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1.
Immunity ; 48(4): 760-772.e4, 2018 04 17.
Article in English | MEDLINE | ID: mdl-29625893

ABSTRACT

Cerebral malaria is a deadly complication of Plasmodium infection and involves blood brain barrier (BBB) disruption following infiltration of white blood cells. During experimental cerebral malaria (ECM), mice inoculated with Plasmodium berghei ANKA-infected red blood cells develop a fatal CM-like disease caused by CD8+ T cell-mediated pathology. We found that treatment with interleukin-15 complex (IL-15C) prevented ECM, whereas IL-2C treatment had no effect. IL-15C-expanded natural killer (NK) cells were necessary and sufficient for protection against ECM. IL-15C treatment also decreased CD8+ T cell activation in the brain and prevented BBB breakdown without influencing parasite load. IL-15C induced NK cells to express IL-10, which was required for IL-15C-mediated protection against ECM. Finally, we show that ALT-803, a modified human IL-15C, mediates similar induction of IL-10 in NK cells and protection against ECM. These data identify a regulatory role for cytokine-stimulated NK cells in the prevention of a pathogenic immune response.


Subject(s)
Interleukin-10/immunology , Interleukin-15/immunology , Killer Cells, Natural/immunology , Malaria, Cerebral/immunology , Plasmodium berghei/immunology , Proteins/pharmacology , Animals , Blood-Brain Barrier/pathology , Brain/immunology , Brain/pathology , CD8-Positive T-Lymphocytes/immunology , Interleukin-10/biosynthesis , Lymphocyte Activation/immunology , Malaria, Cerebral/microbiology , Malaria, Cerebral/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Recombinant Fusion Proteins
2.
Mol Ther ; 30(3): 1171-1187, 2022 03 02.
Article in English | MEDLINE | ID: mdl-35051615

ABSTRACT

Therapy induced senescence (TIS) in tumors and TIS cancer cells secrete proinflammatory senescence-associated secretory phenotype (SASP) factors. SASP factors promote TIS cancer cells to re-enter the growth cycle with stemness characteristics, resulting in chemo-resistance and disease relapse. Herein, we show that the immunotherapeutic HCW9218, comprising transforming growth factor-ß (TGF-ß) receptor II and interleukin (IL)-15/IL-15 receptor α domains, enhances metabolic and cytotoxic activities of immune cells and reduces TIS tumor cells in vivo to improve the efficacy of docetaxel and gemcitabine plus nab-paclitaxel against B16F10 melanoma and SW1990 pancreatic tumors, respectively. Mechanistically, HCW9218 treatment reduces the immunosuppressive tumor microenvironment and enhances immune cell infiltration and cytotoxicity in the tumors to eliminate TIS cancer cells. Immuno-depletion analysis suggests that HCW9218-activated natural killer cells play a pivotal role in TIS cancer cell removal. HCW9218 treatment following docetaxel chemotherapy further enhances efficacy of tumor antigen-specific and anti-programmed death-ligand 1 (PD-L1) antibodies in B16F10 tumor-bearing mice. We also show that HCW9218 treatment decreases TIS cells and lowers SASP factors in off-target tissues caused by chemotherapy of tumor-bearing mice. Collectively, HCW9218 has the potential to significantly enhance anti-tumor efficacy of chemotherapy, therapeutic antibodies, and checkpoint blockade by eliminating TIS cancer cells while reducing TIS-mediated proinflammatory side effects in normal tissues.


Subject(s)
B7-H1 Antigen , Killer Cells, Natural , Animals , B7-H1 Antigen/metabolism , Cell Line, Tumor , Cellular Senescence , Docetaxel/metabolism , Docetaxel/pharmacology , Immunotherapy/methods , Killer Cells, Natural/metabolism , Mice , Tumor Microenvironment
3.
Mol Ther ; 29(10): 2949-2962, 2021 10 06.
Article in English | MEDLINE | ID: mdl-34091051

ABSTRACT

Advances in immunostimulatory and anti-immunosuppressive therapeutics have revolutionized cancer treatment. However, novel immunotherapeutics with these dual functions are not frequently reported. Here we describe the creation of a heterodimeric bifunctional fusion molecule, HCW9218, constructed using our soluble tissue factor (TF)-based scaffold technology. This complex comprises extracellular domains of the human transforming growth factor-ß (TGF-ß) receptor II and a human interleukin-15 (IL-15)/IL-15 receptor α complex. HCW9218 can be readily expressed in CHO cells and purified using antibody-based affinity chromatography in a large-scale manufacturing setting. HCW9218 potently activates mouse natural killer (NK) cells and CD8+ T cells in vitro and in vivo to enhance cell proliferation, metabolism, and antitumor cytotoxic activities. Similarly, human immune cells become activated with increased cytotoxicity following incubation with HCW9218. This fusion complex also exhibits TGF-ß neutralizing activity in vitro and sequesters plasma TGF-ß in vivo. In a syngeneic B16F10 melanoma model, HCW9218 displayed strong antitumor activity mediated by NK cells and CD8+ T cells and increased their infiltration into tumors. Repeat-dose subcutaneous administration of HCW9218 was well tolerated by mice, with a half-life sufficient to provide long-lasting biological activity. Thus, HCW9218 may serve as a novel therapeutic to simultaneously provide immunostimulation and lessen immunosuppression associated with tumors.


Subject(s)
CD8-Positive T-Lymphocytes/metabolism , Interleukin-15/genetics , Killer Cells, Natural/metabolism , Melanoma, Experimental/drug therapy , Receptor, Transforming Growth Factor-beta Type II/chemistry , Receptors, Interleukin-15/genetics , Recombinant Fusion Proteins/administration & dosage , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Female , Humans , Injections, Subcutaneous , Interleukin-15/metabolism , Melanoma, Experimental/immunology , Mice , Receptor, Transforming Growth Factor-beta Type II/genetics , Receptor, Transforming Growth Factor-beta Type II/metabolism , Receptors, Interleukin-15/metabolism , Recombinant Fusion Proteins/pharmacology , Transforming Growth Factor beta/blood , Transforming Growth Factor beta/metabolism , Xenograft Model Antitumor Assays
4.
Blood ; 131(23): 2515-2527, 2018 06 07.
Article in English | MEDLINE | ID: mdl-29463563

ABSTRACT

New therapies for patients with hematologic malignancies who relapse after allogeneic hematopoietic cell transplantation (allo-HCT) are needed. Interleukin 15 (IL-15) is a cytokine that stimulates CD8+ T-cell and natural killer (NK) cell antitumor responses, and we hypothesized this cytokine may augment antileukemia/antilymphoma immunity in vivo. To test this, we performed a first-in-human multicenter phase 1 trial of the IL-15 superagonist complex ALT-803 in patients who relapsed >60 days after allo-HCT. ALT-803 was administered to 33 patients via the IV or subcutaneous (SQ) routes once weekly for 4 doses (dose levels of 1, 3, 6, and 10 µg/kg). ALT-803 was well tolerated, and no dose-limiting toxicities or treatment-emergent graft-versus-host disease requiring systemic therapy was observed in this clinical setting. Adverse events following IV administration included constitutional symptoms temporally related to increased serum IL-6 and interferon-γ. To mitigate these effects, the SQ route was tested. SQ delivery resulted in self-limited injection site rashes infiltrated with lymphocytes without acute constitutional symptoms. Pharmacokinetic analysis revealed prolonged (>96 hour) serum concentrations following SQ, but not IV, injection. ALT-803 stimulated the activation, proliferation, and expansion of NK cells and CD8+ T cells without increasing regulatory T cells. Responses were observed in 19% of evaluable patients, including 1 complete remission lasting 7 months. Thus, ALT-803 is a safe, well-tolerated agent that significantly increased NK and CD8+ T cell numbers and function. This immunostimulatory IL-15 superagonist warrants further investigation to augment antitumor immunity alone and combined with other immunotherapies. This trial was registered at www.clinicaltrials.gov as #NCT01885897.


Subject(s)
Antineoplastic Agents/therapeutic use , Hematologic Neoplasms/therapy , Hematopoietic Stem Cell Transplantation , Interleukin-15/agonists , Neoplasm Recurrence, Local/drug therapy , Proteins/therapeutic use , Adult , Aged , Antineoplastic Agents/adverse effects , Antineoplastic Agents/pharmacokinetics , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Female , Hematologic Neoplasms/immunology , Humans , Interleukin-15/immunology , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Male , Middle Aged , Neoplasm Recurrence, Local/immunology , Proteins/adverse effects , Proteins/pharmacokinetics , Recombinant Fusion Proteins , Young Adult
5.
J Virol ; 92(3)2018 02 01.
Article in English | MEDLINE | ID: mdl-29118125

ABSTRACT

Developing biological interventions to control human immunodeficiency virus (HIV) replication in the absence of antiretroviral therapy (ART) could contribute to the development of a functional cure. As a potential alternative to ART, the interleukin-15 (IL-15) superagonist ALT-803 has been shown to boost the number and function of HIV-specific CD8+ T and NK cell populations in vitro Four simian immunodeficiency virus (SIV)-positive rhesus macaques, three of whom possessed major histocompatibility complex alleles associated with control of SIV and all of whom had received SIV vaccine vectors that had the potential to elicit CD8+ T cell responses, were given ALT-803 in three treatment cycles. The first and second cycles of treatment were separated by 2 weeks, while the third cycle was administered after a 29-week break. ALT-803 transiently elevated the total CD8+ effector and central memory T cell and NK cell populations in peripheral blood, while viral loads transiently decreased by ∼2 logs in all animals. Virus suppression was not sustained as T cells became less responsive to ALT-803 and waned in numbers. No effect on viral loads was observed in the second cycle of ALT-803, concurrent with downregulation of the IL-2/15 common γC and ß chain receptors on both CD8+ T cells and NK cells. Furthermore, populations of immunosuppressive T cells increased during the second cycle of ALT-803 treatment. During the third treatment cycle, responsiveness to ALT-803 was restored. CD8+ T cells and NK cells increased again 3- to 5-fold, and viral loads transiently decreased again by 1 to 2 logs.IMPORTANCE Overall, our data show that ALT-803 has the potential to be used as an immunomodulatory agent to elicit effective immune control of HIV/SIV replication. We identify mechanisms to explain why virus control is transient, so that this model can be used to define a clinically appropriate treatment regimen.


Subject(s)
Proteins/pharmacology , Simian Acquired Immunodeficiency Syndrome/drug therapy , Simian Immunodeficiency Virus/drug effects , Virus Replication/drug effects , Animals , Antibodies, Monoclonal/pharmacology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Cell Line , Disease Models, Animal , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Lymphocyte Activation , Macaca mulatta , Recombinant Fusion Proteins , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus/physiology , Viral Load
6.
Lancet Oncol ; 19(5): 694-704, 2018 05.
Article in English | MEDLINE | ID: mdl-29628312

ABSTRACT

BACKGROUND: Immunotherapy with PD-1 or PD-L1 blockade fails to induce a response in about 80% of patients with unselected non-small cell lung cancer (NSCLC), and many of those who do initially respond then develop resistance to treatment. Agonists that target the shared interleukin-2 (IL-2) and IL-15Rßγ pathway have induced complete and durable responses in some cancers, but no studies have been done to assess the safety or efficacy of these agonists in combination with anti-PD-1 immunotherapy. We aimed to define the safety, tolerability, and activity of this drug combination in patients with NSCLC. METHODS: In this non-randomised, open-label, phase 1b trial, we enrolled patients (aged ≥18 years) with previously treated histologically or cytologically confirmed stage IIIB or IV NSCLC from three academic hospitals in the USA. Key eligibility criteria included measurable disease, eligibility to receive anti-PD-1 immunotherapy, and an Eastern Cooperative Oncology Group performance status of 0 or 1. Patients received the anti-PD-1 monoclonal antibody nivolumab intravenously at 3 mg/kg (then 240 mg when US Food and Drug Administration [FDA]-approved dosing changed) every 14 days (either as new treatment or continued treatment at the time of disease progression) and the IL-15 superagonist ALT-803 subcutaneously once per week on weeks 1-5 of four 6-week cycles for 6 months. ALT-803 was administered at one of four escalating dose concentrations: 6, 10, 15, or 20 µg/kg. The primary endpoint was to define safety and tolerability and to establish a recommended phase 2 dose of ALT-803 in combination with nivolumab. Analyses were per-protocol and included any patients who received at least one dose of study treatment. This trial is registered with ClinicalTrials.gov, number NCT02523469; phase 2 enrolment of patients is ongoing. FINDINGS: Between Jan 18, 2016, and June 28, 2017, 23 patients were enrolled and 21 were treated at four dose levels of ALT-803 in combination with nivolumab. Two patients did not receive treatment because of the development of inter-current illness during enrolment, one patient due to leucopenia and one patient due to pulmonary dysfunction. No dose-limiting toxicities were recorded and the maximum tolerated dose was not reached. The most common adverse events were injection-site reactions (in 19 [90%] of 21 patients) and flu-like symptoms (15 [71%]). The most common grade 3 adverse events, occurring in two patients each, were lymphocytopenia and fatigue. A grade 3 myocardial infarction occurred in one patient. No grade 4 or 5 adverse events were recorded. The recommended phase 2 dose of ALT-803 is 20 µg/kg given once per week subcutaneously in combination with 240 mg intravenous nivolumab every 2 weeks. INTERPRETATION: ALT-803 in combination with nivolumab can be safely administered in an outpatient setting. The promising clinical activity observed with the addition of ALT-803 to the regimen of patients with PD-1 monoclonal antibody relapsed and refractory disease shows evidence of anti-tumour activity for a new class of agents in NSCLC. FUNDING: Altor BioScience (a NantWorks company), National Institutes of Health, and Medical University of South Carolina Hollings Cancer Center.


Subject(s)
Antineoplastic Agents, Immunological/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Non-Small-Cell Lung/drug therapy , Lung Neoplasms/drug therapy , Nivolumab/administration & dosage , Proteins/administration & dosage , Aged , Antineoplastic Agents, Immunological/adverse effects , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Carcinoma, Non-Small-Cell Lung/secondary , Female , Humans , Lung Neoplasms/pathology , Male , Middle Aged , Neoplasm Staging , Nivolumab/adverse effects , Proteins/adverse effects , Recombinant Fusion Proteins , Time Factors , Treatment Outcome , United States
7.
PLoS Pathog ; 12(4): e1005545, 2016 Apr.
Article in English | MEDLINE | ID: mdl-27082643

ABSTRACT

Resting CD4+ T-cells harboring inducible HIV proviruses are a critical reservoir in antiretroviral therapy (ART)-treated subjects. These cells express little to no viral protein, and thus neither die by viral cytopathic effects, nor are efficiently cleared by immune effectors. Elimination of this reservoir is theoretically possible by combining latency-reversing agents (LRAs) with immune effectors, such as CD8+ T-cells. However, the relative efficacy of different LRAs in sensitizing latently-infected cells for recognition by HIV-specific CD8+ T-cells has not been determined. To address this, we developed an assay that utilizes HIV-specific CD8+ T-cell clones as biosensors for HIV antigen expression. By testing multiple CD8+ T-cell clones against a primary cell model of HIV latency, we identified several single agents that primed latently-infected cells for CD8+ T-cell recognition, including IL-2, IL-15, two IL-15 superagonists (IL-15SA and ALT-803), prostratin, and the TLR-2 ligand Pam3CSK4. In contrast, we did not observe CD8+ T-cell recognition of target cells following treatment with histone deacetylase inhibitors or with hexamethylene bisacetamide (HMBA). In further experiments we demonstrate that a clinically achievable concentration of the IL-15 superagonist 'ALT-803', an agent presently in clinical trials for solid and hematological tumors, primes the natural ex vivo reservoir for CD8+ T-cell recognition. Thus, our results establish a novel experimental approach for comparative evaluation of LRAs, and highlight ALT-803 as an LRA with the potential to synergize with CD8+ T-cells in HIV eradication strategies.


Subject(s)
Antiviral Agents/pharmacology , CD4-Positive T-Lymphocytes/virology , HIV Infections/immunology , Proteins/pharmacology , T-Lymphocytes, Cytotoxic/immunology , Virus Latency/drug effects , Enzyme-Linked Immunosorbent Assay , Enzyme-Linked Immunospot Assay , Flow Cytometry , Humans , Polymerase Chain Reaction , Recombinant Fusion Proteins , Virus Activation/drug effects
8.
Cytokine ; 107: 105-112, 2018 07.
Article in English | MEDLINE | ID: mdl-29452720

ABSTRACT

ALT-803 is a fusion protein complex consisting of an interleukin (IL)-15 superagonist and a dimeric IL-15 receptor alpha sushi domain IgG1 Fc fusion protein. When administered to mice, ALT-803 is capable of inducing natural killer (NK) and CD8+ T cell proliferation and activation, and effectively promoting potent anti-tumor responses. Currently, ALT-803 is in clinical trials for treatment of various solid tumors and hematological malignancies. In the initial phase of these clinical studies, intravenous (iv) injection was used according to the route used in pre-clinical efficacy studies. In order to evaluate the possible advantage of subcutaneous (sc) injection versus iv injection, this study compared the biological activity of the two treatment regimens of ALT-803 in pre-clinical in vivo models. The pharmacokinetics, immune stimulation, and anti-tumor efficacy of iv and sc injection routes of ALT-803 in C57BL/6 mice were compared. The half-life of ALT-803 was 7.5 h for iv versus 7.7 h for sc with the maximal detected serum concentration of ALT-803 to be 3926 ng/ml at 0.5 h time-point following iv injection versus 495 ng/ml at 16 h post sc injection. Biodistribution studies indicated that sc ALT-803, similarly to iv ALT-803 as previously reported, has a greater tissue distribution and longer residence time in lymphoid tissues compared to recombinant IL-15. Notably, ALT-803 when administered either iv or sc induced comparable proliferation and activation of CD8+ T and NK cells and resulted in similar reductions of tumor burden. A toxicity study of mice receiving multiple injections of ALT-803 for 4 weeks by iv or sc routes revealed equivalent immune-related changes. The gradual absorbance into the blood stream and lower maximal blood levels of ALT-803 in sc-injected mice, along with similar anti-tumor efficacy support the administration of ALT-803 by sc injection in patients with various malignancies and infectious diseases.


Subject(s)
Interleukin-15/metabolism , Proteins/administration & dosage , Administration, Intravenous/methods , Animals , Antineoplastic Agents/administration & dosage , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Female , Injections, Subcutaneous/methods , Killer Cells, Natural/drug effects , Killer Cells, Natural/metabolism , Lymphocyte Activation/drug effects , Mice , Mice, Inbred C57BL , Recombinant Fusion Proteins , Tissue Distribution , Xenograft Model Antitumor Assays/methods
9.
J Biol Chem ; 291(46): 23869-23881, 2016 Nov 11.
Article in English | MEDLINE | ID: mdl-27650494

ABSTRACT

IL-15 and its receptor α (IL-15Rα) are co-expressed on antigen-presenting cells, allowing transpresentation of IL-15 to immune cells bearing IL-2RßγC and stimulation of effector immune responses. We reported previously that the high-affinity interactions between an IL-15 superagonist (IL-15N72D) and the extracellular IL-15Rα sushi domain (IL-15RαSu) could be exploited to create a functional scaffold for the design of multivalent disease-targeted complexes. The IL-15N72D·IL-15RαSuFc complex, also known as ALT-803, is a multimeric complex constructed by fusing IL-15N72D·IL-15RαSu to the Fc domain of IgG1. ALT-803 is an IL-15 superagonist complex that has been developed as a potent antitumor immunotherapeutic agent and is in clinical trials. Here we describe the creation of a novel fusion molecule, 2B8T2M, using the ALT-803 scaffold fused to four single chains of the tumor-targeting monoclonal antibody rituximab. This molecule displays trispecific binding activity through its recognition of the CD20 molecule on tumor cells, stimulation via IL-2RßγC displayed on immune effector cells, and binding to Fcγ receptors on natural killer cells and macrophages. 2B8T2M activates natural killer cells to enhance antibody-dependent cellular cytotoxicity, mediates complement-dependent cytotoxicity, and induces apoptosis of B-lymphoma cells. Compared with rituximab, 2B8T2M exhibits significantly stronger antitumor activity in a xenograft SCID mouse model and depletes B cells in cynomolgus monkeys more efficiently. Thus, ALT-803 can be modified as a functional scaffold for creating multispecific, targeted IL-15-based immunotherapeutic agents and may serve as a novel platform to improve the antitumor activity and clinical efficacy of therapeutic antibodies.


Subject(s)
Immunity, Cellular/drug effects , Interleukin-15/agonists , Killer Cells, Natural/immunology , Lymphoma, B-Cell/drug therapy , Proteins , Recombinant Fusion Proteins , Rituximab , Animals , Cell Line, Tumor , Female , Humans , Interleukin-15/genetics , Interleukin-15/immunology , Lymphoma, B-Cell/genetics , Lymphoma, B-Cell/immunology , Lymphoma, B-Cell/pathology , Mice , Mice, SCID , Proteins/chemistry , Proteins/genetics , Proteins/pharmacology , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/pharmacology , Rituximab/chemistry , Rituximab/genetics , Rituximab/pharmacology , Xenograft Model Antitumor Assays
10.
Int J Cancer ; 138(1): 187-94, 2016 Jan 01.
Article in English | MEDLINE | ID: mdl-26174883

ABSTRACT

Glioblastoma is the most aggressive primary central nervous system malignancy with a poor prognosis in patients. Despite the need for better treatments against glioblastoma, very little progress has been made in discovering new therapies that exhibit superior survival benefit than the standard of care. Immunotherapy has been shown to be a promising treatment modality that could help improve clinical outcomes of glioblastoma patients by assisting the immune system to overcome the immunosuppressive tumor environment. Interleukin-15 (IL-15), a cytokine shown to activate several effector components of the immune system, may serve as an excellent immunotherapeutic candidate for the treatment of glioblastoma. Thus, we evaluated the efficacy of an IL-15 superagonist complex (IL-15N72D:IL-15RαSu-Fc; also known as ALT-803) in a murine GL261-luc glioblastoma model. We show that ALT-803, as a single treatment as well as in combination with anti-PD-1 antibody or stereotactic radiosurgery, exhibits a robust antitumor immune response resulting in a prolonged survival including complete remission in tumor bearing mice. In addition, ALT-803 treatment results in long-term immune memory against glioblastoma tumor rechallenge. Flow cytometric analysis of tumor infiltrating immune cells shows that ALT-803 leads to increased percentage of CD8+-cell infiltration, but not the NK cells, and IFN-γ production into the tumor microenvironment. Cell depletion studies, in accordance with the flow cytometric results, show that the ALT-803 therapeutic effect is dependent on CD4+ and CD8+ cells. These results provide a rationale for evaluating the therapeutic activity of ALT-803 against glioblastoma in the clinical setting.


Subject(s)
Central Nervous System Neoplasms/immunology , Glioblastoma/immunology , Interleukin-15/agonists , Proteins/pharmacology , Animals , Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Central Nervous System Neoplasms/metabolism , Central Nervous System Neoplasms/mortality , Central Nervous System Neoplasms/pathology , Central Nervous System Neoplasms/therapy , Disease Models, Animal , Female , Glioblastoma/metabolism , Glioblastoma/mortality , Glioblastoma/pathology , Glioblastoma/therapy , Humans , Immunologic Memory , Immunotherapy , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Mice , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/metabolism , Radiosurgery , Recombinant Fusion Proteins , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Xenograft Model Antitumor Assays
11.
J Virol ; 89(12): 6264-74, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25833053

ABSTRACT

UNLABELLED: Natural killer (NK) cells with anti-HIV-1 activity may inhibit HIV-1 replication and dissemination during acute HIV-1 infection. We hypothesized that the capacity of NK cells to suppress acute in vivo HIV-1 infection would be augmented by activating them via treatment with an interleukin-15 (IL-15) superagonist, IL-15 bound to soluble IL-15Rα, an approach that potentiates human NK cell-mediated killing of tumor cells. In vitro stimulation of human NK cells with a recombinant IL-15 superagonist significantly induced their expression of the cytotoxic effector molecules granzyme B and perforin; their degranulation upon exposure to K562 cells, as indicated by cell surface expression of CD107a; and their capacity to lyse K562 cells and HIV-1-infected T cells. The impact of IL-15 superagonist-induced activation of human NK cells on acute in vivo HIV-1 infection was investigated by using hu-spl-PBMC-NSG mice, NOD-SCID-IL2rγ(-/-) (NSG) mice intrasplenically injected with human peripheral blood mononuclear cells (PBMCs) which develop productive in vivo infection after intrasplenic inoculation with HIV-1. IL-15 superagonist treatment potently inhibited acute HIV-1 infection in hu-spl-PBMC-NSG mice even when delayed until 3 days after intrasplenic HIV-1 inoculation. Removal of NK cells from human PBMCs prior to intrasplenic injection into NSG mice completely abrogated IL-15 superagonist-mediated suppression of in vivo HIV-1 infection. Thus, the in vivo activation of NK cells, integral mediators of the innate immune response, by treatment with an IL-15 superagonist increases their anti-HIV activity and enables them to potently suppress acute in vivo HIV-1 infection. These results indicate that in vivo activation of NK cells may represent a new immunotherapeutic approach to suppress acute HIV-1 infection. IMPORTANCE: Epidemiological studies have indicated that NK cells contribute to the control of HIV-1 infection, and in vitro studies have demonstrated that NK cells can selectively kill HIV-1-infected cells. We demonstrated that in vivo activation of NK cells by treatment with an IL-15 superagonist that potently stimulates the antitumor activity of NK cells markedly inhibited acute HIV-1 infection in humanized mice, even when activation of NK cells by IL-15 superagonist treatment is delayed until 3 days after HIV-1 inoculation. NK cell depletion from PBMCs prior to their intrasplenic injection abrogated the suppression of in vivo HIV-1 infection observed in humanized mice treated with the IL-15 superagonist, demonstrating that activated human NK cells were mediating IL-15 superagonist-induced inhibition of acute HIV-1 infection. Thus, in vivo immunostimulation of NK cells, a promising therapeutic approach for cancer therapy, may represent a new treatment modality for HIV-1-infected individuals, particularly in the earliest stages of infection.


Subject(s)
HIV Infections/immunology , HIV Infections/prevention & control , Interleukin-15/antagonists & inhibitors , Killer Cells, Natural/immunology , Lymphocyte Activation , Animals , Disease Models, Animal , Humans , Mice , Mice, SCID
12.
Science ; 383(6687): 1104-1111, 2024 Mar 08.
Article in English | MEDLINE | ID: mdl-38422185

ABSTRACT

The eradication of the viral reservoir represents the major obstacle to the development of a clinical cure for established HIV-1 infection. Here, we demonstrate that the administration of N-803 (brand name Anktiva) and broadly neutralizing antibodies (bNAbs) results in sustained viral control after discontinuation of antiretroviral therapy (ART) in simian-human AD8 (SHIV-AD8)-infected, ART-suppressed rhesus macaques. N-803+bNAbs treatment induced immune activation and transient viremia but only limited reductions in the SHIV reservoir. Upon ART discontinuation, viral rebound occurred in all animals, which was followed by durable control in approximately 70% of all N-803+bNAb-treated macaques. Viral control was correlated with the reprogramming of CD8+ T cells by N-803+bNAb synergy. Thus, complete eradication of the replication-competent viral reservoir is likely not a prerequisite for the induction of sustained remission after discontinuation of ART.


Subject(s)
Anti-Retroviral Agents , Recombinant Fusion Proteins , Simian Acquired Immunodeficiency Syndrome , Simian Immunodeficiency Virus , Animals , Humans , Anti-Retroviral Agents/therapeutic use , Anti-Retroviral Agents/pharmacology , Broadly Neutralizing Antibodies/administration & dosage , CD8-Positive T-Lymphocytes/virology , Immunotherapy , Macaca mulatta , Simian Acquired Immunodeficiency Syndrome/drug therapy , Simian Acquired Immunodeficiency Syndrome/therapy , Viral Load , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/pharmacology , Remission Induction , Drug Therapy, Combination
13.
Aging Cell ; 22(5): e13806, 2023 05.
Article in English | MEDLINE | ID: mdl-36967480

ABSTRACT

Accumulation of senescent cells (SNCs) with a senescence-associated secretory phenotype (SASP) has been implicated as a major source of chronic sterile inflammation leading to many age-related pathologies. Herein, we provide evidence that a bifunctional immunotherapeutic, HCW9218, with capabilities of neutralizing TGF-ß and stimulating immune cells, can be safely administered systemically to reduce SNCs and alleviate SASP in mice. In the diabetic db/db mouse model, subcutaneous administration of HCW9218 reduced senescent islet ß cells and SASP resulting in improved glucose tolerance, insulin resistance, and aging index. In naturally aged mice, subcutaneous administration of HCW9218 durably reduced the level of SNCs and SASP, leading to lower expression of pro-inflammatory genes in peripheral organs. HCW9218 treatment also reverted the pattern of key regulatory circadian gene expression in aged mice to levels observed in young mice and impacted genes associated with metabolism and fibrosis in the liver. Single-nucleus RNA Sequencing analysis further revealed that HCW9218 treatment differentially changed the transcriptomic landscape of hepatocyte subtypes involving metabolic, signaling, cell-cycle, and senescence-associated pathways in naturally aged mice. Long-term survival studies also showed that HCW9218 treatment improved physical performance without compromising the health span of naturally aged mice. Thus, HCW9218 represents a novel immunotherapeutic approach and a clinically promising new class of senotherapeutic agents targeting cellular senescence-associated diseases.


Subject(s)
Cellular Senescence , Senescence-Associated Secretory Phenotype , Mice , Animals , Cellular Senescence/genetics , Aging , Inflammation , Immunotherapy , Phenotype
14.
Cancer Immunol Res ; 9(9): 1071-1087, 2021 09.
Article in English | MEDLINE | ID: mdl-34244297

ABSTRACT

Natural killer (NK) cells are a promising cellular therapy for cancer, with challenges in the field including persistence, functional activity, and tumor recognition. Briefly, priming blood NK cells with recombinant human (rh)IL-12, rhIL-15, and rhIL-18 (12/15/18) results in memory-like NK cell differentiation and enhanced responses against cancer. However, the lack of available, scalable Good Manufacturing Process (GMP)-grade reagents required to advance this approach beyond early-phase clinical trials is limiting. To address this challenge, we developed a novel platform centered upon an inert tissue factor scaffold for production of heteromeric fusion protein complexes (HFPC). The first use of this platform combined IL-12, IL-15, and IL-18 receptor engagement (HCW9201), and the second adds CD16 engagement (HCW9207). This unique HFPC expression platform was scalable with equivalent protein quality characteristics in small- and GMP-scale production. HCW9201 and HCW9207 stimulated activation and proliferation signals in NK cells, but HCW9207 had decreased IL-18 receptor signaling. RNA sequencing and multidimensional mass cytometry revealed parallels between HCW9201 and 12/15/18. HCW9201 stimulation improved NK cell metabolic fitness and resulted in the DNA methylation remodeling characteristic of memory-like differentiation. HCW9201 and 12/15/18 primed similar increases in short-term and memory-like NK cell cytotoxicity and IFNγ production against leukemia targets, as well as equivalent control of leukemia in NSG mice. Thus, HFPCs represent a protein engineering approach that solves many problems associated with multisignal receptor engagement on immune cells, and HCW9201-primed NK cells can be advanced as an ideal approach for clinical GMP-grade memory-like NK cell production for cancer therapy.


Subject(s)
Interleukin-12/pharmacology , Interleukin-15/pharmacology , Interleukin-18/pharmacology , Killer Cells, Natural/immunology , Leukemia/therapy , Animals , Cell Line, Tumor , Humans , Immunologic Memory/drug effects , Leukemia/immunology , Mice , Receptors, Natural Killer Cell/metabolism , Recombinant Fusion Proteins/pharmacology , Remission Induction , Xenograft Model Antitumor Assays
15.
Nat Biotechnol ; 36(8): 707-716, 2018 09.
Article in English | MEDLINE | ID: mdl-29985479

ABSTRACT

Adoptive cell therapy (ACT) with antigen-specific T cells has shown remarkable clinical success; however, approaches to safely and effectively augment T cell function, especially in solid tumors, remain of great interest. Here we describe a strategy to 'backpack' large quantities of supporting protein drugs on T cells by using protein nanogels (NGs) that selectively release these cargos in response to T cell receptor activation. We designed cell surface-conjugated NGs that responded to an increase in T cell surface reduction potential after antigen recognition and limited drug release to sites of antigen encounter, such as the tumor microenvironment. By using NGs that carried an interleukin-15 super-agonist complex, we demonstrated that, relative to systemic administration of free cytokines, NG delivery selectively expanded T cells 16-fold in tumors and allowed at least eightfold higher doses of cytokine to be administered without toxicity. The improved therapeutic window enabled substantially increased tumor clearance by mouse T cell and human chimeric antigen receptor (CAR)-T cell therapy in vivo.


Subject(s)
Drug Delivery Systems , Immunotherapy, Adoptive , Nanoparticles , Receptors, Antigen, T-Cell/metabolism , Signal Transduction , T-Lymphocytes/immunology , Animals , Cell Proliferation , Cytokines/administration & dosage , Humans , Interleukin-15/administration & dosage , Leukocyte Common Antigens/immunology , Lymphocyte Activation , Mice , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , Tumor Microenvironment
16.
Cancer Res ; 78(11): 3067-3074, 2018 06 01.
Article in English | MEDLINE | ID: mdl-29636345

ABSTRACT

Effector CD8+ T cells conditioned with IL12 during activation mediate enhanced antitumor efficacy after adoptive transfer into lymphodepleted hosts; this is due in part to improved IL7 responsiveness. Therefore, we hypothesized that increasing the intensity or type of lymphodepletion would deplete more IL7-consuming host cells and improve the persistence and antitumor activity of IL12-conditioned CD8+ T cells. Using cyclophosphamide, fludarabine, and total body irradiation (TBI, 6 Gy) either individually or in combination, we found that combined lymphodepletion best enhanced T-cell engraftment in mice. This improvement was strongly related to the extent of leukopenia, as posttransfer levels of donor T cells inversely correlated to host cell counts after lymphodepletion. Despite the improvement in engraftment seen with combination lymphodepletion, dual-agent lymphodepletion did not augment the antitumor efficacy of donor T cells compared with TBI alone. Similarly, IL7 supplementation after TBI and transfer of tumor-reactive T cells failed to improve persistence or antitumor immunity. However, IL15 or IL2 supplementation greatly augmented the persistence and antitumor efficacy of donor tumor-reactive T cells. Our results indicate that the amount of host IL7 induced after single agent lymphodepletion is sufficient to potentiate the expansion and antitumor activity of donor T cells, and that the efficacy of future regimens may be improved by providing posttransfer support with IL2 or IL15.Significance: The relationship between lymphodepletion and cytokine support plays a critical role in determining donor T-cell engraftment and antitumor efficacy. Cancer Res; 78(11); 3067-74. ©2018 AACR.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Interleukin-15/immunology , Interleukin-2/immunology , Adoptive Transfer/methods , Animals , Cell Line, Tumor , Female , Immunotherapy, Adoptive/methods , Lymphocyte Activation/immunology , Lymphocyte Depletion/methods , Male , Mice , Mice, Inbred C57BL , T-Lymphocytes, Regulatory/immunology
17.
Blood Adv ; 2(2): 76-84, 2018 01 23.
Article in English | MEDLINE | ID: mdl-29365313

ABSTRACT

Sequestering of latent HIV in follicular helper T cells within B-cell follicles that largely exclude cytotoxic T cells is a major barrier to cellular immune-based approaches to eradicate HIV. Here, we show that the clinical-grade human interleukin-15 (IL-15) superagonist ALT-803 activates and redirects simian immunodeficiency virus (SIV)-specific CD8+ T cells from the peripheral blood into B-cell follicles. In agreement with the increased trafficking of SIV-specific cytotoxic T cells to sites of cryptic viral replication, lymph nodes of elite controlling macaques contained fewer cells expressing SIV RNA or harboring SIV DNA post-ALT-803 treatment. These data establish ALT-803 as an immunotherapeutic for HIV and other chronic viral pathogens that evade host immunity by persisting in B-cell follicles.


Subject(s)
B-Lymphocytes/virology , CD8-Positive T-Lymphocytes/immunology , Proteins/therapeutic use , Simian Immunodeficiency Virus/immunology , Animals , HIV/drug effects , Humans , Immune Evasion/drug effects , Interleukin-15/agonists , Macaca/virology , Recombinant Fusion Proteins , T-Lymphocytes, Cytotoxic/immunology
18.
Oncotarget ; 8(27): 44366-44378, 2017 Jul 04.
Article in English | MEDLINE | ID: mdl-28574833

ABSTRACT

Interleukin-15 (IL-15) is a potent cytokine that increases CD8+ T and NK cell numbers and function in experimental models. However, obstacles remain in using IL-15 therapeutically, specifically its low potency and short in vivo half-life. To help overcome this, a new IL-15 superagonist complex comprised of an IL-15N72D mutation and IL-15RαSu/Fc fusion (IL-15SA, also known as ALT-803) was developed. IL-15SA exhibits a significantly longer serum half-life and increased in vivo activity against various tumors. Herein, we evaluated the effects of IL-15SA in recipients of allogeneic hematopoietic stem cell transplantation. Weekly administration of IL-15SA to transplant recipients significantly increased the number of CD8+ T cells (specifically CD44+ memory/activated phenotype) and NK cells. Intracellular IFN-γ and TNF-α secretion by CD8+ T cells increased in the IL-15SA-treated group. IL-15SA also upregulated NKG2D expression on CD8+ T cells. Moreover, IL-15SA enhanced proliferation and cytokine secretion of adoptively transferred CFSE-labeled T cells in syngeneic and allogeneic models by specifically stimulating the slowly proliferative and nonproliferative cells into actively proliferating cells.We then evaluated IL-15SA's effects on anti-tumor activity against murine mastocytoma (P815) and murine B cell lymphoma (A20). IL-15SA enhanced graft-versus-tumor (GVT) activity in these tumors following T cell infusion. Interestingly, IL-15 SA administration provided GVT activity against A20 lymphoma cells in the murine donor leukocyte infusion (DLI) model without increasing graft versus host disease. In conclusion, IL-15SA could be a highly potent T- cell lymphoid growth factor and novel immunotherapeutic agent to complement stem cell transplantation and adoptive immunotherapy.


Subject(s)
Antineoplastic Agents/pharmacology , Graft vs Tumor Effect/drug effects , Proteins/pharmacology , Adoptive Transfer , Animals , Cell Line, Tumor , Disease Models, Animal , Female , Hematopoietic Stem Cell Transplantation , Interleukin-15/metabolism , Interleukin-15 Receptor alpha Subunit/agonists , Interleukin-15 Receptor alpha Subunit/metabolism , Lymphocyte Count , Mice , Neoplasms/drug therapy , Neoplasms/immunology , Neoplasms/metabolism , Neoplasms/pathology , Recombinant Fusion Proteins , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Transplantation, Homologous , Xenograft Model Antitumor Assays
19.
JCI Insight ; 2(23)2017 12 07.
Article in English | MEDLINE | ID: mdl-29212951

ABSTRACT

NK cell activation has been shown to be metabolically regulated in vitro; however, the role of metabolism during in vivo NK cell responses to infection is unknown. We examined the role of glycolysis in NK cell function during murine cytomegalovirus (MCMV) infection and the ability of IL-15 to prime NK cells during CMV infection. The glucose metabolism inhibitor 2-deoxy-ᴅ-glucose (2DG) impaired both mouse and human NK cell cytotoxicity following priming in vitro. Similarly, MCMV-infected mice treated with 2DG had impaired clearance of NK-specific targets in vivo, which was associated with higher viral burden and susceptibility to infection on the C57BL/6 background. IL-15 priming is known to alter NK cell metabolism and metabolic requirements for activation. Treatment with the IL-15 superagonist ALT-803 rescued mice from otherwise lethal infection in an NK-dependent manner. Consistent with this, treatment of a patient with ALT-803 for recurrent CMV reactivation after hematopoietic cell transplant was associated with clearance of viremia. These studies demonstrate that NK cell-mediated control of viral infection requires glucose metabolism and that IL-15 treatment in vivo can reduce this requirement and may be effective as an antiviral therapy.


Subject(s)
Cytotoxicity, Immunologic/immunology , Herpesviridae Infections/prevention & control , Killer Cells, Natural/immunology , Muromegalovirus/isolation & purification , Actins/metabolism , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Blood Glucose/metabolism , Cell Proliferation/drug effects , Cells, Cultured , Cytomegalovirus/physiology , Cytomegalovirus Infections/drug therapy , Cytomegalovirus Infections/immunology , Cytomegalovirus Infections/virology , Cytotoxicity, Immunologic/drug effects , Deoxyglucose/pharmacology , Female , Glycolysis/drug effects , Glycolysis/immunology , Granzymes/metabolism , Herpesviridae Infections/drug therapy , Herpesviridae Infections/immunology , Herpesviridae Infections/virology , Humans , Interferon-gamma/biosynthesis , Interleukin-15/agonists , Interleukin-15/immunology , Killer Cells, Natural/drug effects , Male , Mice, Inbred C57BL , Proteins/pharmacology , Proteins/therapeutic use , Recombinant Fusion Proteins , Viral Load/drug effects , Virus Activation , Young Adult
20.
Oncotarget ; 7(1): 814-30, 2016 Jan 05.
Article in English | MEDLINE | ID: mdl-26625316

ABSTRACT

Shedding of the human NKG2D ligand MIC (MHC class I-chain-related molecule) from tumor cell surfaces correlates with progression of many epithelial cancers. Shedding-derived soluble MIC (sMIC) enables tumor immune escape through multiple immune suppressive mechanisms, such as disturbing natural killer (NK) cell homeostatic maintenance, impairing NKG2D expression on NK cells and effector T cells, and facilitating the expansion of arginase I+ myeloid suppressor cells. Our recent study has demonstrated that sMIC is an effective cancer therapeutic target. Whether targeting tumor-derived sMIC would enhance current active immunotherapy is not known. Here, we determined the in vivo therapeutic effect of an antibody co-targeting sMIC with the immunostimulatory IL-15 superagonist complex, ALT-803, using genetically engineered transplantable syngeneic sMIC+ tumor models. We demonstrate that combined therapy of a nonblocking antibody neutralizing sMIC and ALT-803 improved the survival of animals bearing sMIC+ tumors in comparison to monotherapy. We further demonstrate that the enhanced therapeutic effect with combined therapy is through concurrent augmentation of NK and CD8 T cell anti-tumor responses. In particular, expression of activation-induced surface molecules and increased functional potential by cytokine secretion are improved greatly by the administration of combined therapy. Depletion of NK cells abolished the cooperative therapeutic effect. Our findings suggest that administration of the sMIC-neutralizing antibody can enhance the anti-tumor effects of ALT-803. With ALT-803 currently in clinical trials to treat progressive solid tumors, the majority of which are sMIC+, our findings provide a rationale for co-targeting sMIC to enhance the therapeutic efficacy of ALT-803 or other IL-15 agonists.


Subject(s)
Antibodies, Neutralizing/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Histocompatibility Antigens Class I/metabolism , Interleukin-15/agonists , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Neoplasms/drug therapy , Proteins/pharmacology , Animals , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/immunology , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/immunology , Cells, Cultured , Coculture Techniques , Flow Cytometry , Histocompatibility Antigens Class I/immunology , Humans , Interferon-gamma/immunology , Interferon-gamma/metabolism , Interleukin-15/metabolism , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Ligands , Male , Mice, Knockout , Mice, Transgenic , Neoplasms/immunology , Neoplasms/metabolism , Proteins/administration & dosage , Recombinant Fusion Proteins , Spleen/cytology , Spleen/immunology , Spleen/metabolism , Tumor Burden/drug effects , Tumor Burden/immunology
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