564 Potency-reduced and extended half-life IL12 heterodimeric Fc-fusions exhibit strong anti-tumor activity with potentially improved therapeutic index compared to native IL12 agents

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A598-A598
Author(s):  
Matthew Bernett ◽  
Rajat Varma ◽  
Ke Liu ◽  
Christine Bonzon ◽  
Rumana Rashid ◽  
...  
Keyword(s):  
Nk Cells ◽  
Half Life ◽  
Single Agent ◽  
Therapeutic Index ◽  
Immune Checkpoints ◽  
Therapeutic Window ◽  
Interleukin 12 ◽  
Severe Toxicity ◽  
Anti Tumor Activity

BackgroundInterleukin-12 (IL12) is a proinflammatory cytokine produced by activated antigen-presenting cells that induces differentiation of Th1 cells and increased proliferation and cytotoxicity of T and NK cells. Stimulation of these cells by IL12 leads to production of high levels of IFNγ. These immune-stimulating aspects of IL12 may help to establish an inflammatory tumor microenvironment critical for anti-tumor responses. Preclinical studies in mice revealed that native IL12 can dramatically shrink syngeneic tumors, however clinical studies in humans resulted in severe toxicity and a small therapeutic window, limiting response rates. Prior work at Xencor demonstrated that reduced-potency IL15/IL15Rα-Fc fusion proteins exhibited superior pharmacokinetics, pharmacodynamics, and safety in non-human primates through reduction of receptor-mediated clearance. Applying similar principles to IL12, we created IL12 heterodimeric Fc-fusions (IL12-Fc) with reduced potency to improve tolerability, slow receptor-mediated clearance, and extend half-life.MethodsIL12 is a heterodimeric protein consisting of two subunits, so we engineered IL12-Fc fusions by fusing the IL12p35 subunit to one side of a heterodimeric (and inactive) Fc domain, and the IL12p40 subunit to the other side. These Fc-fusions were tuned for optimal activity by introducing amino acid substitutions at putative receptor-interface positions and screening for reductions of in vitro potency. In vitro activity was assessed on human PBMCs by measuring signaling in a STAT4 phosphorylation assay and IFNγ production in a mixed-lymphocyte reaction (MLR). In vivo anti-tumor activity was assessed by engrafting MCF-7 cells into PBMC engrafted NSG MHC class I and II double-knockout mice and by measuring tumor volume, lymphocyte activation/proliferation, and IFNγ production over time.ResultsIL12-Fc were produced with good yield and purity. An IL12-Fc potency series was created, and variants had up to a 10,000-fold reduction in STAT4 signaling potency and IFNγ production in an MLR assay compared to native IL12-Fc. Anti-tumor activity in the huPBMC-MCF7 model was achieved with potency-reduced IL12-Fc as a single-agent and in combination with anti-PD1, with weaker variants maintaining anti-tumor activity at higher dose levels. Analysis of peripheral lymphocytes indicated increased numbers of T and NK cells as well as activation of CD8+ T cells, as evidenced by upregulation of CD25. Increased expression of immune checkpoints including PD1 was also observed. Analysis of serum indicated up to 200-fold increases in IFNγ levels.ConclusionsCombined, these data indicate that potency-reduced IL12-Fc retain strong anti-tumor activity, while potentially overcoming safety and tolerability issues related to small therapeutic index associated with recombinant native IL12 or IL12-Fc agents.

scholarly journals 707 IL12 Fc-fusions engineered for reduced potency and extended half-life exhibit strong anti-tumor activity and improved therapeutic index compared to wild-type IL12 agents

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A736-A736
Author(s):  
Matthew Bernett ◽  
Ke Liu ◽  
Christine Bonzon ◽  
Michael Hackett ◽  
Katrina Bykova ◽  
...  
Keyword(s):  
Nk Cells ◽  
Therapeutic Index ◽  
Immune Checkpoints ◽  
Therapeutic Window ◽  
Interleukin 12 ◽  
Severe Toxicity ◽  
Wild Type ◽  
Anti Tumor Activity

BackgroundInterleukin-12 (IL12) is a proinflammatory cytokine that induces differentiation of Th1 cells and increased cytotoxicity of T and NK cells. Stimulation by IL12 leads to production of IFNγ and an inflammatory tumor microenvironment critical for anti-tumor responses. Studies in mice revealed IL12 can dramatically shrink syngeneic tumors, however human clinical studies resulted in severe toxicity and a small therapeutic window, limiting response rates. Prior work at Xencor demonstrated that reduced-potency IL15/IL15Rα-Fc fusion proteins exhibited superior therapeutic index (TI) in non-human primates (NHP) by reducing receptor-mediated clearance. Applying similar principles to IL12, we created IL12 heterodimeric Fc-fusions (IL12-Fc) with reduced potency to improve TI.MethodsIL12 is a heterodimer of two subunits, so we engineered IL12-Fc fusions by fusing the IL12p35 subunit to one side of a heterodimeric (and inactive) Fc domain, and IL12p40 to the other side. These Fc-fusions were tuned for optimal activity by introducing amino acid substitutions at putative receptor-interface positions and screening for reductions of in vitro potency. In vitro activity was assessed on human PBMCs by measuring signaling in a STAT4 phosphorylation assay and IFNγ production in a mixed-lymphocyte reaction (MLR). In vivo anti-tumor activity of human IL12-Fc was assessed in huPBMC-NSG-DKO and huCD34+ MCF7 xenograft models. Surrogate mouse potency-reduced IL12-Fc were evaluated in syngeneic tumor models. Tolerability and pharmacodynamic activity were assessed in NHP.ResultsAn IL12-Fc potency series was created, and variants had up to a 10,000-fold reduction in STAT4 signaling and IFNγ production in an MLR assay compared to wild-type IL12-Fc. Anti-tumor activity was achieved with potency-reduced IL12-Fc as single-agents and in combination with anti-PD1, with weaker variants maintaining anti-tumor activity at higher dose levels. Analysis of peripheral lymphocytes indicated increased numbers of T and NK cells as well as activation of CD8+ T cells. Increased expression of immune checkpoints including PD1 was also observed. Analysis of serum indicated up to 200-fold increases in IFNγ levels. Surrogate potency-reduced IL12-Fc had improved tolerability and greater selectivity of IFNγ production in tumors compared to spleen and less production of IL10 compared to wild-type IL12-Fc. In NHP, potency-reduced IL12-Fc had superior exposure with slower, more sustained accumulation of IFNγ and IP10, and a more gradual dose-dependent peak response, as well as more sustained margination of T and NK cells compared to wild-type IL12-Fc.ConclusionsPotency-reduced IL12-Fc retain strong anti-tumor activity, while potentially overcoming safety and tolerability issues related to narrow TI associated with wild-type IL12 or IL12-Fc agents.

scholarly journals 858 TILKine-2: a novel best-in-class tumor infiltrating lymphocyte (TIL) targeting engineered IL-2 with superior pre-clinical efficacy and safety for immunotherapy of cancer

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A899-A899
Author(s):  
Sreerupa Challa ◽  
Jonathan Carnino ◽  
Andrea Umana ◽  
Yuesheng Li ◽  
Jing Xu ◽  
...  
Keyword(s):  
Nk Cells ◽  
Half Life ◽  
Intracellular Signaling ◽  
Functional Characterization ◽  
Interleukin 2 ◽  
Systemic Toxicity ◽  
Fine Tuning ◽  
Therapeutic Window ◽  
High Dose ◽  
Severe Toxicity

BackgroundHigh-dose Interleukin-2 is the earliest FDA-approved immunotherapy for metastatic melanoma and renal cell carcinoma. Unfortunately, its application is limited due to its short half-life and severe toxicity at the therapeutic dose. To limit systemic toxicity, tumor-targeting antibody-based delivery of IL-2 has been developed, however with poor outcomes. We here deploy a novel strategy to deliver IL-2 to the tumor microenvironment by binding to Tumor-Infiltrating Lymphocytes (TILs). TILKine-2 is a recombinant bifunctional protein comprised of an antibody directed against TILs (TILAb) fused to an engineered IL-2, which simultaneously revives and expands antigen-primed exhausted T cells. The IL-2 portion of TILKine-2 was engineered to have improved tolerability, slower receptor-mediated clearance, and prolonged half-life.MethodsTarget binding of TILKine-2 was evaluated by cell-free and cell-based methods. In vitro functional characterization was performed using human peripheral blood mononuclear cells (PBMCs). Pharmacokinetics (PK), pharmacodynamics (PD), and anti-tumor activity of murine TILKine-2 surrogate (TILKine-2s) were evaluated in various syngeneic models. The safety and immune cell activation of TILKine-2 were assessed in non-human primates (NHPs).ResultsStructure-based design and activity-guided fine-tuning resulted in an optimized IL-2 variant that was fused to TILAb to generate TILKine-2. TILKine-2 demonstrated TIL-target antigen binding and blocking activity with sub-nM potency. TILKine-2 has a binding activity abolished to IL-2Rα and fine-tuned to IL-2Rβγ. In PBMCs, TILKine-2 potently induced intracellular signaling and cell proliferation in IL-2Rβγ dominant effector CD8+T and NK cells along with IFN-γ secretion. In vivo, TILKine-2 displayed significantly prolonged half-life with sustained proliferation, expansion, and Granzyme B expression on CD8+T and NK cells. Notably, the effects were more pronounced in the tumor than periphery, leading to massive immune hot tumors. Consequently, TILKine-2s exhibited robust anti-tumor primary and memory response in both cold and hot tumor models (MC38, CT26, B16F10, PAN02). Furthermore, TILKine-2s demonstrated superior and synergistic anti-tumor efficacy compared to TILAb alone, engineered IL-2 alone, or their combination, with 100% tumor regression resulting in ~80% tumor free mice in MC38 and Pan02 models. In NHPs, TILKine-2 preferentially induced memory CD8+T, total CD8+T, and NK cell expansion. TILKine-2 was safe and well-tolerated in NHPs with no notable changes in body weight, temperature, clinical pathology, or signs of vascular leakage after repeated dosing.ConclusionsBy targeting TILs, TILKine-2 demonstrated robust anti-tumor efficacy by preferentially inducing proliferation, expansion, and activation of intra-tumoral lymphocytes while reducing systemic toxicity and improving therapeutic window. In conclusion, TILKine-2 is a promising therapeutic agent for clinical development.Ethics ApprovalFor mouse studies, the practices and procedures used were reviewed and approved by Brandeis University IACUC committee (Protocol #22001). For monkey studies, the practices and procedures used were in accordance with the safety and Quality Assurance guidelines set out in the Guideline for Experiments document of Kunming Biomed International (KBI--01-GEv2.0).

Fibronectin maintains survival of mouse natural killer (NK) cells via CD11b/Src/β-catenin pathway

Blood ◽  
2009 ◽  
Vol 114 (19) ◽  
pp. 4081-4088 ◽  
Author(s):  
Ting Zhang ◽  
Shuxun Liu ◽  
Pengyuan Yang ◽  
Chaofeng Han ◽  
Jianli Wang ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Nuclear Translocation ◽  
Nk Cell ◽  
Ex Vivo ◽  
Interleukin 12 ◽  
Erk Phosphorylation ◽  
Extracellular Signal ◽  
B Cell Leukemia

Abstract Tissue microenvironment and stroma-derived extracellular matrix (ECM) molecules play important roles in the survival and differentiation of cells. Mouse natural killer (NK) cells usually die within 24 hours once isolated ex vivo. Exogenous cytokines such as interleukin-12 (IL-12) and IL-15 are required to maintain the survival and activity of mouse NK cells cultured in vitro. Whether and how ECM molecules such as fibronectin can support the survival of NK cells remain unknown. We demonstrate that fibronectin, just like IL-15, can maintain survival of mouse NK cells in vitro. Furthermore, we show that fibronectin binds to the CD11b on NK cells, and then CD11b recruits and activates Src. Src can directly interact with β-catenin and trigger nuclear translocation of β-catenin. The activation of β-catenin promotes extracellular signal-related kinase (ERK) phosphorylation, resulting in the increased expression of antiapoptotic protein B-cell leukemia 2 (Bcl-2), which may contribute to the maintenance of NK-cell survival. Consistently, fibronectin cannot maintain the survival of CD11b− NK cells and β-catenin–deficient NK cells in vitro, and the number of NK cells is dramatically decreased in the β-catenin–deficient mice. Therefore, fibronectin can maintain survival of mouse NK cells by activating ERK and up-regulating Bcl-2 expression via CD11b/Src/β-catenin pathway.

scholarly journals Elacestrant (RAD1901) exhibits anti-tumor activity in multiple ER+ breast cancer models resistant to CDK4/6 inhibitors

2019 ◽  
Vol 21 (1) ◽  
Author(s):  
Hitisha K. Patel ◽  
Nianjun Tao ◽  
Kyung-Min Lee ◽  
Mariela Huerta ◽  
Heike Arlt ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Estrogen Receptor ◽  
De Novo ◽  
Single Agent ◽  
Metastatic Breast ◽  
Cancer Resistance ◽  
Treatment Paradigm ◽  
Anti Tumor Activity

Abstract Background Addition of CDK4/6 inhibitors (CDK4/6i) to endocrine therapy significantly increased progression-free survival, leading to their approval and incorporation into the metastatic breast cancer treatment paradigm. With these inhibitors being routinely used for patients with advanced estrogen receptor-positive (ER+) breast cancer, resistance to these agents and its impact on subsequent therapy needs to be understood. Considering the central role of ER in driving the growth of ER+ breast cancers, and thus endocrine agents being a mainstay in the treatment paradigm, the effects of prior CDK4/6i exposure on ER signaling and the relevance of ER-targeted therapy are important to investigate. The objective of this study was to evaluate the anti-tumor activity of elacestrant, a novel oral selective estrogen receptor degrader (SERD), in preclinical models of CDK4/6i resistance. Methods Elacestrant was evaluated as a single agent, and in combination with alpelisib or everolimus, in multiple in vitro models and patient-derived xenografts that represent acquired and “de novo” CDK4/6i resistance. Results Elacestrant demonstrated growth inhibition in cells resistant to all three approved CDK4/6i (palbociclib, abemaciclib, ribociclib) in both ESR1 wild-type and mutant backgrounds. Furthermore, we demonstrated that elacestrant, as a single agent and in combination, inhibited growth of patient-derived xenografts that have been derived from a patient previously treated with a CDK4/6i or exhibit de novo resistance to CDK4/6i. While the resistant lines demonstrate distinct alterations in cell cycle modulators, this did not affect elacestrant’s anti-tumor activity. In fact, we observe that elacestrant downregulates several key cell cycle players and halts cell cycle progression in vitro and in vivo. Conclusions We demonstrate that breast cancer tumor cells continue to rely on ER signaling to drive tumor growth despite exposure to CDK4/6i inhibitors. Importantly, elacestrant can inhibit this ER-dependent growth despite previously reported mechanisms of CDK4/6i resistance observed such as Rb loss, CDK6 overexpression, upregulated cyclinE1 and E2F1, among others. These data provide a scientific rationale for the evaluation of elacestrant in a post-CDK4/6i patient population. Additionally, elacestrant may also serve as an endocrine backbone for rational combinations to combat resistance.

A phase II study of combined methotrexate and teniposide infusions prior to reinduction therapy in relapsed childhood acute lymphoblastic leukemia: a Pediatric Oncology Group study.

1991 ◽  
Vol 9 (1) ◽  
pp. 139-144 ◽  
Author(s):  
J Ochs ◽  
J Rodman ◽  
M Abromowitch ◽  
R Kavanagh ◽  
M Harris ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Phase Ii ◽  
Phase Ii Study ◽  
Lymphoblastic Leukemia ◽  
Therapeutic Index ◽  
Small Sample ◽  
Therapeutic Window ◽  
Infusion Schedule ◽  
Small Sample Sizes

Teniposide (VM-26) can increase intracellular methotrexate (MTX) and its polyglutamate derivatives in vitro and thus has the potential to improve the therapeutic index of regimens containing MTX. In this phase II study, children and adolescents with acute lymphoblastic leukemia (ALL) in first or second marrow relapse were randomly assigned to receive either simultaneous (n = 11) or sequential (n = 12) continuous infusions of MTX and VM-26 prior to reinduction. Infusions of VM-26 were begun 12 hours after completion of MTX infusion in the sequential group. Dosages were individually adjusted to maintain plasma concentration levels of 10 microns for MTX and 15 microns for VM-26; total infusion times were 24 and 72 hours, respectively. Significant toxicity in the first six patients who received the scheduled 72-hour VM-26 infusion (including one drug-related death) prompted a 50% reduction in infusion duration. The reduced dose was associated with similar but more manageable toxicity. Examination of bone marrow aspirates 10 days after therapy was begun showed one complete and two partial marrow remissions; a fourth patient who had an aplastic marrow on day 10 received no further chemotherapy and had a complete remission (CR) documented on day 31. There was no obvious clinical advantage associated with either infusion schedule, although small sample sizes preclude definitive conclusions. The 17% response rate to the MTX/VM-26 therapeutic window in patients with refractory disease suggests the need for further investigation to evaluate alternative schedules and concomitant therapy for this drug combination.

Molecular modification of a recombinant anti-CD3ε-directed immunotoxin by inducing terminal cysteine bridging enhances anti-GVHD efficacy and reduces organ toxicity in a lethal murine model

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 1157-1165 ◽  
Author(s):  
Daniel A. Vallera ◽  
David W. Kuroki ◽  
Angela Panoskaltsis-Mortari ◽  
Donald J. Buchsbaum ◽  
Buck E. Rogers ◽  
...  
Keyword(s):  
Therapeutic Index ◽  
Maximum Tolerated Dose ◽  
Cysteine Residue ◽  
Genetically Engineered ◽  
Therapeutic Window ◽  
Single Chain ◽  
Molecular Modification ◽  
Organ Toxicity

Abstract Immunotoxin (IT) therapy shows potential for selectively eliminating GVHD-causing T cells in vivo, but the field has been hampered by toxicity. Previously, we showed that a genetically engineered IT consisting of a single-chain protein, including the anti-CD3sFv spliced to a portion of diphtheria-toxin (DT390) has anti-GVHD effects, but pronounced organ toxicity common to this class of agent. A recombinant DT390 anti-CD3sFv protein previously shown to have anti-GVHD activity was modified to reduce its filtration into kidney by genetically inserting a cysteine residue downstream of the sFv moiety at the c-terminus of the protein. This modification produced an intermolecular disulfide bridge, resulting in a bivalent, rather than a monovalent IT, termed SS2, that selectively inhibited T-cell proliferation in vitro. Although monomer and SS2 were similar in in vitro activity, SS2 had a superior therapeutic index in vivo with at least 8-fold more being tolerated with reduced kidney toxicity. Most importantly, in a lethal model of GVHD, 40 μg SS2 given for 1 day, protected 100% of the mice from lethal GVHD for 3 months, whereas the maximum tolerated dose (MTD) of monomer protected only 33%. To our knowledge, this is the first time disulfide bonded ITs have been created in this way and this simple molecular modification may address several problems in the IT field because it (1) markedly increased efficacy curing mice of GVHD after a single daily treatment, (2) markedly decreased organ toxicity, (3) increased the tolerated dosage, and (4) created a therapeutic window where none existed before.

Contribution of Natural Killer Cells to Inhibition of Angiogenesis by Interleukin-12

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1612-1621 ◽  
Author(s):  
Lei Yao ◽  
Cecilia Sgadari ◽  
Keizo Furuke ◽  
Eda T. Bloom ◽  
Julie Teruya-Feldstein ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Function ◽  
Nk Cell ◽  
Primary Cultures ◽  
Interleukin 12 ◽  
Ifn Γ

Abstract Interleukin-12 (IL-12) inhibits angiogenesis in vivo by inducing interferon-γ (IFN-γ) and other downstream mediators. Here, we report that neutralization of natural killer (NK) cell function with antibodies to either asialo GM1 or NK 1.1 reversed IL-12 inhibition of basic fibroblast growth factor (bFGF)-induced angiogenesis in athymic mice. By immunohistochemistry, those sites where bFGF-induced neovascularization was inhibited by IL-12 displayed accumulation of NK cells and the presence of IP-10–positive cells. Based on expression of the cytolytic mediators perforin and granzyme B, the NK cells were locally activated. Experimental Burkitt lymphomas treated locally with IL-12 displayed tumor tissue necrosis, vascular damage, and NK-cell infiltration surrounding small vessels. After activation in vitro with IL-12, NK cells from nude mice became strongly cytotoxic for primary cultures of syngeneic aortic endothelial cells. Cytotoxicity was neutralized by antibodies to IFN-γ. These results document that NK cells are required mediators of angiogenesis inhibition by IL-12, and provide evidence that NK-cell cytotoxicity of endothelial cells is a potential mechanism by which IL-12 can suppress neovascularization.

Natural-killer cells and dendritic cells: “l'union fait la force”

Blood ◽  
2005 ◽  
Vol 106 (7) ◽  
pp. 2252-2258 ◽  
Author(s):  
Thierry Walzer ◽  
Marc Dalod ◽  
Scott H. Robbins ◽  
Laurence Zitvogel ◽  
Eric Vivier
Keyword(s):  
Dendritic Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Activation ◽  
Nk Cell ◽  
Interleukin 12 ◽  
Cell Contact ◽  
Ifn Γ

AbstractSeveral recent publications have focused on the newly described interactions between natural-killer (NK) cells and dendritic cells (DCs). Activated NK cells induce DC maturation either directly or in synergy with suboptimal levels of microbial signals. Immature DCs appear susceptible to autologous NK-cell-mediated cytolysis while mature DCs are protected. NK-cell-induced DC activation is dependent on both tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ) secretion and a cell-cell contact involving NKp30. In vitro, interleukin-12 (IL-12)/IL-18, IL-15, and IFN-α/β production by activated DCs enhance, in turn, NK-cell IFN-γ production, proliferation, and cytotoxic potential, respectively. In vivo, NK-cell/DC interactions may occur in lymphoid organs as well as in nonlymphoid tissues, and their consequences are multiple. By inducing DC activation, NK-cell activation induced by tumor cells can indirectly promote antitumoral T-cell responses. Reciprocally, DCs activated through Toll-like receptors (TLRs) induce potent NK-cell activation in antiviral responses. Thus, DCs and NK cells are equipped with complementary sets of receptors that allow the recognition of various pathogenic agents, emphasizing the role of NK-cell/DC crosstalk in the coordination of innate and adaptive immune responses.

Molecular modification of a recombinant anti-CD3ε-directed immunotoxin by inducing terminal cysteine bridging enhances anti-GVHD efficacy and reduces organ toxicity in a lethal murine model

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 1157-1165 ◽  
Author(s):  
Daniel A. Vallera ◽  
David W. Kuroki ◽  
Angela Panoskaltsis-Mortari ◽  
Donald J. Buchsbaum ◽  
Buck E. Rogers ◽  
...  
Keyword(s):  
Therapeutic Index ◽  
Maximum Tolerated Dose ◽  
Cysteine Residue ◽  
Genetically Engineered ◽  
Therapeutic Window ◽  
Single Chain ◽  
Molecular Modification ◽  
Organ Toxicity

Immunotoxin (IT) therapy shows potential for selectively eliminating GVHD-causing T cells in vivo, but the field has been hampered by toxicity. Previously, we showed that a genetically engineered IT consisting of a single-chain protein, including the anti-CD3sFv spliced to a portion of diphtheria-toxin (DT390) has anti-GVHD effects, but pronounced organ toxicity common to this class of agent. A recombinant DT390 anti-CD3sFv protein previously shown to have anti-GVHD activity was modified to reduce its filtration into kidney by genetically inserting a cysteine residue downstream of the sFv moiety at the c-terminus of the protein. This modification produced an intermolecular disulfide bridge, resulting in a bivalent, rather than a monovalent IT, termed SS2, that selectively inhibited T-cell proliferation in vitro. Although monomer and SS2 were similar in in vitro activity, SS2 had a superior therapeutic index in vivo with at least 8-fold more being tolerated with reduced kidney toxicity. Most importantly, in a lethal model of GVHD, 40 μg SS2 given for 1 day, protected 100% of the mice from lethal GVHD for 3 months, whereas the maximum tolerated dose (MTD) of monomer protected only 33%. To our knowledge, this is the first time disulfide bonded ITs have been created in this way and this simple molecular modification may address several problems in the IT field because it (1) markedly increased efficacy curing mice of GVHD after a single daily treatment, (2) markedly decreased organ toxicity, (3) increased the tolerated dosage, and (4) created a therapeutic window where none existed before.

scholarly journals The Protozoan Neospora caninum Directly Triggers Bovine NK Cells To Produce Gamma Interferon and To Kill Infected Fibroblasts

2006 ◽  
Vol 74 (2) ◽  
pp. 953-960 ◽  
Author(s):  
Preben Boysen ◽  
Siv Klevar ◽  
Ingrid Olsen ◽  
Anne K. Storset
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Neospora Caninum ◽  
Gamma Interferon ◽  
Functional Study ◽  
Interleukin 12 ◽  
Protozoan Infection ◽  
Autologous Fibroblasts ◽  
Ifn Γ

ABSTRACT Natural killer (NK) cells are considered to be key players in the early innate responses to protozoan infections, primarily indirectly by producing gamma interferon (IFN-γ) in response to cytokines, like interleukin 12 (IL-12). We demonstrate that live, as well as heat-inactivated, tachyzoites of Neospora caninum, a Toxoplasma-like protozoan, directly trigger production of IFN-γ from purified, IL-2-activated bovine NK cells. This response occurred independently of IL-12 but was increased by the addition of the cytokine. A similar IFN-γ response was measured in cocultures of NK cells and N. caninum-infected autologous fibroblasts. However, no NK cell-derived IFN-γ response was detected when cells were cultured with soluble antigens from the organism, indicating that intact tachyzoites or nonsoluble components are necessary for NK cell triggering. Furthermore, N. caninum-infected autologous fibroblasts had increased susceptibility to NK cell cytotoxicity compared to uninfected fibroblasts. This cytotoxicity was largely mediated by a perforin-mediated mechanism. The activating receptor NKp46 was involved in cytotoxicity against fibroblasts but could not explain the increased cytotoxicity against infected targets. Interestingly, N. caninum tachyzoites were able to infect cultured NK cells, in which tachyzoites proliferated inside parasitophorous vacuoles. Together, these findings underscore the role of NK cells as primary responders during a protozoan infection, describe intracellular protozoan infection of NK cells in vitro for the first time, and represent the first functional study of purified bovine NK cells in response to infection.

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