Sandip Panicker

AF-353, a novel, potent and orally bioavailable P2X3/P2X2/3 receptor antagonist

Background and purpose:  Purinoceptors containing the P2X3 subunit (P2X3 homotrimeric and P2X2/3 heterotrimeric) are members of the P2X family of ion channels gated by ATP and may participate in primary afferent sensitization in a variety of pain‐related diseases. The current work describes the in vitro pharmacological characteristics of AF‐353, a novel, orally bioavailable, highly potent and selective P2X3/P2X2/3 receptor antagonist.

TNT003, an inhibitor of the serine protease C1s, prevents complement activation induced by cold agglutinins

Activation of the classical pathway (CP) of complement is often associated with autoimmune disorders in which disease pathology is linked to the presence of an autoantibody. One such disorder is cold agglutinin disease (CAD), an autoimmune hemolytic anemia in which autoantibodies (cold agglutinins) bind to red blood cells (RBCs) at low temperatures. Anemia occurs as a result of autoantibody-mediated CP activation on the surface of the erythrocyte, leading to the deposition of complement opsonins that drive extravascular hemolysis in the liver. Here we test the effects of TNT003, a mouse monoclonal antibody targeting the CP-specific serine protease C1s, on CP activity induced by cold agglutinins on human RBCs. We collected 40 individual CAD patient samples and showed that TNT003 prevented cold agglutinin-mediated deposition of complement opsonins that promote phagocytosis of RBCs. Furthermore, we show that by preventing CP activation, TNT003 also prevents cold agglutinin-driven generation of anaphylatoxins. Finally, we provide evidence that CP activity in CAD patients terminates prior to activation of the terminal cascade, supporting the hypothesis that the primary route of RBC destruction in these patients occurs via extravascular hemolysis. Our results support the development of a CP inhibitor for the treatment of CAD.

Characterization of a novel CRAC inhibitor that potently blocks human T cell activation and effector functions

Store operated calcium entry (SOCE) downstream of T cell receptor (TCR) activation in T lymphocytes has been shown to be mediated mainly through the Calcium Release Activated Calcium (CRAC) channel. Here, we compared the effects of a novel, potent and selective CRAC current inhibitor, 2,6-Difluoro-N-{5-[4-methyl-1-(5-methyl-thiazol-2-yl)-1,2,5,6-tetrahydro-pyridin-3-yl]-pyrazin-2-yl}-benzamide (RO2959), on T cell effector functions with that of a previously reported CRAC channel inhibitor, YM-58483, and a calcineurin inhibitor Cyclosporin A (CsA). Using both electrophysiological and calcium-based fluorescence measurements, we showed that RO2959 is a potent SOCE inhibitor that blocked an IP3-dependent current in CRAC-expressing RBL-2H3 cells and CHO cells stably expressing human Orai1 and Stim1, as well as SOCE in human primary CD4(+) T cells triggered by either TCR stimulation or thapsigargin treatment. Furthermore, we demonstrated that RO2959 completely inhibited cytokine production as well as T cell proliferation mediated by TCR stimulation or MLR (mixed lymphocyte reaction). Lastly, we showed by gene expression array analysis that RO2959 potently blocked TCR triggered gene expression and T cell functional pathways similar to CsA and another calcineurin inhibitor FK506. Thus, both from a functional and transcriptional level, our data provide evidence that RO2959 is a novel and selective CRAC current inhibitor that potently inhibits human T cell functions.

Synaptic AMPA receptor subunit trafficking is independent of the C terminus in the GluR2-lacking mouse

Glutamate is the primary excitatory neurotransmitter in the brain, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors mediate most fast synaptic transmission. AMPA receptors are tetrameric assemblies composed from four possible subunits (GluR1–4). In hippocampal pyramidal cells, AMPA receptors are heteromeric receptors containing the GluR2 subunit and either GluR1 or GluR3. It is generally accepted that the trafficking of GluR1/GluR2 receptors to synapses requires activity, whereas GluR2/GluR3 receptors traffic constitutively. It has been suggested that the trafficking is governed by the cytoplasmic C termini of the subunits. Because the basis for this theory relied on the introduction of unnatural, homomeric, calcium-permeable AMPA receptors, we have used the GluR2−/− knock out mouse to determine whether the expression of mutated forms of GluR2 can rescue WT synaptic responses. We find that GluR2, lacking its entire C terminus, or a GluR2 chimera containing the C terminus of GluR1, is capable of trafficking to the synapse in the absence of activity. These findings suggest that the GluR2 C terminus is not required for GluR2 synaptic insertion.

An Anti-C1s Monoclonal, TNT003, Inhibits Complement Activation Induced by Antibodies Against HLA.

Antibody‐mediated rejection (AMR) of solid organ transplants (SOT) is characterized by damage triggered by donor‐specific antibodies (DSA) binding donor Class I and II HLA (HLA‐I and HLA‐II) expressed on endothelial cells. While F(ab′)2 portions of DSA cause cellular activation and proliferation, Fc regions activate the classical complement cascade, resulting in complement deposition and leukocyte recruitment, both hallmark features of AMR. We characterized the ability of an anti‐C1s monoclonal antibody, TNT003, to inhibit HLA antibody (HLA‐Ab)‐induced complement activation. Complement deposition induced by HLA‐Ab was evaluated using novel cell‐ and bead‐based assays. Human aortic endothelial cells (HAEC) were cultured with HLA‐Ab and human complement; production of activated complement proteins was measured by flow cytometry. Additionally, C3d deposition was measured on single antigen beads (SAB) mixed with HLA‐Ab and human complement. TNT003 inhibited HLA‐Ab mediated complement deposition on HAEC in a concentration‐dependent manner; C3a, C4a and C5a anaphylatoxin production was also diminished by TNT003. Finally, TNT003 blocked C3d deposition induced by Class I (HLAI‐Ab)‐ and Class II (HLAII‐Ab)‐specific antibodies on SAB. These data suggest TNT003 may be useful for modulating the effects of DSA, as TNT003 inhibits complement deposition and split product formation generated by HLA‐I/II‐Ab in vitro.

Minimal Structural Rearrangement of the Cytoplasmic Pore during Activation of the 5-HT3A Receptor

Ligand-gated ion channel receptors mediate the response of fast neurotransmitters by opening in less than a millisecond. Here, we investigated the activation mechanism of a serotonin-gated receptor (5-HT3A) by systematically introducing cysteine substitutions throughout the pore-lining M1-M2 loop and M2 transmembrane domain. We hypothesized that multiple cysteines in the narrowest region of the pore, which together can form a high affinity binding site for metal cations, would reveal changes in pore structure during gating. Using cadmium (Cd2+) as a probe, two cysteine substitutions in the cytoplasmic selectivity filter, S2′C and, to a lesser extent, G-2′C, showed high affinity inhibition with Cd2+ when applied extracellularly in the open state. Cd2+ inhibition in S2′C was attenuated if applied in the presence of an open-channel inhibitor and showed voltage-dependent recovery, indicating a direct effect of Cd2+ in the pore. When applied intracellularly, Cd2+ appeared to bind S2′C receptors in the closed state. The ability of cysteine side chains at the 2′ and –2′ positions to coordinate Cd2+ in both the native open and closed states of the channel suggests that the cytoplasmic selectivity filter of 5-HT3A receptors maintains a narrow pore during channel gating.

Altered nuclear localization of bax protein in BCNU-resistant glioma cells.

To investigate the role of apoptosis suppression in glioma chemotherapy resistance, protein levels and subcellular localization of bcl-2 family members were investigated in 2 pairs of sensitive cell lines and their in vitro generated resistant derivatives. The alkylating agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), induced apoptosis in both sensitive cell strains and apoptosis was suppressed in both resistant derivatives. Both resistant cell lines contained altered regulation of a bcl-2 related protein consistent with the suppression of apoptosis. Independent of which bcl-2 family member was dysregulated, resistance was associated with altered regulation in the subcellular localization of bax protein. Following BCNU treatment, bax accumulated in nucleoli and a nuclei containing fraction of sensitive cells but not their resistant derivatives. Nuclear accumulation was an early event in apotosis induction. These data indicates altered subcellular localization of bax may play a role in resistance. In addition, the association between an early, nucleolar localization of bax and the induction of apoptosis suggests that localization of bax to nucleoli may play a role in apoptosis-induction of glioma cells.

Uncoupling complement C1s activation from C1q binding in apoptotic cell phagocytosis and immunosuppressive capacity

Complement activation contributes to inflammation in many diseases, yet it also supports physiologic apoptotic cells (AC) clearance and its downstream immunosuppressive effects. The roles of individual complement components in AC phagocytosis have been difficult to dissect with artificially depleted sera. Using human in vitro systems and the novel antibody complement C1s inhibitor TNT003, we uncoupled the role of the enzymatic activation of the classical pathway from the opsonizing role of C1q in mediating a) the phagocytosis of early and late AC, and b) the immunosuppressive capacity of early AC. We found that C1s inhibition had a small impact on the physiologic clearance of early AC, leaving their immunosuppressive properties entirely unaffected, while mainly inhibiting the phagocytosis of late apoptotic/secondary necrotic cells. Our data suggest that C1s inhibition may represent a valuable therapeutic strategy to control classical pathway activation without causing significant AC accumulation in diseases without defects in AC phagocytosis.

Anti-C1s Monoclonal Antibody BIVV009 in Late Antibody-Mediated Kidney Allograft Rejection - Results from a First-in-Patient Phase 1 Trial

The classical pathway (CP) of complement may contribute to the pathogenesis of antibody‐mediated rejection (ABMR). Selective CP blockade may be a promising strategy to counteract rejection. The objective of this first‐in‐patient phase 1b trial was to evaluate the safety/tolerability and CP‐blocking potential of 4 weekly doses (60 mg/kg) of the anti‐C1s antibody BIVV009 in complement‐mediated disorders. Here we describe the results in a cohort of 10 stable kidney transplant recipients (median of 4.3 years posttransplantation) with late active ABMR and features of CP activation, such as capillary C4d or complement‐fixing donor‐specific antibodies (DSA). During 7 weeks follow‐up, no severe adverse events were reported, and BIVV009 profoundly inhibited overall and DSA‐triggered CP activation in serum. Five of 8 C4d‐positive recipients turned C4d‐negative in 5‐week follow‐up biopsies, while another 2 recipients showed a substantial decrease in C4d scores. There was, however, no change in microcirculation inflammation, gene expression patterns, DSA levels, or kidney function. In conclusion, we demonstrate that BIVV009 effectively blocks alloantibody‐triggered CP activation, even though short‐course treatment had no effect on indices of activity in late ABMR. This initial trial provides a valuable basis for future studies designed to clarify the therapeutic value of CP blockade in transplantation. ClinicalTrials.gov NCT#02502903.

Classical complement pathway activation in immune thrombocytopenia purpura: inhibition by a novel C1s inhibitor.

Immune thrombocytopenia purpura (ITP) is an autoimmune disorder whose pathophysiology is characterized by immune mediated platelet destruction and decreased platelet production (Johnsen 2012). Platelet destruction in ITP occurs by a variety of immune mediated mechanisms, including humoral and cell mediated immunity. Increasing evidence suggests a role for complement activation in ITP (Peerschke, et al 2009, Najaoui, et al 2011). Although early work by Frank et al (1975) demonstrated that an intact classical complement pathway was required for damage of antibody sensitized mammalian cell membranes and the development of thrombocytopenia in a guinea pig model, the role for classical pathway (CP) complement activation in human ITP has not been definitively established. The lectin pathway has been reported also to be activated by antibodies and autoantibodies, generating the same activated complement proteins downstream of C1, as the classical pathway (Malhotra et al, 1995). In the present study, we performed an in vitro trial of a novel classical pathway complement inhibitor (TNT003), a murine monoclonal antibody directed against C1s, to evaluate the role of CP activation in ITP, and provide in vitro proof of principle that CP inhibition prevents complement activation in ITP patient plasma. TNT003 is a novel C1s inhibitor that has been shown to inhibit cold agglutinin mediated complement deposition on the surface of red blood cells in vitro (Shi, et al 2014). Patients (n=55) consisted of males (n=21), age 39 ± 24 years (mean ± S.D.) (range 8–87 years) and females (n=34), age 48 ± 23 years (range 17–86 years), with a median ITP duration of 106 months and 99.5 months, respectively. At the time of blood collection, patients were undergoing treatment with a variety of modalities including Rituximab, IVIG, Eltrombopag, Romiplostin,, Veltuzumab, Cyclopsorin, Danazol, Azathioprine, Prednisone, Dexamethasone, and Mycophenolate mofetil, either alone or in combination. Fifteen patients had undergone splenectomy. Results of CP activation were compared to platelet count, obtained as part of the patient’s clinical laboratory assessment, and the presence of antiplatelet antibodies (IgG, IgM, IgA) directed against major platelet membrane glycoprotein antigens, IIb/IIIa, Ia/IIa, and Ib/IX, using the Lifecodes Pak12 assay (Immucor GTI Diagnostics, Inc. Waukesha, WI). This study was approved by the Institutional Review Boards of Weill Cornell Medical School and Memorial Sloan Kettering Cancer Center. CP activation was evaluated using a previously described assay (Peerschke, et al 2009). Since complement activation occurs spontaneously on activated or immobilized platelets (Peerschke, et al 2010), CP activation by patient plasma was expressed as a ratio relative to pooled normal control plasma, in order to detect enhanced complement activation. Enhanced complement activation was defined as a ratio of ≥ 1.5, representing values greater than 3 S.D. above the reference interval (97.5% confidence limit). Increased complement activation was noted in 26/55 patients with ITP (~47%). Elevated C1q deposition was found in 42% of patients (23/55 patient plasma samples). Enhanced C4d deposition was demonstrated in ~13% of patients (7/55 plasma samples). The latter was associated with a statistically significant inverse correlation (p=0.042) with platelet count (Figure). In 6 of 7 patients with heightened C4d deposition, the circulating whole blood platelet count was below 100K/μl, including 5 patients with platelet counts below 50K/μl. Figure Correlation between classical pathway (CP) complement activation, represented here by C4d deposition, and platelet count (Advia 2120, Siemens Healthcare Systems, Tarrytown, NY) in patients with chronic ITP. CP activation is expressed as a ratio relative ... Increased C4d deposition was associated with the presence of autoantibodies directed against major platelet antigens in all 5 patients with platelet counts below 50K/μl. In these patients (5/5), antibodies directed against GPIIb-IIIa were identified. Three patients additionally demonstrated antibodies against GPIa/IIa. A single patient exhibited detectable autoantibodies also against GPIb/IX. These findings are consistent with previous reports summarized by McMillan (2009), who described anti platelet antibodies in approximately 58% of patients, with reactivity to GPIIb-IIIa being the most common. CP activation was inhibited by TNT003, as demonstrated by reduced C4d deposition, and markedly reduced downstream C3b and C5b-9 deposition from patient plasma (n=55)(Table). More complete complement inhibition was achieved by chelation of divalent cations with 10 mM EDTA, confirming the participation also of the alternative pathway in complement activation on platelets (Peerschke et al, 2009). TNT003 appears to impact CP activation predominantly downstream of C1 binding, and supports the notion that CP activation plays a major role in terminal complement pathway activation in ITP plasma. Indeed, in vitro platelet lysis has been described following normal platelet exposure to autoantibodies from ITP patient sera (McMillan, et al 1981). Table Inhibition of classical pathway (CP) complement activation in plasma from patients with chronic ITP (n=55) In addition to direct cellular damage via C5b-9 lytic complexes, autoantibody mediated complement deposition promotes platelet clearance by the reticuloendothelial system (Johnsen, 2012). Interestingly, ITP patients with a high degree of platelet associated complement deposition/fixation have been reported to benefit significantly from splenectomy (Bell, 2002). In the present study, increased plasma CP activation was noted in 10 of 15 patients who had undergone splenectomy (p= 0.032, Fisher’s Exact Test). These preliminary in vitro findings may suggest that inhibition of CP activation could be a potential alternative to splenectomy for select patients with ITP. Taken together, the present study provides direct evidence of increased CP activation in ITP and proof of principle that CP inhibition may effectively target this process. CP blockade using C1 esterase inhibitor (C1 INH) has been used clinically, predominantly in patients with hereditary angioedema. Although C1 INH therapy is well tolerated in humans, it exerts effects beyond regulation of the classical complement pathway, including modulation of the lectin pathway and kinin, coagulation, and fibrinolytic systems. Targeted inhibition of C1s, therefore, may represent more specific inhibition of CP activation. Indeed, TNT003, when tested in vitro against C1 INH was found to be >3 orders of magnitude more potent for inhibiting antibody dependent complement activation in hemolysis based assays (data not shown). Evaluation of CP complement inhibition in ITP and the potential risk for infection by pyogenic bacteria with chronic use awaits clinical trails.