KIR Blockade Research Articles

The difference in endothelium-dependent relaxation components in proximal and distal thoracic aorta regions of male rats.

Aorta, the largest vessel in the body, is generally considered anatomically homogeneous, yet spatial functional differences exist. In our study, we conducted a comprehensive analysis by reexamining public RNA-SEQ data, comparing expression patterns between thoracic and abdominal aorta. Additionally, we measured acetylcholine-induced relaxations of the different regions of thoracic aorta in Wistar Rats. Our results revealed a distinct percentage difference in acetylcholine-induced relaxation in the proximal and distal segments of the thoracic aorta (p = 1.14e-4). To explain this variation, we performed differential expression analysis of previously published RNA-sequencing data between thoracic and abdominal aorta, which showed 497 differentially expressed genes between these locations. From results of RNA-Seq analysis, we draw a hypothesis that differential expressions of the potassium inward rectifying channels (KIR) and voltage gated calcium channels (VGCC) presumably located on SMC, with higher expression in the distal thoracic segments in comparison with the proximal thoracic segments of aorta, can explain differences in acetylcholine-induced relaxation. Notably, specific blockade of KIR eliminated differences between the proximal and distal regions of thoracic aorta, underscoring their significance in understanding the spatial nuances in aortic behavior, also blockade of VGCC, shows a higher effect on basal tone, in distal region of thoracic aorta in comparison with proximal.

K+ -independent Kir blockade by external Cs+ and Ba2.

Cations such as Cs+ and Ba2+ are known to block K+ currents by entering an open channel and binding to the selectivity filter, where they obstruct the pore and block diffusion of the permeant ion. This obstruction is voltage‐ and K+‐dependent and is relieved by the trans permeant ion flux. The present patch‐clamp study on Xenopus muscle cells shows that, unlike the voltage‐activated K+ (Kv) channels, blockade of the inward rectifier K+ (Kir) channels by external foreign cations results from the combination of pore obstruction with a new and independent mechanism. This new blockade is independent of the K+ concentrations and flux and acts indiscriminately on both the outward and the inward Kir components. External Cs+ and Ba2+ compete for this blockade with free access to common channel sites. These features suggest that the blocking cations do not need to enter the channel for this new mechanism, and should bind to the extracellular side of the channel. When K+ fluxes are flowing outward, the pore obstruction is relieved for both Kir and Kv currents, and the K+‐independent blockade here described is responsible for a selective Kir inhibition, justifying the use of these external cations as tools in cell physiology studies.

Impact of Nrf2 Deficiency on Potassium Channel Function in Rat Soleus Arterioles

Introduction Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and its response element are essential in regulation of cellular oxidative stress. Nrf2 expression has also been linked to potassium (K+) channel function. We hypothesized that vasoactive responses that are enhanced by exercise training and also modulated by K+channel function would be impaired in arterioles from skeletal muscle of rats in which Nrf2 (Nrf2 KO) is deleted. Methods Exercise training was performed in male wildtype (WT) Sprague-Dawley rats, and their littermates lacking the Nrf2 gene (Nrf2 KO). Rats were exercise trained using a mixed moderate and high-intensity interval treadmill program for 10-12 weeks. After exercise training, soleus resistance arterioles were isolated and cannulated for the assessment of vascular reactivity. Vasodilatory responses to low concentrations of KCl [10 and 20mM} were assessed. Vasoconstrictor responses to higher concentrations of KCl [30–100mM] and myogenic responses to increasing intraluminal pressure (pressure 0cm H2O – 130cm H2O) were also assessed. The contribution of inward rectifying K+ channels (Kir) to vasodilation during low concentrations of KCl was assessed by treatment with barium chloride (BaCl2, 100mM). Results Myogenic responsiveness was reduced in resistance arterioles from soleus muscle of Nrf2 KO rats as compared to those from WT rats (p<0.01). Vasodilatory responses to low concentrations of KCl were also reduced in resistance arterioles from soleus muscle of Nrf2 KO rats as compared to those from WT rats (p<0.05); however, there were no differences in vasoconstrictor responses to higher concentrations of KCL between arterioles from WT and KO rats. Inhibition of Kir channels with BaCl2 eliminated the differences in vasodilatory responses to low concentrations KCl between genotypes. Conclusion These data indicate that vasodilatory responses to low concentrations of KCl and myogenic responsiveness, which may be modulated by Kir activity, are impaired in soleus muscle arterioles from Nrf2KO rats. The finding that blockade of Kir eliminated genotypic differences in vasodilatory responsiveness to low concentrations of KCl suggests that altered redox homeostasis and/or direct transcriptional regulation by Nrf2 impacts Kir function and related vascular tone.

A phase 1b study of dual PD-1 and CTLA-4 or KIR blockade in patients with relapsed/refractory lymphoid malignancies

Simultaneously targeting other pathways could increase the activity of PD-1 blockade in lymphoid malignancies not sensitive to single-agent blockade. We explored the safety and efficacy of combined PD-1 and CTLA-4 or KIR blockade in patients with relapsed/refractory (R/R) lymphoid malignancies. This phase 1b trial enrolled adult patients with R/R classical Hodgkin lymphoma (cHL), non-Hodgkin lymphoma (NHL), or multiple myeloma (MM). Patients received nivolumab plus ipilimumab (nivo/ipi) or lirilumab (nivo/liri) until complete response (CR), progression, or unacceptable toxicity. The primary endpoint was safety and tolerability, while secondary endpoints included overall (ORR) and CR rates (CRR), progression-free and overall survival. Sixty-five patients were treated with nivo/ipi, and 72 with nivo/liri. Twenty-nine percent of patients experienced grade 3–4 treatment-related adverse events with nivo/ipi, and 15% with nivo/liri. In cHL, ORR was 74% for nivo/ipi and 76% for nivo/liri, CRRs were 23% and 24%, respectively. In B-NHL and T-NHL, ORR range was 9–22% and CRR was 0–6%. No patient with MM had an objective response. While both combinations were active in cHL, the toxicity of nivo/ipi was higher than expected from nivolumab alone. These data suggest no meaningful improvement in the efficacy of the combinations over single-agent nivolumab in the diseases studied.

An assessment of KIR channel function in human cerebral arteries.

In the rodent cerebral circulation, inward rectifying K+ (KIR) channels set resting tone and the distance over which electrical phenomena spread along the arterial wall. The present study sought to translate these observations into human cerebral arteries obtained from resected brain tissue. Computational modeling and a conduction assay first defined the impact of KIR channels on electrical communication; patch-clamp electrophysiology, quantitative PCR, and immunohistochemistry then characterized KIR2.x channel expression/activity. In keeping with rodent observations, computer modeling highlighted that KIR blockade should constrict cerebral arteries and attenuate electrical communication if functionally expressed. Surprisingly, Ba2+ (a KIR channel inhibitor) had no effect on human cerebral arterial tone or intercellular conduction. In alignment with these observations, immunohistochemistry and patch-clamp electrophysiology revealed minimal KIR channel expression/activity in both smooth muscle and endothelial cells. This absence may be reflective of chronic stress as dysphormic neurons, leukocyte infiltrate, and glial fibrillary acidic protein expression was notable in the epileptic cortex. In closing, KIR2.x channel expression is limited in human cerebral arteries from patients with epilepsy and thus has little impact on resting tone or the spread of vasomotor responses. NEW & NOTEWORTHY KIR2.x channels are expressed in rodent cerebral arterial smooth muscle and endothelial cells. As they are critical to setting membrane potential and the distance signals conduct, we sought to translate this work into humans. Surprisingly, KIR2.x channel activity/expression was limited in human cerebral arteries, a paucity tied to chronic brain stress in the epileptic cortex. Without substantive expression, KIR2.x channels were unable to govern arterial tone or conduction.

A phase 1 study of lirilumab (antibody against killer immunoglobulin-like receptor antibody KIR2D; IPH2102) in patients with solid tumors and hematologic malignancies.

PurposeAnti-KIR monoclonal antibodies (mAbs) can enhance the antitumor responses of natural killer (NK) cells. We evaluated the safety of the anti-KIR2D mAb lirilumab in patients with various cancers.Experimental designThirty-seven patients with hematological malignancies (n = 22) or solid tumors (n = 15) were included in the study. Dose escalation (0.015 to 10 mg/kg) was conducted following a 3 + 3 design. Patients were scheduled to receive four cycles of treatment. In a second (extension) phase 17 patients were treated at 0.015 (n = 9) or 3 mg/kg (n = 8).ResultsNo dose-limiting toxicity was recorded. The most frequent lirilumab-related adverse events were pruritus (19%), asthenia (16%), fatigue (14%), infusion-related reaction (14%), and headache (11%), mostly mild or moderate. Pharmacokinetics was dose-dependent and linear, with minimal accumulation resulting from the 4-weekly repeated administrations. Full KIR occupancy (>95%) was achieved with all dosages, and the duration of occupancy was dose-related. No significant changes were observed in the number or distribution of lymphocyte subpopulations, nor was any reduction in the distribution of KIR2D-positive NK cells.ConclusionsThis phase 1 trial demonstrated the satisfactory safety profile of lirilumab up to doses that enable full and sustained blockade of KIR.

Sympatholytic effect of intravascular ATP is independent of nitric oxide, prostaglandins, Na+ /K+ -ATPase and KIR channels in humans.

Intravascular ATP attenuates sympathetic vasoconstriction (sympatholysis) similar to what is observed in contracting skeletal muscle of humans, and may be an important contributor to exercise hyperaemia. Similar to exercise, ATP-mediated vasodilatation occurs via activation of inwardly rectifying potassium channels (KIR ), and synthesis of nitric oxide (NO) and prostaglandins (PG). However, recent evidence suggests that these dilatatory pathways are not obligatory for sympatholysis during exercise; therefore, we tested the hypothesis that the ability of ATP to blunt α1 -adrenergic vasoconstriction in resting skeletal muscle would be independent of KIR , NO, PGs and Na+ /K+ -ATPase activity. Blockade of KIR channels alone or in combination with NO, PGs and Na+ /K+ -ATPase significantly reduced the vasodilatatory response to ATP, although intravascular ATP maintained the ability to attenuate α1 -adrenergic vasoconstriction. This study highlights similarities in the vascular response to ATP and exercise, and further supports a potential role of intravascular ATP in blood flow regulation during exercise in humans. Exercise and intravascular ATP elicit vasodilatation that is dependent on activation of inwardly rectifying potassium (KIR ) channels, with a modest reliance on nitric oxide (NO) and prostaglandin (PG) synthesis. Both exercise and intravascular ATP attenuate sympathetic α-adrenergic vasoconstriction (sympatholysis). However, KIR channels, NO, PGs and Na+ /K+ -ATPase activity are not obligatory to observe sympatholysis during exercise. To further determine similarities between exercise and intravascular ATP, we tested the hypothesis that inhibition of KIR channels, NO and PG synthesis, and Na+ /K+ -ATPase would not alter the ability of ATP to blunt α1 -adrenergic vasoconstriction. In healthy subjects, we measured forearm blood flow (Doppler ultrasound) and calculated changes in vascular conductance (FVC) to intra-arterial infusion of phenylephrine (PE; α1 -agonist) during ATP or control vasodilatator infusion, before and after KIR channel inhibition alone (barium chloride; n=7; Protocol 1); NO (l-NMMA) and PG (ketorolac) inhibition alone, or combined NO, PGs, Na+ /K+ -ATPase (ouabain) and KIR channel inhibition (n=6; Protocol 2). ATP attenuated PE-mediated vasoconstriction relative to adenosine (ADO) and sodium nitroprusside (SNP) (PE-mediated ΔFVC: ATP: -16±2; ADO: -38±6; SNP: -59±6%; P<0.05 vs. ADO and SNP). Blockade of KIR channels alone or combined with NO, PGs and Na+ /K+ -ATPase, attenuated ATP-mediated vasodilatation (∼35 and ∼60% respectively; P<0.05 vs. control). However, ATP maintained the ability to blunt PE-mediated vasoconstriction (PE-mediated ΔFVC: KIR blockade alone: -6±5%; combined blockade:-4±14%; P>0.05 vs. control). These findings demonstrate that intravascular ATP modulates α1 -adrenergic vasoconstriction via pathways independent of KIR channels, NO, PGs and Na+ /K+ -ATPase in humans, consistent with a role for endothelium-derived hyperpolarization in functional sympatholysis.

Targeting KIR Blockade in Multiple Myeloma: Trouble in Checkpoint Paradise?

Findings within the current issue indicate that treatment with IPH2101 when used as a monotherapy in smoldering multiple myeloma, meant to enhance natural killer (NK) cell function through inhibitory KIR blockade, results in a surprising reduction of NK-cell function mediated through monocyte trogocytosis. The significance of these findings is discussed. Clin Cancer Res; 22(21); 5161-3. ©2016 AACRSee related article by Carlsten et al., p. 5211.

KIR channels tune electrical communication in cerebral arteries

The conducted vasomotor response reflects electrical communication in the arterial wall and the distance signals spread is regulated by three factors including resident ion channels. This study defined the role of inward-rectifying K+ channels (KIR) in governing electrical communication along hamster cerebral arteries. Focal KCl application induced a vasoconstriction that conducted robustly, indicative of electrical communication among cells. Inhibiting dominant K+ conductances had no attenuating effect, the exception being Ba2+ blockade of KIR. Electrophysiology and Q-PCR analysis of smooth muscle cells revealed a Ba2+-sensitive KIR current comprised of KIR2.1/2.2 subunits. This current was surprisingly small and when incorporated into a model, failed to account for the observed changes in conduction. We theorized a second population of KIR channels exist and consistent with this idea, a robust Ba2+-sensitive KIR2.1/2.2 current was observed in endothelial cells. When both KIR currents were incorporated into, and then inhibited in our model, conduction decay was substantive, aligning with experiments. Enhanced decay was ascribed to the rightward shift in membrane potential and the increased feedback arising from voltage-dependent-K+ channels. In summary, this study shows that two KIR populations work collaboratively to govern electrical communication and the spread of vasomotor responses along cerebral arteries.

Identification and Localization of Gold Nanoparticles in Potassium Ion Pores: Implications for Kir Blockade.

IntroductionIn our previous study, we found that negatively charged gold nanoparticles with spermidine have the potential of blocking inwardly rectifying potassium channels (Kir), both at the cellular and the tissue level.MethodsFor the purpose of the present study, we purified the cytoplasmic domain of the Kir 3.1 receptor from Escherichia coli. Using single particles with surface coating by transmission electron microscopy, we identified the gold nanoparticles at the cytoplasmic side of the human Kir channel.ResultsEnergy-dispersive X-ray spectroscopy showed the presence of the gold deposits in the cytoplasmic domain of the Kir receptor.ConclusionIn conclusion, we could identify undecagold in the ion pore of the Kir3.1 channel in order to clarify its direct blocking effect in the Kir ion pore by undecagold.Electronic supplementary materialThe online version of this article (doi:10.1007/s40119-016-0060-8) contains supplementary material, which is available to authorized users.

Immune checkpoint inhibitors enhance cytotoxicity of cytokine-induced killer cells against human myeloid leukaemic blasts.

We studied whether blockade of inhibitory receptors on cytokine-induced killer (CIK) cells by immune checkpoint inhibitors could increase its anti-tumour potency against haematological malignancies. CIK cultures were generated from seven normal donors and nine patients with acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL) or multiple myeloma (MM). The inhibitory receptors B and T lymphocyte attenuator, CD200 receptor, lymphocyte activation gene-3 (LAG-3) and T cell immunoglobulin and mucin-domain-containing-3 (TIM-3) were present at variable percentages in most CIK cultures, while cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed death-1 (PD-1) and killer cell immunoglobulin-like receptors (KIR2DL1/2/3) were expressed at low level in most cultures. Without blockade, myeloid leukaemia cells were susceptible to autologous and allogeneic CIK-mediated cytotoxicity. Blockade of KIR, LAG-3, PD-1 and TIM-3 but not CTLA-4 resulted in remarkable increase in killing against these targets, even in those with poor baseline cytotoxicity. ALL and MM targets were resistant to CIK-mediated cytotoxicity, and blockade of receptors did not increase cytotoxicity to a meaningful extent. Combination of inhibitors against two receptors did not further increase cytotoxicity. Interestingly, potentiation of CIK killing by blocking antibodies was not predicted by expression of receptors on CIK and their respective ligands on the targets. Compared to un-activated T and NK cells, blockade potentiated the cytotoxicity of CIK cells to a greater degree and at a lower E:T ratio, but without significant increase in cytotoxicity against normal white cell. Our findings provide the basis for clinical trial combining autologous CIK cells with checkpoint inhibitors for patients with AML.

A Phase I Trial Of Anti-KIR Monoclonal Antibody IPH2101 and Lenalidomide For Multiple Myeloma

IntroductionMultiple myeloma (MM) remains an essentially incurable plasma cell malignancy. MM utilizes specific immunoevasive strategies to avoid natural killer (NK) cell immune surveillance and cytotoxicity. Immunomodulatory agents such as lenalidomide (LEN) may exert indirect anti-MM efficacy via expansion and activation of NK cells. However, these favorable effects may be diminished when LEN is co-administered with high doses of dexamethasone (DEX). IPH2101 is a monoclonal anti-inhibitory KIR antibody which prevents negative signaling in NK cells and enhances NK cell recognition and killing of MM cells. A single-agent, phase I study of IPH2101 demonstrated full KIR blockade with encouraging safety and tolerability, and 34% of heavily pre-treated patients achieved disease stabilization (Blood 2012;120:4324-33). Preclinical data demonstrate that LEN and IPH2101 exert anti-MM effects via complementary NK-cell immunomodulatory mechanisms (Blood 2011;118:6397-91). Herein, data are presented from the first clinical experience with IPH2101 and LEN in combination in patients with MM. MethodsA 3+3 phase I dose-escalation trial was conducted. Patients (age 18-80) with measurable, progressive MM were enrolled having received one or two prior lines of therapy. Prior LEN exposure was permitted unless resistance or intolerance was observed. Patients must have had ECOG performance status ≤ 2, creatinine clearance ≥ 60 ml/min, platelets ≥ 75,000/uL (or ≥ 30,000/uL if > 50% bone marrow plasma cells), absolute neutrophil count ≥ 1,000/uL, bilirubin < 1.5 ULN, and ALT / AST < 3 ULN. Patients must have adhered to standard prescribing guidelines for LEN. Three dose levels included: IPH2101 0.2mg/kg IV q 28 days + LEN 10 mg PO days 1-21; IPH2101 0.2 mg/kg + LEN 25 mg, and IPH2101 1mg/kg + LEN 25 mg for 4 cycles. Responding patients were allowed to receive 4 additional cycles. Patients completing all 8 cycles were maintained on LEN thereafter. No administration of DEX or other systemic corticosteroids was permitted. Dose reductions of LEN were permitted per prescribing information. The primary objective was to determine the safety and tolerability of IPH2101 + LEN, the secondary objectives included pharmacokinetics (PK) and pharmacodynamics (PD) of IPH2101 and biologic correlates with LEN as well as to determine clinical activity by standard IMWG uniform response criteria. Results15 patients (10 M, 5 F, median age 60) were enrolled, 8 in first relapse and 9 in second relapse. 9 had prior LEN exposure. Cohorts 1 and 3 were expanded to n=6 patients respectively due to occurrence of possible dose-limiting toxicity. In both cases, a patient experienced a similar, apparent infusion reaction on cycle 1, day 1, characterized by fever, chills, cytokine release, and leucopenia. Events resolved with supportive care and both patients continued on trial without recurrence. The protocol was amended to include premedication with anti-histamine and acetaminophen,and no further infusion reactions were observed. Most other observed adverse events were of low grade and generally investigator-attributed as possibly or probably related to LEN. IPH2101 PD were not affected by co-administration of LEN. Full KIR occupancy was achieved in cohort 3 across the dosing interval. Five patients achieved a response (2 VGPR, 3 PR) with a median duration of 15+ months (3-26+). ConclusionThe combination of IPH2101 + LEN appears to be a safe and well tolerated, and steroid-free combination in MM patients. Infusion reactions have not been observed since the addition of premedication prior to IPH2101 dosing. IPH2101 PD do not appear to be altered by co-administration of LEN, and full KIR blockade over the dosing interval has been achieved. Although the study is small, response rate and response duration are encouraging. These findings support further investigation of antiKIR therapy with LEN as the first, steroid-sparing, dual immunotherapy for MM. Disclosures:Benson:Innate Pharma: Research Funding. Off Label Use: Lenalidomide without concomitant dexamethasone. Zerbib:Innate Pharma: Employment. Andre:Innate Pharma: Employment. Caligiuri:Innate Pharma: Membership on an entity's Board of Directors or advisory committees.

Potassium Buffering in the Neurovascular Unit: Models and Sensitivity Analysis

Astrocytes are critical regulators of neural and neurovascular network communication. Potassium transport is a central mechanism behind their many functions. Astrocytes encircle synapses with their distal processes, which express two potassium pumps (Na-K and NKCC) and an inward rectifying potassium channel (Kir), whereas the vessel-adjacent endfeet express Kir and BK potassium channels. We provide a detailed model of potassium flow throughout the neurovascular unit (synaptic region, astrocytes, and arteriole) for the cortex of the young brain. Our model reproduces several phenomena observed experimentally: functional hyperemia, in which neural activity triggers astrocytic potassium release at the perivascular endfoot, inducing arteriole dilation; K+ undershoot in the synaptic space after periods of neural activity; neurally induced astrocyte hyperpolarization during Kir blockade. Our results suggest that the dynamics of the vascular response during functional hyperemia are governed by astrocytic Kir for the fast onset and astrocytic BK for maintaining dilation. The model supports the hypothesis that K+ undershoot is caused by excessive astrocytic uptake through Na-K and NKCC pumps, whereas the effect is balanced by Kir. We address parametric uncertainty using high-dimensional stochastic sensitivity analysis and identify possible model limitations.

ATP‐mediated vasodilatation: all thanks to potassium?

ATP‐mediated vasodilatation: all thanks to potassium?

Imbalance of activating and inhibitory receptor expression on effector cells in multiple myeloma patients results in immune suppression (127.35)

Abstract Multiple myeloma (MM) is an incurable plasma cell malignancy of the bone marrow (BM). Recent evidence suggests that natural killer (NK) cells are intricately involved in the immunosurveillance of MM and can specifically target malignant plasma cells in the absence of the immunosuppressive BM microenvironment. Immune suppression in MM patients is systemic with total CD4 T cell counts in the peripheral blood significantly lower than healthy donors. We have data showing that the major NK activating receptor NKG2D is significantly reduced in MM patients, whereas inhibitory NKG2A, KIR2DL1 and KIR2DL3 are up-regulated. These inhibitory receptors target HLA-E and C respectively, and dominate over activating signals. Interestingly, we report that NKG2A, KIR2DL1 and KIR2DL3 were also up-regulated on T cells in MM patients. Our subsequent investigations showed that expression of KIR receptors could be induced on CD4 T cells independent of direct cell contact with MM cells. Throughout treatment using an empirical MM drug regimen including an immunomodulatory drug and a corticosteroid, we saw restoration of NKG2D but no change in the expression of inhibitory receptors on NK or T cells in MM patients. Together, this data reveals a novel model of T cell tolerance to MM and highlights to therapeutic potential of KIR blockade to reverse immune suppression.

Relationship between glial potassium regulation and axon excitability: a role for glial Kir4.1 channels.

Uptake of K(+) released by axons during action potential propagation is a major function of astrocytes. Here, we demonstrate the importance of glial inward rectifying potassium channels (Kir) in regulating extracellular K(+) ([K(+)](o)) and axonal electrical activity in CNS white matter of the mouse optic nerve. Increasing optic nerve stimulation frequency from 1 Hz to 10-35 Hz for 120 s resulted in a rise in [K(+)](o) and consequent decay in the compound action potential (CAP), a measure of reduced axonal activity. On cessation of high frequency stimulation, rapid K(+) clearance resulted in a poststimulus [K(+)](o) undershoot, followed by a slow recovery of [K(+)](o) and the CAP, which were more protracted with increasing stimulation frequency. Blockade of Kir (100 μM BaCl(2)) slowed poststimulus recovery of [K(+)](o) and the CAP at all stimulation frequencies, indicating a primary function of glial Kir was redistributing K(+) to the extracellular space to offset active removal by Na(+)-K(+) pumps. At higher levels of axonal activity, Kir blockade also increased [K(+)](o) accumulation, exacerbating the decline in the CAP and impeding its subsequent recovery. In the Kir4.1-/- mouse, astrocytes displayed a marked reduction of inward currents and were severely depolarized, resulting in retarded [K(+)](o) regulation and reduced CAP. The results demonstrate the importance of glial Kir in K(+) spatial buffering and sustaining axonal activity in the optic nerve. Glial Kir have increasing importance in K(+) clearance at higher levels of axonal activity, helping to maintain the physiological [K(+)](o) ceiling and ensure the fidelity of signaling between the retina and brain.

IPH2101, a Novel Anti-Inhibitory KIR Monoclonal Antibody for Multiple Myeloma: Interm Phase 1 Trial Results and Correlative Biologic and Safety Data

AbstractAbstract 1966 Introduction:A potent natural killer (NK) cell versus multiple myeloma (MM) effect has been reported in clinical observations and characterized mechanistically in translational studies. However, the NK cell versus MM effect is lost as the disease methodically progresses due, in part, to upregulation of MHC Class 1 antigens on MM tumor cells which serve as ligands to inhibitory KIR on NK cells. Our group has shown that IPH-2101, a novel IgG4 monoclonal blocking antibody against commonly expressed inhibitory KIR (KIR2DL-1, -2 and -3), may augment NK cell function against MM tumor cells. A single-agent phase 1 open-label, dose escalation study of IPH-2101 is nearing completion to determine the safety, tolerability and biologic effects of inhibitory KIR blockade as a novel form of therapy for MM. Methods:A single-agent, open-label, Phase 1 dose escalation study is being performed with 7 cohorts in standard 3+3 design. The protocol was amended to include 7 additional patients with less heavily pretreated disease in the final cohort. Doses from 0.0003 mg/kg to 3mg/kg are being evaluated. Patients enrolled on the 7 cohorts had heavily pre-treated, relapsed/refractory MM with at least one prior therapy, whereas 7 additional patients accrued to the final cohort are limited to one prior therapy only. IPH-2101 is administered intravenously once every 28 days, with 4 cycles of therapy possible as determined by safety, tolerability and disease characteristics. Extensive pharmacodynamic (PD), pharmacokinetic (PK), KIR occupancy, and correlative biologic data are being collected. Results:Currently, 32 patients have been enrolled, and 5 patients remain on study in the final cohort. IPH-2101 has been well tolerated without determination of a dose limiting toxicity (DLT). 1 patient at dose level 1 was replaced and 3 additional patients were enrolled at dose level 4 due to an SAE (acute renal failure) deemed possibly related to drug. PK and PD data closely approximate those predicted by preclinical modeling. Full KIR occupancy across the dosing interval appears to be achieved in most cases with the 1mg/kg dose. No evidence of autoimmunity and no deleterious effect on NK cell maturation has been observed to date. Ongoing correlative studies will characterize NK cell phenotype, cytotoxicity, and effects of IPH-2101 on immunomodulation. To date, no objective responses have been observed; however, preliminary analyses suggest that several patients may have achieved disease stabilization. Conclusions:IPH-2101 improves autologous NK cell-mediated cytotoxicity against MM tumor cells and appears to be well tolerated and safe with achievement of biologic endpoints independent of DLT or maximally tolerated dose. Preliminary data suggest that several patients may have disease stabilization on therapy. Updated correlative, biologic data will be presented at the meeting. Disclosures:No relevant conflicts of interest to declare.

Effect of tempol on inward‐rectifier K+ channel regulation of afferent arteriolar tone in diabetic rat kidney

We previously reported that inward‐rectifier K+ channels (Kir) contribute to the decreased afferent arteriolar tone that accompanies streptozotocin (STZ)‐induced diabetes in the rat. We hypothesized that the oxidative stress characteristic of diabetes underlies this phenomenon. Sham and STZ rats were chronically‐treated with tempol (SOD mimetic; 9 ± 1 mg/day) or left untreated for 27 days. The in vitro blood‐perfused juxtamedullary nephron technique was used to quantify afferent arteriolar lumen diameter responses to Kir blockade (10, 30, 100 μM Ba2+). In untreated animals, lumen diameter averaged 23.8 ± 1.4 µm in STZ and 18.6 ± 1.9 μm in Sham rats (P=0.05) and the concentration‐dependent response to Ba2+ was exaggerated in the STZ group (100 nM Ba2+: Sham δ = ‐2.0 ± 0.7 μm, STZ δ = ‐7.9 ± 1.4 μm; P&lt;0.001). In tempol‐treated animals, baseline lumen diameter did not differ between Sham and STZ rats, averaging 21.4 ± 1.4 and 21.1 ± 1.6 μm, respectively. Moreover, the afferent arteriolar diameter response to Ba2+ was attenuated in tempol‐treated STZ rats (100 nM Ba2+: δ = ‐2.4 ± 1.3 μm; P&lt;0.05 vs untreated STZ) and did not differ from either untreated or tempol‐treated Sham rats. Thus, tempol normalized baseline diameter and the response to Ba2+ in afferent arterioles from STZ rats. We conclude that oxidative stress plays a role in the Kir‐dependent tonic afferent arteriolar dilation that occurs during STZ‐induced diabetes.

Blocking Inhibitory KIR Is Insufficient for Optimal Killing of AML and ALL Targets: Additional Requirements for NKG2A and LIR-1 Blockade

NK cell alloreactivity is determined by the interaction between inhibitory NK cell receptors and their MHC class I cognate ligands. The best characterized NK cell receptor that recognizes MHC I is KIR. Mismatching donors and recipients for KIR ligands has led to transplant strategies that can result in less relapse and better survival in myeloid leukemia patients. However, other MHC recognizing receptors may also be involved in leukemia eradication. We developed a model using primary AML and ALL targets to explore the hierarchy of different class I MHC recognizing inhibitory NK cell receptors. In addition to KIR, NKG2A is expressed on 54±14% of PB NK cells and is the dominant NK cell receptor expressed in the first six months after transplant. LIR-1 is expressed on 35±17% of PB NK cells and is more likely to be co-expressed on KIR+ NK cells than KIR- NK cells. The goal of our study was to determine optimal conditions to enhance NK alloreactivity against primary AML and ALL targets. A pan-HLA blocking antibody (HP-1F7, binding to HLA, B, C, E and G) was used as a positive control to interrupt all signals from MHC class I. Receptor blockade with anti-KIR (1-7F9, blocking KIR 2DL1/L2/L3, Novo Nordisk), anti-NKG2A and anti-LIR-1 was tested either alone or in combination to understand the hierarchy of inhibitory receptors that determine NK cell reactivity of leukemia targets. Resting NK cells were tested fresh or activated with low concentrations of IL-2 (5 IU for 42 hours) to mimic conditions achievable by pharmacologic IL-2 administration. Six of ten primary AML targets were sensitive to allogeneic NK cell killing only when HLA-A, B, C, E and G were blocked (7.8±5.7 vs. 24±9.6, n=6; p<0.005). Six of seven primary ALL targets also responded to resting allogeneic NK cells but only after class I blockade (8.2±5.7 vs. 25±9.7, n=6; p<0.005). Low concentrations of IL-2 enhanced mean killing with class I blockade by approximately 10% for AML and ALL targets. CD107a degranulation yielded similar results. We next evaluated the class I recognizing inhibitory receptors on NK cells responsible for the enhanced killing of primary leukemia targets. Experiments were performed with KIR, NKG2A and LIR-1 blockade. In both cytotoxicity and CD107a degranulation assays, single inhibitory receptor blockade resulted in enhanced killing slightly. However, addition of LIR-1 blockade to either KIR blockade or NKG2A blockade consistently increased killing of all targets sensitive to pan-HLA blockade. Purification of KIR- NK cells, expressing NKG2A or LIR-1, showed they were potently alloreactive against primary leukemia targets only upon dual blockade with NKG2A and LIR-1 mAbs. Blocking of more than one receptor was always more effective than single receptor blockade. All leukemia targets expressed HLA-E (the ligand for NKG2A) but none expressed HLA-G (the high affinity ligand for LIR-1) by Western Blot analysis, suggesting that other HLA class I (low affinity ligands for LIR-1) are involved. Our data show that:Both KIR+ and KIR- NK cells exhibit significant potential for cytotoxicity of killing primary AML and ALL blasts but only when more than one class I receptor was blocked,KIR- NK cells can be licensed through receptors other than KIR, andMore than half of primary AML and ALL targets tested are susceptible to allogeneic NK cell killing following multiple inhibitory receptor blockade.Therefore, it is not surprising that the current strategies of adoptive NK cell therapy may be limited. Clinical trials with mAb blocking of multiple MHC class I inhibitor receptors (KIR, NKG2A, or LIR-1) to enhance NK cell killing of AML and ALL is warranted.

Kir4.1 channels regulate swelling of astroglial processes in experimental spinal cord edema

In glial cells, inwardly rectifying K(+) channels (Kir) control extracellular [K(+) ](o) homeostasis by uptake of K(+) from the extracellular space and release of K(+) into the microvasculature. Kir channels were also recently implicated in K(+) -associated water influx and cell swelling. We studied the time-dependent expression and functional implication of the glial Kir4.1 channel for astroglial swelling in a spinal cord edema model. In this CNS region, Kir4.1 is expressed on astrocytes from the second postnatal week on and co-localizes with aquaporin 4 (AQP4). Swelling of individual astrocytes in response to osmotic stress and to pharmacological Kir blockade were analyzed by time-lapse-two-photon laser-scanning microscopy in situ. Application of 30% hypotonic solution induced astroglial soma swelling whereas no swelling was observed on astroglial processes or endfeet. Co-application of hypotonic solution and Ba(2+) , a Kir channel blocker, induced prominent swelling of astroglial processes. In Kir4.1-/- mice, however, somatic as well as process swelling was observed upon application of 30% hypotonic solutions. No additional effect was provoked upon co-application with Ba(2+) . Our experiments show that Kir channels prevent glial process swelling under osmotic stress. The underlying Kir channel subunit that controls glial process swelling is Kir4.1, whereas changes of the glial soma are not substantially related to Kir4.1.