Interleukin-15 Complex Treatment Protects Mice from Cerebral Malaria by Inducing Interleukin-10-Producing Natural Killer Cells.

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.

A "Prime and Expand" strategy using the multifunctional fusion proteins to generate memory-like NK cells for cell therapy.

Adoptive cellular therapy (ACT) using memory-like (ML) natural killer (NK) cells, generated through overnight ex vivo activation with IL-12, IL-15, and IL-18, has shown promise for treating hematologic malignancies. We recently reported that a multifunctional fusion molecule, HCW9201, comprising IL-12, IL-15, and IL-18 domains could replace individual cytokines for priming human ML NK cell programming ("Prime" step). However, this approach does not include ex vivo expansion, thereby limiting the ability to test different doses and schedules. Here, we report the design and generation of a multifunctional fusion molecule, HCW9206, consisting of human IL-7, IL-15, and IL-21 cytokines. We observed > 300-fold expansion for HCW9201-primed human NK cells cultured for 14 days with HCW9206 and HCW9101, an IgG1 antibody, recognizing the scaffold domain of HCW9206 ("Expand" step). This expansion was dependent on both HCW9206 cytokines and interactions of the IgG1 mAb with CD16 receptors on NK cells. The resulting "Prime and Expand" ML NK cells exhibited elevated metabolic capacity, stable epigenetic IFNG promoter demethylation, enhanced antitumor activity in vitro and in vivo, and superior persistence in NSG mice. Thus, the "Prime and Expand" strategy represents a simple feeder cell-free approach to streamline manufacturing of clinical-grade ML NK cells to support multidose and off-the-shelf ACT.

Immunotherapeutic HCW9218 augments anti-tumor activity of chemotherapy via NK cell-mediated reduction of therapy-induced senescent cells.

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.

Bifunctional TGF-ß trap/IL-15 protein complex elicits potent NK cell and CD8+ T cell immunity against solid tumors.

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.

T cell sensing of antigen dose governs interactive behavior with dendritic cells and sets a threshold for T cell activation.

After homing to lymph nodes, CD8+ T cells are primed by dendritic cells (DCs) in three phases. During phase one, T cells undergo brief serial contacts with DCs for several hours, whereas phase two is characterized by stable T cell-DC interactions. We show here that the duration of phase one and T cell activation kinetics correlated inversely with the number of complexes of cognate peptide and major histocompatibility complex (pMHC) per DC and with the density of antigen-presenting DCs per lymph node. Very few pMHC complexes were necessary for the induction of full-fledged T cell activation and effector differentiation. However, neither T cell activation nor transition to phase two occurred below a threshold antigen dose determined in part by pMHC stability. Thus, phase one permits T cells to make integrated 'measurements' of antigen dose that determine subsequent T cell participation in immune responses.

First-in-human phase 1 clinical study of the IL-15 superagonist complex ALT-803 to treat relapse after transplantation.

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.

ALT-803 Transiently Reduces Simian Immunodeficiency Virus Replication in the Absence of Antiretroviral Treatment.

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.

ALT-803, an IL-15 superagonist, in combination with nivolumab in patients with metastatic non-small cell lung cancer: a non-randomised, open-label, phase 1b trial.

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.

An IL-15 superagonist/IL-15Rα fusion complex protects and rescues NK cell-cytotoxic function from TGF-ß1-mediated immunosuppression.

Natural killer (NK) cells are innate cytotoxic lymphocytes that play a fundamental role in the immunosurveillance of cancers. NK cells of cancer patients exhibit impaired function mediated by immunosuppressive factors released from the tumor microenvironment (TME), such as transforming growth factor (TGF)-ß1. An interleukin (IL)-15 superagonist/IL-15 receptor α fusion complex (IL-15SA/IL-15RA; ALT-803) activates the IL-15 receptor on CD8 T cells and NK cells, and has shown significant anti-tumor activity in several in vivo studies. This in vitro study investigated the efficacy of IL-15SA/IL-15RA on TGF-ß1-induced suppression of NK cell-cytotoxic function. IL-15SA/IL-15RA inhibited TGF-ß1 from decreasing NK cell lysis of four of four tumor cell lines (H460, LNCap, MCF7, MDA-MB-231). IL-15SA/IL-15RA rescued healthy donor and cancer patient NK cell-cytotoxicity, which had previously been suppressed by culture with TGF-ß1. TGF-ß1 downregulated expression of NK cell-activating markers and cytotoxic granules, such as CD226, NKG2D, NKp30, granzyme B, and perforin. Smad2/3 signaling was responsible for this TGF-ß1-induced downregulation of NK cell-activating markers and cytotoxic granules. IL-15SA/IL-15RA blocked Smad2/3-induced transcription, resulting in the rescue of NK cell-cytotoxic function from TGF-ß1-induced suppression. These findings suggest that in addition to increasing NK cell function via promoting the IL-15 signaling pathway, IL-15SA/IL-15RA can function as an inhibitor of TGF-ß1 signaling, providing a potential remedy for NK cell dysfunction in the immunosuppressive tumor microenvironment.

A Subset of Latency-Reversing Agents Expose HIV-Infected Resting CD4+ T-Cells to Recognition by Cytotoxic T-Lymphocytes.

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.

Evaluation of the biological activities of the IL-15 superagonist complex, ALT-803, following intravenous versus subcutaneous administration in murine models.

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.

A Novel Fusion of ALT-803 (Interleukin (IL)-15 Superagonist) with an Antibody Demonstrates Antigen-specific Antitumor Responses.

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.

ImmunoPET and Near-Infrared Fluorescence Imaging of Pancreatic Cancer with a Dual-Labeled Bispecific Antibody Fragment.

Dual-targeted imaging agents have shown improved targeting efficiencies in comparison to single-targeted entities. The purpose of this study was to quantitatively assess the tumor accumulation of a dual-labeled heterobifunctional imaging agent, targeting two overexpressed biomarkers in pancreatic cancer, using positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging modalities. A bispecific immunoconjugate (heterodimer) of CD105 and tissue factor (TF) Fab' antibody fragments was developed using click chemistry. The heterodimer was dual-labeled with a radionuclide (64Cu) and fluorescent dye. PET/NIRF imaging and biodistribution studies were performed in four-to-five week old nude athymic mice bearing BxPC-3 (CD105/TF+/+) or PANC-1 (CD105/TF-/-) tumor xenografts. A blocking study was conducted to investigate the specificity of the tracer. Ex vivo tissue staining was performed to compare TF/CD105 expression in tissues with PET tracer uptake to validate in vivo results. PET imaging of 64Cu-NOTA-heterodimer-ZW800 in BxPC-3 tumor xenografts revealed enhanced tumor uptake (21.0 ± 3.4%ID/g; n = 4) compared to the homodimer of TRC-105 (9.6 ± 2.0%ID/g; n = 4; p < 0.01) and ALT-836 (7.6 ± 3.7%ID/g; n = 4; p < 0.01) at 24 h postinjection. Blocking studies revealed that tracer uptake in BxPC-3 tumors could be decreased by 4-fold with TF blocking and 2-fold with CD105 blocking. In the negative model (PANC-1), heterodimer uptake was significantly lower than that found in the BxPC-3 model (3.5 ± 1.1%ID/g; n = 4; p < 0.01). The specificity was confirmed by the successful blocking of CD105 or TF, which demonstrated that the dual targeting with 64Cu-NOTA-heterodimer-ZW800 provided an improvement in overall tumor accumulation. Also, fluorescence imaging validated the PET imaging, allowing for clear delineation of the xenograft tumors. Dual-labeled heterodimeric imaging agents, like 64Cu-NOTA-heterodimer-ZW800, may increase the overall tumor accumulation in comparison to single-targeted homodimers, leading to improved imaging of cancer and other related diseases.

Therapeutic administration of IL-15 superagonist complex ALT-803 leads to long-term survival and durable antitumor immune response in a murine glioblastoma model.

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.

In Vivo Activation of Human NK Cells by Treatment with an Interleukin-15 Superagonist Potently Inhibits Acute In Vivo HIV-1 Infection in Humanized Mice.

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.

ImmunoPET of tissue factor expression in triple-negative breast cancer with a radiolabeled antibody Fab fragment.

PURPOSE: To date, there is no effective therapy for triple-negative breast cancer (TNBC), which has a dismal clinical outcome. Upregulation of tissue factor (TF) expression leads to increased patient morbidity and mortality in many solid tumor types, including TNBC. Our goal was to employ the Fab fragment of ALT-836, a chimeric anti-human TF mAb, for PET imaging of TNBC, which can be used to guide future TNBC therapy. METHODS: ALT-836-Fab was generated by enzymatic papain digestion. SDS-PAGE and FACS studies were performed to evaluate the integrity and TF binding affinity of ALT-836-Fab before NOTA conjugation and (64)Cu-labeling. Serial PET imaging and biodistribution studies were carried out to evaluate the tumor targeting efficacy and pharmacokinetics in the MDA-MB-231 TNBC model, which expresses high levels of TF on the tumor cells. Blocking studies, histological assessment, as well as RT-PCR were performed to confirm TF specificity of (64)Cu-NOTA-ALT-836-Fab. RESULTS: ALT-836-Fab was produced with high purity, which exhibited superb TF binding affinity and specificity. Serial PET imaging revealed rapid and persistent tumor uptake of (64)Cu-NOTA-ALT-836-Fab (5.1 ± 0.5 %ID/g at 24 h post-injection; n = 4) and high tumor/muscle ratio (7.0 ± 1.2 at 24 h post-injection; n = 4), several-fold higher than that of the blocking group and tumor models that do not express significant level of TF, which was confirmed by biodistribution studies. TF specificity of the tracer was also validated by histology and RT-PCR. CONCLUSION: (64)Cu-NOTA-ALT-836-Fab exhibited prominent tissue factor targeting efficiency in MDA-MB-231 TNBC model. The use of a Fab fragment led to fast tumor uptake and good tissue/muscle ratio, which may be translated into same-day immunoPET imaging in the clinical setting to improve TNBC patient management.

Induction of durable remission by dual immunotherapy in SHIV-infected ART-suppressed macaques.

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.

A novel interleukin-2-based fusion molecule, HCW9302, differentially promotes regulatory T cell expansion to treat atherosclerosis in mice.

Atherosclerosis is a chronic inflammatory disease caused by deposition of oxidative low-density lipoprotein (LDL) in the arterial intima which triggers the innate immune response through myeloid cells such as macrophages. Regulatory T cells (Tregs) play an important role in controlling the progression or regression of atherosclerosis by resolving macrophage-mediated inflammatory functions. Interleukin-2 (IL-2) signaling is essential for homeostasis of Tregs. Since recombinant IL-2 has an unfavorable pharmacokinetic profile limiting its therapeutic use, we constructed a fusion protein, designated HCW9302, containing two IL-2 domains linked by an extracellular tissue factor domain. We found that HCW9302 exhibited a longer serum half-life with an approximately 1000-fold higher affinity for the IL-2Rα than IL-2. HCW9302 could be administered to mice at a dosing range that expanded and activated Tregs but not CD4+ effector T cells. In an ApoE-/- mouse model, HCW9302 treatment curtailed the progression of atherosclerosis through Treg activation and expansion, M2 macrophage polarization and myeloid-derived suppressor cell induction. HCW9302 treatment also lessened inflammatory responses in the aorta. Thus, HCW9302 is a potential therapeutic agent to expand and activate Tregs for treatment of inflammatory and autoimmune diseases.

Immunotherapeutic approach to reduce senescent cells and alleviate senescence-associated secretory phenotype in mice.

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.

Predicting Ca2+ -binding sites using refined carbon clusters.

Identifying Ca(2+) -binding sites in proteins is the first step toward understanding the molecular basis of diseases related to Ca(2+) -binding proteins. Currently, these sites are identified in structures either through X-ray crystallography or NMR analysis. However, Ca(2+) -binding sites are not always visible in X-ray structures due to flexibility in the binding region or low occupancy in a Ca(2+) -binding site. Similarly, both Ca(2+) and its ligand oxygens are not directly observed in NMR structures. To improve our ability to predict Ca(2+) -binding sites in both X-ray and NMR structures, we report a new graph theory algorithm (MUG(C) ) to predict Ca(2+) -binding sites. Using carbon atoms covalently bonded to the chelating oxygen atoms, and without explicit reference to side-chain oxygen ligand co-ordinates, MUG(C) is able to achieve 94% sensitivity with 76% selectivity on a dataset of X-ray structures composed of 43 Ca(2+) -binding proteins. Additionally, prediction of Ca(2+) -binding sites in NMR structures was obtained by MUG(C) using a different set of parameters, which were determined by the analysis of both Ca(2+) -constrained and unconstrained Ca(2+) -loaded structures derived from NMR data. MUG(C) identified 20 of 21 Ca(2+) -binding sites in NMR structures inferred without the use of Ca(2+) constraints. MUG(C) predictions are also highly selective for Ca(2+) -binding sites as analyses of binding sites for Mg(2+) , Zn(2+) , and Pb(2+) were not identified as Ca(2+) -binding sites. These results indicate that the geometric arrangement of the second-shell carbon cluster is sufficient not only for accurate identification of Ca(2+) -binding sites in NMR and X-ray structures but also for selective differentiation between Ca(2+) and other relevant divalent cations.