology
- Abstract
T cells engineered to express chimeric antigen receptors (CARs) that are specific for tumour antigens have led to high complete response rates in patients with haematologic malignancies. Despite this early success, major challenges to the broad application of CAR-T cells as cancer therapies remain, including treatment-associated toxicities and cancer relapse with antigen-negative tumours. Targeting solid tumours with CAR-T cells poses additional obstacles because of the paucity of tumour-specific antigens and the immunosuppressive effects of the tumour microenvironment. To overcome these challenges, T cells can be programmed with genetic modules that increase their therapeutic potency and specificity. In this Review Article, we survey major advances in the engineering of next-generation CAR-T therapies for haematologic cancers and solid cancers, with particular emphasis on strategies for the control of CAR specificity and activity and on approaches for improving CAR-T-cell persistence and overcoming immunosuppression. We also lay out a roadmap for the development of off-the-shelf CAR-T cells.
- Published
- 2018
- Full Text
- View/download PDF
25. Potent antitumor efficacy of anti-GD2 CAR T cells in H3-K27M + diffuse midline gliomas.
- Author
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Mount CW, Majzner RG, Sundaresh S, Arnold EP, Kadapakkam M, Haile S, Labanieh L, Hulleman E, Woo PJ, Rietberg SP, Vogel H, Monje M, and Mackall CL
- Subjects
- Animals, Humans, Methylation, Mice, Xenograft Model Antitumor Assays, Brain Neoplasms immunology, Gangliosides metabolism, Glioma immunology, Histones metabolism, Immunotherapy, Adoptive, Lysine metabolism, Receptors, Antigen, T-Cell metabolism
- Abstract
Diffuse intrinsic pontine glioma (DIPG) and other diffuse midline gliomas (DMGs) with mutated histone H3 K27M (H3-K27M)
1-5 are aggressive and universally fatal pediatric brain cancers6 . Chimeric antigen receptor (CAR)-expressing T cells have mediated impressive clinical activity in B cell malignancies7-10 , and recent results suggest benefit in central nervous system malignancies11-13 . Here, we report that patient-derived H3-K27M-mutant glioma cell cultures exhibit uniform, high expression of the disialoganglioside GD2. Anti-GD2 CAR T cells incorporating a 4-1BBz costimulatory domain14 demonstrated robust antigen-dependent cytokine generation and killing of DMG cells in vitro. In five independent patient-derived H3-K27M+ DMG orthotopic xenograft models, systemic administration of GD2-targeted CAR T cells cleared engrafted tumors except for a small number of residual GD2lo glioma cells. To date, GD2-targeted CAR T cells have been well tolerated in clinical trials15-17 . Although GD2-targeted CAR T cell administration was tolerated in the majority of mice bearing orthotopic xenografts, peritumoral neuroinflammation during the acute phase of antitumor activity resulted in hydrocephalus that was lethal in a fraction of animals. Given the precarious neuroanatomical location of midline gliomas, careful monitoring and aggressive neurointensive care management will be required for human translation. With a cautious multidisciplinary clinical approach, GD2-targeted CAR T cell therapy for H3-K27M+ diffuse gliomas of pons, thalamus and spinal cord could prove transformative for these lethal childhood cancers.- Published
- 2018
- Full Text
- View/download PDF
26. Engineering cell sensing and responses using a GPCR-coupled CRISPR-Cas system.
- Author
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Kipniss NH, Dingal PCDP, Abbott TR, Gao Y, Wang H, Dominguez AA, Labanieh L, and Qi LS
- Subjects
- Clustered Regularly Interspaced Short Palindromic Repeats, HEK293 Cells, Humans, Ligands, Models, Theoretical, CRISPR-Cas Systems, Cell Engineering methods, Receptors, G-Protein-Coupled
- Abstract
G-protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes and detect a wide array of cues in the human body. Here we describe a molecular device that couples CRISPR-dCas9 genome regulation to diverse natural and synthetic extracellular signals via GPCRs. We generate alternative architectures for fusing CRISPR to GPCRs utilizing the previously reported design, Tango, and our design, ChaCha. Mathematical modeling suggests that for the CRISPR ChaCha design, multiple dCas9 molecules can be released across the lifetime of a GPCR. The CRISPR ChaCha is dose-dependent, reversible, and can activate multiple endogenous genes simultaneously in response to extracellular ligands. We adopt the design to diverse GPCRs that sense a broad spectrum of ligands, including synthetic compounds, chemokines, mitogens, fatty acids, and hormones. This toolkit of CRISPR-coupled GPCRs provides a modular platform for rewiring diverse ligand sensing to targeted genome regulation for engineering cellular functions.
- Published
- 2017
- Full Text
- View/download PDF
27. High-throughput screening technologies for enzyme engineering.
- Author
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Longwell CK, Labanieh L, and Cochran JR
- Subjects
- Cells metabolism, Enzymes metabolism, High-Throughput Screening Assays methods, Protein Engineering methods
- Abstract
Emerging technologies are enabling ultra-high-throughput screening of combinatorial enzyme libraries to identify variants with improved properties such as increased activity, altered substrate specificity, and increased stability. Each of these enzyme engineering platforms relies on compartmentalization of reaction components, similar to microtiter plate-based assays which have been commonly used for testing the activity of enzyme variants. The technologies can be broadly divided into three categories according to their spatial segregation strategy: (1) cells as reaction compartments, (2) in vitro compartmentalization via synthetic droplets, and (3) microchambers. Here, we discuss these emerging platforms, which in some cases enable the screening of greater than 10 million enzyme variants, and highlight benefits and limitations of each technology., (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
28. Digital quantification of miRNA directly in plasma using integrated comprehensive droplet digital detection.
- Author
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Zhang K, Kang DK, Ali MM, Liu L, Labanieh L, Lu M, Riazifar H, Nguyen TN, Zell JA, Digman MA, Gratton E, Li J, and Zhao W
- Subjects
- Colonic Neoplasms blood, Humans, Limit of Detection, Blood Chemical Analysis instrumentation, Lab-On-A-Chip Devices, MicroRNAs blood, Systems Integration
- Abstract
Quantification of miRNAs in blood can be potentially used for early disease detection, surveillance monitoring and drug response evaluation. However, quantitative and robust measurement of miRNAs in blood is still a major challenge in large part due to their low concentration and complicated sample preparation processes typically required in conventional assays. Here, we present the 'Integrated Comprehensive Droplet Digital Detection' (IC 3D) system where the plasma sample containing target miRNAs is encapsulated into microdroplets, enzymatically amplified and digitally counted using a novel, high-throughput 3D particle counter. Using Let-7a as a target, we demonstrate that IC 3D can specifically quantify target miRNA directly from blood plasma at extremely low concentrations ranging from 10s to 10 000 copies per mL in ≤3 hours without the need for sample processing such as RNA extraction. Using this new tool, we demonstrate that target miRNA content in colon cancer patient blood is significantly higher than that in healthy donor samples. Our IC 3D system has the potential to introduce a new paradigm for rapid, sensitive and specific detection of low-abundance biomarkers in biological samples with minimal sample processing.
- Published
- 2015
- Full Text
- View/download PDF
29. Floating Droplet Array: An Ultrahigh-Throughput Device for Droplet Trapping, Real-time Analysis and Recovery.
- Author
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Labanieh L, Nguyen TN, Zhao W, and Kang DK
- Abstract
We describe the design, fabrication and use of a dual-layered microfluidic device for ultrahigh-throughput droplet trapping, analysis, and recovery using droplet buoyancy. To demonstrate the utility of this device for digital quantification of analytes, we quantify the number of droplets, which contain a β-galactosidase-conjugated bead among more than 100,000 immobilized droplets. In addition, we demonstrate that this device can be used for droplet clustering and real-time analysis by clustering several droplets together into microwells and monitoring diffusion of fluorescein, a product of the enzymatic reaction of β-galactosidase and its fluorogenic substrate FDG, between droplets.
- Published
- 2015
- Full Text
- View/download PDF
30. Nucleic acid aptamers in cancer research, diagnosis and therapy.
- Author
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Ma H, Liu J, Ali MM, Mahmood MA, Labanieh L, Lu M, Iqbal SM, Zhang Q, Zhao W, and Wan Y
- Subjects
- Antineoplastic Agents therapeutic use, Biomarkers, Drug Design, Humans, Aptamers, Nucleotide chemistry, Neoplasms diagnosis, Neoplasms therapy
- Abstract
Aptamers are single-stranded DNA or RNA oligomers, identified from a random sequence pool, with the ability to form unique and versatile tertiary structures that bind to cognate molecules with superior specificity. Their small size, excellent chemical stability and low immunogenicity enable them to rival antibodies in cancer imaging and therapy applications. Their facile chemical synthesis, versatility in structural design and engineering, and the ability for site-specific modifications with functional moieties make aptamers excellent recognition motifs for cancer biomarker discovery and detection. Moreover, aptamers can be selected or engineered to regulate cancer protein functions, as well as to guide anti-cancer drug design or screening. This review summarizes their applications in cancer, including cancer biomarker discovery and detection, cancer imaging, cancer therapy, and anti-cancer drug discovery. Although relevant applications are relatively new, the significant progress achieved has demonstrated that aptamers can be promising players in cancer research.
- Published
- 2015
- Full Text
- View/download PDF
31. Aptamer technology for tracking cells' status & function.
- Author
-
Wiraja C, Yeo D, Lio D, Labanieh L, Lu M, Zhao W, and Xu C
- Abstract
In fields such as cancer biology and regenerative medicine, obtaining information regarding cell bio-distribution, tropism, status, and other cellular functions are highly desired. Understanding cancer behaviors including metastasis is important for developing effective cancer treatments, while assessing the fate of therapeutic cells following implantation is critical to validate the efficacy and efficiency of the therapy. For visualization purposes with medical imaging modalities (e.g. magnetic resonance imaging), cells can be labeled with contrast agents (e.g. iron-oxide nanoparticles), which allows their identification from the surrounding environment. Despite the success of revealing cell biodistribution in vivo, most of the existing agents do not provide information about the status and functions of cells following transplantation. The emergence of aptamers, single-stranded RNA or DNA oligonucleotides of 15 to 60 bases in length, is a promising solution to address this need. When aptamers bind specifically to their cognate molecules, they undergo conformational changes which can be transduced into a change of imaging contrast (e.g. optical, magnetic resonance). Thus by monitoring this signal change, researchers can obtain information about the expression of the target molecules (e.g. mRNA, surface markers, cell metabolites), which offer clues regarding cell status/function in a non-invasive manner. In this review, we summarize recent efforts to utilize aptamers as biosensors for monitoring the status and function of transplanted cells. We focus on cancer cell tracking for cancer study, stem cell tracking for regenerative medicine, and immune cell (e.g. dendritic cells) tracking for immune therapy.
- Published
- 2014
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