400 Persistence and tissue distribution of agent-797 – a native allogeneic iNKT cell-therapy drug product

BackgroundInvariant Natural Killer T (iNKT) cells are key effectors and regulators of immune responses, making them an ideal immunotherapy. There is a paucity of evidence describing the persistence and trafficking of these cells in humans to inform the optimal clinical application. Here, we describe the development of a murine Xenograft model for the study of an unmodified human iNKT cell therapy (Agent-797) and present data on the persistence and tissue distribution of human iNKT cells in this model. We further describe the development and validation of a digital PCR-based methodology to track unmodified allogeneic human iNKT cells in blood and tissue and present exploratory clinical data on iNKT cell persistence in patients with cancer and viral ARDS treated with Agent-797.MethodsPersistence and tissue distribution of ex-vivo expanded human iNKT cells was investigated in immune compromised mice (NOG), as well as in NOG mice expressing human IL15 (NOG-hIL15), a key cytokine promoting iNKT cell survival. Persistence of iNKT cells was determined over a 35-day period, with takedowns on day 1, 7, 14, 21 and 35. iNKT cells were phenotyped for activation markers by flow cytometry. An assay based on Imegen Quimera digital PCR technology was developed and validated to quantify human iNKT in an allogeneic setting. We employed this assay to measure persistence of Agent-797 drug product in patients participating in clinical trials using iNKT cell-based immunotherapy in viral ARDS (NCT04582201) or multiple myeloma (NCT04754100).ResultsHuman IL15 was essential for the engraftment and persistence of human iNKT cells in NOG mice. Following injection, iNKT cells located to the blood, lung, liver, spleen, and bone marrow. iNKT cells persisted most prominently in bone marrow, where they demonstrated an activated phenotype. In mice challenged with hematological tumor cells (ALL cell line NALM6 expressing CD1d) persistence of iNKT cells in blood was prolonged. Initial data from human trials confirmed rapid translocation from peripheral blood of this tissue resident immune cell population following infusion of Agent-797.ConclusionsWe established a murine xenograft model and digital PCR-based methodology to characterize the persistence, trafficking, and efficacy of native allogeneic human iNKT cell-based products. Our models recapitulated the human iNKT distribution and demonstrated iNKTs induced preclinical efficacy in a tumor model. We further successfully developed a validated methodology to track unmodified allogeneic iNKT cells in humans.Trial RegistrationNCT04582201 and NCT04754100Ethics ApprovalAll procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards

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205 AgenT-797, a native allogeneic ‘off-the-shelf’ iNKT cell therapy product shows anti-tumor activity in preclinical xenograft models

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.205 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A216-A216

Author(s):

Burcu Yigit ◽

Darrian Moskowitz ◽

Xavier Michelet ◽

Antoine Tanne ◽

Marc Van Dijk

Keyword(s):

Cell Therapy ◽

Tumor Growth ◽

Ex Vivo ◽

Inkt Cell ◽

Ethical Standards ◽

Tumor Infiltration ◽

Inkt Cells ◽

Cell Therapy Product ◽

Xenograft Models

BackgroundagenT-797 is an allogeneic, native invariant natural killer T (iNKT) cell therapy product currently in phase I clinical trials for cancer (heme and solid). iNKT cells are a distinct population of T cells that can recognize tumors via direct recognition of CD1d (an MHC-I like molecule presenting glycolipids) through the TCR or recognition of NK cell receptor ligands via various NK receptors. We developed agenT-797 from isolated and ex-vivo expanded peripheral blood iNKT cells. Here we describe in vivo xenograft models to demonstrate the overall tissue distribution, tumor infiltration and efficacy of agenT-797 in liquid as well as solid tumors.MethodsWe utilized NOG-hIL15 (human IL-15) transgenic mice to ensure persistence/maintenance of ex-vivo expanded human iNKT cells throughout the studies. For studying efficacy in liquid tumors, we used NALM6, an acute lymphoblastic leukemia (ALL) cell line and for solid tumors selected A375, a melanoma cell line. Both cell lines were engineered to overexpress CD1d. Upon injection of iNKT cells, tumor growth and iNKT cell tissue/tumor infiltration as well as phenotype were studied.ResultsInjection of iNKT cells in NALM6- engrafted NOG-hIL15 mice resulted in an overall reduction in leukemic burden as measured by luminescence-based imaging. Flow cytometric analysis revealed infiltration of iNKT cells at the site of leukemic expansion, namely blood, spleen, bone marrow and liver. Cells were activated when reaching the site of the tumor. In addition, iNKT cells produced IFNγ and TNFα and low levels of IL-13/IL-4, consistent with a Th1 response. When iNKT cells were injected into A375 engrafted mice we observed infiltration of iNKT cells into the tumor, where they become activated and proliferate overtime. We observed an overall reduction in tumor size when iNKT cells were injected compared to control group, demonstrating the impact of iNKT cells on tumor growth.ConclusionsWe established xenograft mouse models to address various biological questions around human iNKT cells as a cell therapy product. We have demonstrated homing and infiltration of iNKT cells at the site of tumor and relative proliferation and expansion. These models provide a suitable platform for in-vivo preclinical studies on agenT -797 in cancer.Ethics ApprovalAll procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

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Inhibition of tumor growth of cervical cancer cell line in a murine xenograft model by thalidomide

Geburtshilfe und Frauenheilkunde ◽

10.1055/s-2006-952205 ◽

2006 ◽

Vol 66 (S 01) ◽

Author(s):

LC Horn ◽

M Hermes ◽

M Wobus ◽

J Schwock ◽

M Brulport ◽

...

Keyword(s):

Cervical Cancer ◽

Tumor Growth ◽

Cell Line ◽

Cancer Cell ◽

Xenograft Model ◽

Cancer Cell Line ◽

Cervical Cancer Cell ◽

Cervical Cancer Cell Line ◽

Murine Xenograft Model ◽

Inhibition Of Tumor Growth

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Intratumoral Canine Distemper Virus Infection Inhibits Tumor Growth by Modulation of the Tumor Microenvironment in a Murine Xenograft Model of Canine Histiocytic Sarcoma

International Journal of Molecular Sciences ◽

10.3390/ijms22073578 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3578

Author(s):

Federico Armando ◽

Adnan Fayyad ◽

Stefanie Arms ◽

Yvonne Barthel ◽

Dirk Schaudien ◽

...

Keyword(s):

Tumor Microenvironment ◽

Virus Infection ◽

Tumor Growth ◽

Canine Distemper Virus ◽

Xenograft Model ◽

Histiocytic Sarcoma ◽

Oncolytic Viruses ◽

Canine Distemper ◽

Murine Xenograft Model ◽

The Impact

Histiocytic sarcomas refer to highly aggressive tumors with a poor prognosis that respond poorly to conventional treatment approaches. Oncolytic viruses, which have gained significant traction as a cancer therapy in recent decades, represent a promising option for treating histiocytic sarcomas through their replication and/or by modulating the tumor microenvironment. The live attenuated canine distemper virus (CDV) vaccine strain Onderstepoort represents an attractive candidate for oncolytic viral therapy. In the present study, oncolytic virotherapy with CDV was used to investigate the impact of this virus infection on tumor cell growth through direct oncolytic effects or by virus-mediated modulation of the tumor microenvironment with special emphasis on angiogenesis, expression of selected MMPs and TIMP-1 and tumor-associated macrophages in a murine xenograft model of canine histiocytic sarcoma. Treatment of mice with xenotransplanted canine histiocytic sarcomas using CDV induced overt retardation in tumor progression accompanied by necrosis of neoplastic cells, increased numbers of intratumoral macrophages, reduced angiogenesis and modulation of the expression of MMPs and TIMP-1. The present data suggest that CDV inhibits tumor growth in a multifactorial way, including direct cell lysis and reduction of angiogenesis and modulation of MMPs and their inhibitor TIMP-1, providing further support for the concept of its role in oncolytic therapies.

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Establishment of a Patient-Derived Xenograft Model of Colorectal Cancer in CIEA NOG Mice and Exploring Smartfish Liquid Diet as a Source of Omega-3 Fatty Acids

Biomedicines ◽

10.3390/biomedicines9030282 ◽

2021 ◽

Vol 9 (3) ◽

pp. 282

Author(s):

Helle Samdal ◽

Lene C Olsen ◽

Knut S Grøn ◽

Elin S Røyset ◽

Therese S Høiem ◽

...

Keyword(s):

Colorectal Cancer ◽

Fatty Acid ◽

Fish Oil ◽

Fatty Acid Content ◽

Xenograft Model ◽

Liquid Diet ◽

Lymphoid Cells ◽

Omega 3 ◽

Pdx Model ◽

Nog Mice

Cancer patient-derived xenografts (PDXs) better preserve tumor characteristics and microenvironment than traditional cancer cell line derived xenografts and are becoming a valuable model in translational cancer research and personalized medicine. We have established a PDX model for colorectal cancer (CRC) in CIEA NOG mice with a 50% engraftment rate. Tumor fragments from patients with CRC (n = 5) were engrafted in four mice per tumor (n = 20). Mice with established PDXs received a liquid diet enriched with fish oil or placebo, and fatty acid profiling was performed to measure fatty acid content in whole blood. Moreover, a biobank consisting of tissue and blood samples from patients was established. Histology, immunohistochemistry and in situ hybridization procedures were used for staining of tumor and xenograft tissue slides. Results demonstrate that key histological characteristics of the patients’ tumors were retained in the established PDXs, and the liquid diets were consumed as intended by the mice. Some of the older mice developed lymphomas that originated from human Ki67+, CD45+, and EBV+ lymphoid cells. We present a detailed description of the process and methodology, as well as possible issues that may arise, to refine the method and improve PDX engraftment rate for future studies. The established PDX model for CRC can be used for exploring different cancer treatment regimes, and liquid diets enriched with fish oil may be successfully delivered to the mice through the drinking flasks.

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Innate and cytokine-driven signals, rather than microbial antigens, dominate in natural killer T cell activation during microbial infection

Journal of Experimental Medicine ◽

10.1084/jem.20102555 ◽

2011 ◽

Vol 208 (6) ◽

pp. 1163-1177 ◽

Cited By ~ 186

Author(s):

Manfred Brigl ◽

Raju V.V. Tatituri ◽

Gerald F.M. Watts ◽

Veemal Bhowruth ◽

Elizabeth A. Leadbetter ◽

...

Keyword(s):

T Cells ◽

Natural Killer ◽

T Cell Activation ◽

Cell Activation ◽

Constitutive Expression ◽

Microbial Pathogens ◽

Inkt Cell ◽

Microbial Infection ◽

Inkt Cells ◽

Natural Killer T

Invariant natural killer T cells (iNKT cells) are critical for host defense against a variety of microbial pathogens. However, the central question of how iNKT cells are activated by microbes has not been fully explained. The example of adaptive MHC-restricted T cells, studies using synthetic pharmacological α-galactosylceramides, and the recent discovery of microbial iNKT cell ligands have all suggested that recognition of foreign lipid antigens is the main driver for iNKT cell activation during infection. However, when we compared the role of microbial antigens versus innate cytokine-driven mechanisms, we found that iNKT cell interferon-γ production after in vitro stimulation or infection with diverse bacteria overwhelmingly depended on toll-like receptor–driven IL-12. Importantly, activation of iNKT cells in vivo during infection with Sphingomonas yanoikuyae or Streptococcus pneumoniae, pathogens which are known to express iNKT cell antigens and which require iNKT cells for effective protection, also predominantly depended on IL-12. Constitutive expression of high levels of IL-12 receptor by iNKT cells enabled instant IL-12–induced STAT4 activation, demonstrating that among T cells, iNKT cells are uniquely equipped for immediate, cytokine-driven activation. These findings reveal that innate and cytokine-driven signals, rather than cognate microbial antigen, dominate in iNKT cell activation during microbial infections.

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CD34+ Cord Blood DC-induced Antitumor Lymphoid Cells Have Efficacy in a Murine Xenograft Model of Human ALL

Journal of Immunotherapy ◽

10.1097/cji.0b013e31821b7230 ◽

2011 ◽

Vol 34 (4) ◽

pp. 362-371 ◽

Cited By ~ 2

Author(s):

Hannah Cullup ◽

Andy K.W. Hsu ◽

Andrew J. Kassianos ◽

Kylie McDonald ◽

Kristen J. Radford ◽

...

Keyword(s):

Cord Blood ◽

Xenograft Model ◽

Lymphoid Cells ◽

Murine Xenograft Model

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Glycyrol Alone or in Combination with Gefitinib Is Effective against Gefitinib-Resistant HCC827GR Lung Cancer Cells

Applied Sciences ◽

10.3390/app112210526 ◽

2021 ◽

Vol 11 (22) ◽

pp. 10526

Author(s):

Shuang Zhao ◽

Shangyun Lu ◽

Lihong Fan ◽

Hongbo Hu

Keyword(s):

Lung Cancer ◽

Cancer Cells ◽

Xenograft Model ◽

Lung Cancer Cells ◽

Clinical Utilization ◽

Murine Xenograft Model ◽

Coumarin Compounds ◽

Line Setting

Gefitinib has been clinically demonstrated to be effective in the first-line setting for patients with advanced EGFR-mutated non-small cell lung cancer (NSCLC). However, acquired therapeutic resistance to gefitinib almost unavoidably develops, posing a major hurdle for its clinical utilization. Our previous study showed that glycyrol (GC), a representative of coumarin compounds isolated from the medicinal plant licorice, was effective against A549 lung cancer cells in both cell culture and a murine xenograft model. In this follow-up study, we evaluated the effect of glycyrol against gefitinib-resistant NSCLC and its ability to overcome the resistance using gefitinib-resistant HCC827GR cells. Results showed that glycyrol was effective against HCC827GR cells in both in vitro and in vivo. Moreover, glycyrol was able to significantly increase the sensitivity of HCC827GR cells to gefitinib, mechanistically associated with inactivating MET, which is a known important contributor to the resistance of HCC827GR cells to gefitinib. The findings of the present study suggest that glycyrol holds potential to be developed as a novel agent against gefitinib-resistant NSCLC.

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Exogenous Activation of Invariant Natural Killer T Cells by α-Galactosylceramide Reduces Pneumococcal Outgrowth and Dissemination Postinfluenza

mBio ◽

10.1128/mbio.01440-16 ◽

2016 ◽

Vol 7 (6) ◽

Cited By ~ 11

Author(s):

Adeline Barthelemy ◽

Stoyan Ivanov ◽

Maya Hassane ◽

Josette Fontaine ◽

Béatrice Heurtault ◽

...

Keyword(s):

Mouse Model ◽

Influenza A Virus ◽

Bacterial Pneumonia ◽

Influenza A ◽

Bacterial Infections ◽

Pneumococcal Infection ◽

Clinical Development ◽

Inkt Cell ◽

Inkt Cells ◽

Invariant Natural Killer

ABSTRACT Influenza A virus infection can predispose to potentially devastating secondary bacterial infections. Invariant natural killer T (iNKT) cells are unconventional, lipid-reactive T lymphocytes that exert potent immunostimulatory functions. Using a mouse model of postinfluenza invasive secondary pneumococcal infection, we sought to establish whether α-galactosylceramide (α-GalCer [a potent iNKT cell agonist that is currently in clinical development]) could limit bacterial superinfection. Our results highlighted the presence of a critical time window during which α-GalCer treatment can trigger iNKT cell activation and influence resistance to postinfluenza secondary pneumococcal infection. Intranasal treatment with α-GalCer during the acute phase (on day 7) of influenza virus H3N2 and H1N1 infection failed to activate (gamma interferon [IFN-γ] and interleukin-17A [IL-17A]) iNKT cells; this effect was associated with a strongly reduced number of conventional CD103 + dendritic cells in the respiratory tract. In contrast, α-GalCer treatment during the early phase (on day 4) or during the resolution phase (day 14) of influenza was associated with lower pneumococcal outgrowth and dissemination. Less intense viral-bacterial pneumonia and a lower morbidity rate were observed in superinfected mice treated with both α-GalCer (day 14) and the corticosteroid dexamethasone. Our results open the way to alternative (nonantiviral/nonantibiotic) iNKT-cell-based approaches for limiting postinfluenza secondary bacterial infections. IMPORTANCE Despite the application of vaccination programs and antiviral drugs, influenza A virus (IAV) infection is responsible for widespread morbidity and mortality (500,000 deaths/year). Influenza infections can also result in sporadic pandemics that can be devastating: the 1918 pandemic led to the death of 50 million people. Severe bacterial infections are commonly associated with influenza and are significant contributors to the excess morbidity and mortality of influenza. Today’s treatments of secondary bacterial (pneumococcal) infections are still not effective enough, and antibiotic resistance is a major issue. Hence, there is an urgent need for novel therapies. In the present study, we set out to evaluate the efficacy of α-galactosylceramide (α-GalCer)—a potent agonist of invariant NKT cells that is currently in clinical development—in a mouse model of postinfluenza, highly invasive pneumococcal pneumonia. Our data indicate that treatment with α-GalCer reduces susceptibility to superinfections and, when combined with the corticosteroid dexamethasone, reduces viral-bacterial pneumonia.

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