Richard M. Walsh

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

The immunomodulatory properties of yeast β-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 β-glucans to human immune cells mainly via complement receptor 3 (CR3) or Dectin-1, few have focused on understanding the binding characteristics of soluble β-glucans. Using a well-characterized, pharmaceutical-grade, soluble yeast β-glucan, this study evaluated and characterized the binding of soluble β-glucan to human neutrophils and monocytes. The results demonstrated that soluble β-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 β-glucan in these cells. Binding of soluble β-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 β-glucan was demonstrated by detection of iC3b, the complement opsonin on β-glucan-bound cells, as well as by the direct binding of iC3b to β-glucan in the absence of cells. Binding of β-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.

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

Imprime PGG (Imprime), an intravenously-administered, soluble β-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-β 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.

Abstract B008: Imprime PGG, an intravenously administered beta glucan PAMP activates the innate immune system: A phase I clinical study to evaluate immunopharmacodynamic responses

Imprime PGG (Imprime), in combination with both tumor-targeting and anti-angiogenic antibodies, has shown promising efficacy in multiple phase 2 clinical trials. In numerous pre-clinical in vivo tumor models, Imprime also enhances the efficacy of immune checkpoint inhibitor antibodies in addition to tumor-targeting and anti-angiogenic antibodies. Imprime is a yeast-derived, soluble β-1,3/1,6 glucan that acts as a Pathogen Associated Molecular Pattern (PAMP) to trigger activation of innate immune effector cells (macrophages, monocytes, neutrophils, dendritic cells (DC)), which orchestrate a coordinated anti-cancer immune response with cells of the adaptive immune system. Ex vivo studies with human whole blood have shown that Imprime forms an immune complex with endogenous anti-beta glucan antibodies (ABA) to trigger a constellation of innate immune functions. These include complement activation via the classical complement pathway, select chemokine production, phenotypic activation and enhanced tumor cell killing by neutrophils and macrophages. Imprime also activates antigen-presenting cells (e.g. macrophages, DC), enabling T cell expansion and activation. In vivo, intravenous (IV) injection of Imprime in C57BL/6 mice increases select chemokine expression, triggers neutrophil and monocyte mobilization into circulation and secondary lymphoid organs, and also enhances DC maturation and antigen-specific T-cell priming. In this study, we show that the immunopharmacodynamic (IPD) responses elicited by IV administration of Imprime in healthy human subjects are consistent with the innate immune responses observed in ex vivo human and in vivo mouse studies. Healthy human volunteers (18-65 yr) were administered single (Cohort 1) or multiple (once weekly for 3 wks-Cohort 2) doses of Imprime PGG (4 mg/kg) by IV infusion over 2-3 hrs. Physical examination with vital signs, adverse event solicitation and timed blood sampling for IPD changes were performed. IPD endpoints included complement protein levels, circulating blood cell lineage counts, ABA concentrations, circulating immune complex (CIC) levels, cytokine and chemokine concentrations, as well as binding and activation of blood leukocytes. Cohort 1 and 2 results show that the complement activation proteins C5a and SC5b-9 were significantly increased in the plasma at the end of infusion (EOI) of Imprime. The formation of Imprime:ABA complexes was evident in a substantial drop of free ABA and a concomitant increase in CIC in the serum also at the EOI. IL-8 and MCP-1 were consistently detected between EOI and 1 hr post infusion. Additional chemokines, including MIP-1α, MIP-1β, and IP-10 were also detected in some of the subjects. A significant increase in the neutrophil and monocyte counts was seen in the blood after infusion. Cellular analyses showed Imprime binding to neutrophils, monocytes, and subsets of DC (classical and inflammatory) 15-30 mins after the start of infusion. Additionally, 24 hrs after completion of Imprime administration, a population of non-classical monocytes (CD14/CD16 positive), which are known to have higher antigen presentation capability and thus express higher levels of the activation markers CD86, PD-L1, and HLA-DR, was observed. Importantly, these IPD responses were evident only in subjects with higher ABA levels. Collectively, these data provide the first evidence that, when dosed IV in healthy human subjects, Imprime elicits a constellation of innate immune activating events that are consistent with efficacy in preclinical tumor models. Importantly, these human data also provide the first evidence linking pre-treatment ABA levels and Imprime induced IPD changes, suggesting the plausibility of using pre-treatment ABA levels in the selection of patients most likely to benefit from Imprime-based therapy. Citation Format: Nadine C. Ottoson, Richard D. Huhn, Jamie Lowe, Ben Harrison, Jose Iglesias, Blaine Rathmann, Takashi Kangas, Lindsay R. Wurst, Xiaohong Qiu, Anissa Chan, Adria Bykowski Jonas, Kathryn Fraser, Richard M. Walsh, Katie Ertelt, Steven M. Leonardo, Ross Fulton, Keith Gorden, Mark A. Matson, Mark Uhlik, Jeremy Graff, Nandita Bose. Imprime PGG, an intravenously administered beta glucan PAMP activates the innate immune system: A phase I clinical study to evaluate immunopharmacodynamic responses [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B008.

Abstract 5627: Antitumor activity of soluble beta-1,3/1,6 glucans: Structure matters

Beta-glucan is a pathogen-associated molecular pattern recognized by a variety of innate immune cells. Beta-1,3/1,6 glucans possess numerous immune potentiating activities. In particular, antitumor activity has been demonstrated with the soluble beta glucan Imprime PGG used in combination with complement-activating antitumor monoclonal antibodies (MAb) in several tumor models. Structurally, beta-1,3/1,6 glucans can vary on the basis of chain length, types of linkages, branching, and tertiary structure. Previously, the antitumor activity of soluble beta-1,3/1,6 glucans in combination with antitumor MAb has only been reported using beta glucans derived from yeast. The primary objective of this study was to compare the antitumor activity of three structurally distinct soluble beta-1,3/1,6 glucans in vivo in a mouse syngeneic lymphoma model. Tumor-bearing mice were administered antitumor MAb plus beta glucan 2x/week for 4 weeks. Antitumor activity was based on inhibition of tumor growth at the tumor implantation site and enhanced long-term survival. In addition, critical innate immune cells responsible for antitumor activity were identified focusing on the role of neutrophils. Also, characterization of relevant human immune cells and their beta glucan receptors will be shown focusing on complement receptor 3 (CR3) and dectin-1. Beta glucan binding to immune cells and identification of relevant receptors were determined by flow cytometry using beta glucan-specific antibody and fluorescently-labeled beta glucans. Key findings are that soluble beta glucans, which differ structurally, do not induce similar antitumor activity, and antitumor activity is abrogated in granulocyte-deficient mice. Finally, soluble glucans bind to human neutrophils in a CR3-dependent manner. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5627.