Inhibitory affinity modulation of FcγRIIA ligand binding by glycosphingolipids by inside-out signaling.

The interaction of the human FcγRIIA with immune complexes (ICs) promotes neutrophil activation and thus must be tightly controlled to avoid damage to healthy tissue. Here, we demonstrate that a fungal-derived soluble ß-1,3/1,6-glucan binds to the glycosphingolipid long-chain lactosylceramide (LacCer) to reduce FcγRIIA-mediated recruitment to immobilized ICs under flow, a process requiring high-affinity FcγRIIA-immunoglobulin G (IgG) interactions. The inhibition requires Lyn phosphorylation of SHP-1 phosphatase and the FcγRIIA immunotyrosine-activating motif. ß-glucan reduces the effective 2D affinity of FcγRIIA for IgG via Lyn and SHP-1 and, in vivo, inhibits FcγRIIA-mediated neutrophil recruitment to intravascular IgG deposited in the kidney glomeruli in a glycosphingolipid- and Lyn-dependent manner. In contrast, ß-glucan did not affect FcγR functions that bypass FcγR affinity for IgG. In summary, we have identified a pathway for modulating the 2D affinity of FcγRIIA for ligand that relies on LacCer-Lyn-SHP-1-mediated inhibitory signaling triggered by ß-glucan, a previously described activator of innate immunity.

A randomized, controlled trial evaluating the efficacy and safety of BTH1677 in combination with bevacizumab, carboplatin, and paclitaxel in first-line treatment of advanced non-small cell lung cancer.

BACKGROUND: BTH1677, a beta-glucan pathogen-associated molecular pattern molecule, drives an anti-cancer immune response in combination with oncology antibody therapies. This phase II study explored the efficacy, pharmacokinetics (PK), and safety of BTH1677 combined with bevacizumab/carboplatin/paclitaxel in patients with untreated advanced non-small cell lung cancer (NSCLC). METHODS: Patients were randomized to the BTH1677 arm (N = 61; intravenous [IV] BTH1677, 4 mg/kg, weekly; IV bevacizumab, 15 mg/kg, once each 3-week cycle [Q3W]; IV carboplatin, 6 mg/mL/min Calvert formula area-under-the-curve, Q3W; and IV paclitaxel, 200 mg/m2, Q3W) or Control arm (N = 31; bevacizumab/carboplatin/paclitaxel as above). Carboplatin/paclitaxel was discontinued after 4-6 cycles and patients who responded or remained stable received maintenance therapy with BTH1677/bevacizumab (BTH1677 arm) or bevacizumab (Control arm). Efficacy assessments, based on blinded central radiology review, included objective response rate (ORR; primary endpoint), disease control rate, duration of objective response, and progression-free survival. Overall survival and adverse events (AEs) were also assessed. RESULTS: ORR was higher in the BTH1677 vs Control arm but the difference between groups was not statistically significant (60.4% vs 43.5%; P = .2096). All other clinical endpoints also favored the BTH1677 arm but none statistically differed between arms. PK was consistent with previous studies. Although a higher incidence of Grade 3/4 AEs occurred in the BTH1677 vs Control arm (93.2% vs 66.7%), no unexpected AEs were observed. Serious AEs and discontinuations due to AEs were lower in the BTH1677 vs Control arm. CONCLUSIONS: Improvements in tumor assessments and survival were observed with BTH1677/bevacizumab/carboplatin/paclitaxel compared with control treatment in patients with advanced NSCLC. TRIAL REGISTRATION: ClinicalTrials.gov registration ID: NCT00874107 . Registered 2 April 2009. First participant was enrolled on 29 September 2009.

Imprime PGG-Mediated Anti-Cancer Immune Activation Requires Immune Complex Formation.

Imprime PGG (Imprime), an intravenously-administered, soluble ß-glucan, has shown compelling efficacy in multiple phase 2 clinical trials with tumor targeting or anti-angiogenic antibodies. Mechanistically, Imprime acts as pathogen-associated molecular pattern (PAMP) directly activating innate immune effector cells, triggering a coordinated anti-cancer immune response. Herein, using whole blood from healthy human subjects, we show that Imprime-induced anti-cancer functionality is dependent on immune complex formation with naturally-occurring, anti-ß glucan antibodies (ABA). The formation of Imprime-ABA complexes activates complement, primarily via the classical complement pathway, and is opsonized by iC3b. Immune complex binding depends upon Complement Receptor 3 and Fcg Receptor IIa, eliciting phenotypic activation of, and enhanced chemokine production by, neutrophils and monocytes, enabling these effector cells to kill antibody-opsonized tumor cells via the generation of reactive oxygen species and antibody-dependent cellular phagocytosis. Importantly, these innate immune cell changes were not evident in subjects with low ABA levels but could be rescued with exogenous ABA supplementation. Together, these data indicate that pre-existing ABA are essential for Imprime-mediated anti-cancer immune activation and suggest that pre-treatment ABA levels may provide a plausible patient selection biomarker to delineate patients most likely to benefit from Imprime-based therapy.

A Phase II Efficacy and Safety, Open-Label, Multicenter Study of Imprime PGG Injection in Combination With Cetuximab in Patients With Stage IV KRAS-Mutant Colorectal Cancer.

BACKGROUND: Imprime PGG (ß(1,6)-[poly-(1,3)-D-glucopyranosyl]-poly-ß(1,3)-D-glucopyranose) is an innate immune cell modulator that primes neutrophils and monocytes/macrophages to exert antitumor activity against complement opsonized tumor cells. In patients with KRAS-mutant colorectal cancer (CRC), cetuximab alone is ineffective; however, it can bind to tumor cells and induce opsonization for recognition by Imprime PGG-bound innate immune cells. The primary objective of this study was to determine the antitumor activity of Imprime PGG in combination with cetuximab in patients with KRAS-mutant metastatic CRC. PATIENTS AND METHODS: The study had a 2-stage Simon optimal design with 80% power to detect a target objective response rate (ORR) of ≥10% at a 10% significance level. Patients received weekly Imprime PGG (4 mg/kg) and cetuximab (loading dose, 400 mg/m(2), then 250 mg/m(2)) intravenously. The primary end point was ORR; secondary end points included duration of response (DOR), time to progression (TTP), overall survival (OS), disease control rate, progression-free survival, and safety. Stage 1 of the study was to enroll 17 evaluable patients. RESULTS: One partial response (5.6%) was observed among 18 patients enrolled into stage 1. Median DOR was 4.2 months, TTP 2.7 months, and OS 6.6 months. Overall, observed toxicity was as expected from cetuximab alone. The most common (≥20%) adverse events related to Imprime PGG were fatigue (7 patients; 38.9%), infusion reaction (4 patients; 22.2%), and headache (4 patients; 22.2%). There was no Grade 4 toxicity nor treatment-related deaths. CONCLUSION: Imprime PGG in combination with cetuximab treatment in patients with KRAS-mutant CRC showed compelling, albeit modest, clinical activity. This study provides proof of principle that Imprime PGG, in combination with complement-activating antibodies, is associated with clinical activity.

Differential regulation of oxidative burst by distinct ß-glucan-binding receptors and signaling pathways in human peripheral blood mononuclear cells.

ß-Glucans possess broad immunomodulatory properties, including activation of innate immune functions such as oxidative burst activity. The differential roles of complement receptor type 3 (CR3) and Dectin-1, the known ß-glucan receptors, and their associated signaling pathways in the generation of oxidative burst induced by different physical forms of Saccharomyces cerevisiae-derived ß-glucan were examined in human peripheral blood mononuclear cells (PBMC). In this study whole glucan particle (WGP) or immobilized soluble ß-glucan (ISG) was used to represent the phagocytizable or the nonphagocytizable form of a fungus, respectively. Oxidative burst as measured by the formation of superoxide (SO) was detected in PBMC in response to WGP and ISG. SO induction with WGP was concluded to be Dectin-1-mediated and required Src family kinases, phosphatidylinositol-3 kinase and protein kinase B/Akt. In contrast, the SO induction generated by ISG was CR3-mediated and required focal adhesion kinase, spleen tyrosine kinase, phosphatidylinositol-3 kinase, Akt, p38 mitogen activated protein kinase, phospholipase C and protein kinase C. The study results support the hypothesis that human PBMC, specifically monocytes, utilize distinct receptors and overlapping, but distinct, signaling pathways for the oxidative burst in response to challenge by different physical forms of ß-glucan.

Binding of Soluble Yeast ß-Glucan to Human Neutrophils and Monocytes is Complement-Dependent.

The immunomodulatory properties of yeast ß-1,3/1,6 glucans are mediated through their ability to be recognized by human innate immune cells. While several studies have investigated binding of opsonized and unopsonized particulate ß-glucans to human immune cells mainly via complement receptor 3 (CR3) or Dectin-1, few have focused on understanding the binding characteristics of soluble ß-glucans. Using a well-characterized, pharmaceutical-grade, soluble yeast ß-glucan, this study evaluated and characterized the binding of soluble ß-glucan to human neutrophils and monocytes. The results demonstrated that soluble ß-glucan bound to both human neutrophils and monocytes in a concentration-dependent and receptor-specific manner. Antibodies blocking the CD11b and CD18 chains of CR3 significantly inhibited binding to both cell types, establishing CR3 as the key receptor recognizing the soluble ß-glucan in these cells. Binding of soluble ß-glucan to human neutrophils and monocytes required serum and was also dependent on incubation time and temperature, strongly suggesting that binding was complement-mediated. Indeed, binding was reduced in heat-inactivated serum, or in serum treated with methylamine or in serum reacted with the C3-specific inhibitor compstatin. Opsonization of soluble ß-glucan was demonstrated by detection of iC3b, the complement opsonin on ß-glucan-bound cells, as well as by the direct binding of iC3b to ß-glucan in the absence of cells. Binding of ß-glucan to cells was partially inhibited by blockade of the alternative pathway of complement, suggesting that the C3 activation amplification step mediated by this pathway also contributed to binding.

SPI-0211 activates T84 cell chloride transport and recombinant human ClC-2 chloride currents.

The purpose of this study was to determine the mechanism of action of SPI-0211 (lubiprostone), a novel bicyclic fatty acid in development for the treatment of bowel dysfunction. Adult rabbit intestine was shown to contain mRNA for ClC-2 using RT-PCR, Northern blot analysis, and in situ hybridization. T84 cells grown to confluence on permeable supports were shown to express ClC-2 channel protein in the apical membrane. SPI-0211 increased electrogenic Cl- transport across the apical membrane of T84 cells, with an EC50 of approximately 18 nM measured by short-circuit current (Isc) after permeabilization of the basolateral membrane with nystatin. SPI-0211 effects on Cl- currents were also measured by whole cell patch clamp using the human embryonic kidney (HEK)-293 cell line stably transfected with either recombinant human ClC-2 or recombinant human cystic fibrosis transmembrane regulator (CFTR). In these studies, SPI-0211 activated ClC-2 Cl- currents in a concentration-dependent manner, with an EC50 of approximately 17 nM, and had no effect in nontransfected HEK-293 cells. In contrast, SPI-0211 had no effect on CFTR Cl- channel currents measured in CFTR-transfected HEK-293 cells. Activation of ClC-2 by SPI-0211 was independent of PKA. Together, these studies demonstrate that SPI-0211 is a potent activator of ClC-2 Cl- channels and suggest a physiologically relevant role for ClC-2 Cl- channels in intestinal Cl- transport after SPI-0211 administration.