Imprime PGG Enhances Anti-Tumor Effects of Tumor-Targeting, Anti-Angiogenic, and Immune Checkpoint Inhibitor Antibodies.

Imprime PGG (Imprime) is in late-stage clinical development as a combinatorial agent with several therapeutic modalities. Here we present pre-clinical mechanistic data supportive of Imprime, a soluble yeast ß-1,3/1,6-glucan pathogen-associated molecular pattern able to prime innate immune cells in a Dectin-1dependent manner. In tumor-free mice, Imprime evoked broad innate immune responses (type I interferon signature, mobilization of myeloid cells, dendritic cell and monocyte/macrophage expression of co-stimulatory ligands like CD86, and activation of natural killer cells). Imprime-mediated activation of myeloid cells also resulted in functional priming of antigen-specific CD8 T cell response. In tumor-bearing mice, Imprime monotherapy further resulted in activation of systemic and tumor infiltrating macrophages and enhanced cytotoxic CD8 T cell trafficking. Imprime enhanced the anti-tumor activity of several combinatorial agents in mouse cancer models; anti-tyrosinase-related protein 1 antibody in B16F10 melanoma experimental lung metastasis model, anti-vascular endothelial growth factor receptor 2 antibody in H1299 and H441 lung cancer, and anti-programmed cell death protein 1 antibody in MC38 colon cancer models. Mechanistically, combining Imprime with these combinatorial therapeutic agents elicited enhanced innate immune activation, supporting immunological synergy. Finally, Imprime treatment induced similar in vitro phenotypic and functional activation of human innate immune cells. Collectively, these data demonstrate Imprime's potential to orchestrate a broad, yet coordinated, anti-cancer immune response and complement existing cancer immunotherapies.

Immune Pharmacodynamic Responses of the Novel Cancer Immunotherapeutic Imprime PGG in Healthy Volunteers.

Imprime PGG (Imprime) is an i.v. administered, yeast ß-1,3/1,6 glucan in clinical development with checkpoint inhibitors. Imprime-mediated innate immune activation requires immune complex formation with naturally occurring IgG anti-ß glucan Abs (ABA). We administered Imprime to healthy human volunteers to assess the necessity of ABA for Imprime-mediated immunopharmacodynamic (IPD) changes. Imprime (4 mg/kg) was administered i.v. in single and multiple infusions. Subsets of subjects were premedicated with antihistamine and corticosteroid. Peripheral blood was measured before, during and after Imprime administration for IPD changes (e.g., ABA, circulating immune complexes, complement activation, complete blood counts, cytokine/chemokine, and gene expression changes). IPD changes were analyzed based on pretreatment serum ABA levels: low-ABA (<20 µg/ml), mid-ABA (≥20-50 µg/ml), and high-ABA (≥50 µg/ml). At the end of infusion, free serum ABA levels decreased, circulating immune complex levels increased, and complement activation was observed. At ∼1-4 h after end of infusion, increased expression of cytokines/chemokines, a 1.5-4-fold increase in neutrophil and monocyte counts and a broad activation of innate immune genes were observed. Low-ABA subjects typically showed minimal IPD changes except when ABA levels rose above 20 µg/ml after repeated Imprime dosing. Mild-to-moderate infusion-related reactions occurred in subjects with ABA ≥20 µg/ml. Premedications alleviated some of the infusion-related reactions, but also inhibited cytokine responses. In conclusion, ABA levels, being critical for Imprime-mediated immune activation may provide a plausible, mechanism-based biomarker to identify patients most likely to respond to Imprime-based anticancer immunotherapy.

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.

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.

Distinct indirect pathways govern human NK-cell activation by TLR-7 and TLR-8 agonists.

NK cells limit the emergence of cancers and viral infections by surveillance of 'missing-self' and 'induced-self' ligands, and by direct recognition of pathogen-associated molecules. We examined individual roles for Toll-like receptors (TLRs)-7 and -8 in human NK-cell activation using synthetic, small molecule agonists of either TLR-7 (imiquimod and 3M-001), TLR-8 (3M-002) or both TLR-7/8 (3M-003 and R-848) for comparison with known ligands of TLR-2 to -9. Tracking cytokine production in PBMC initially revealed that a subset of TLR agonists including polyinosinic-polycytidylic acid (poly I:C), 3M-002, 3M-003, R-848 and single-stranded RNA trigger relatively high levels of IFN-gamma expression by NK cells. Isolated NK cells did not express TLR-7 or TLR-8. Unlike MALP-2 and poly I:C, 3M-001-3 did not induce expression of either CD69 or IFN-gamma by purified NK cells suggesting indirect activation. IL-18 and IL-12p70 were primarily required for induction of IFN-gamma by both synthetic and natural TLR-8 ligands, while type I IFN was required for induction of CD69 on NK cells by the TLR-7 agonist 3M-001. In addition to expression of IFN-gamma and CD69, relative induction of NK-cell cytotoxicity by TLR-7 and TLR-8 agonists was compared. Immune response modifiers (IRMs) with a TLR-8 agonist component (3M-002 and 3M-003) stimulated greater levels of K562 cytolysis than achieved with 3M-001 or IL-2 (1000 units ml(-1)). In vivo NK-cell cytotoxicity was also enhanced by R-848, but not in type I IFNR-deficient mice. We conclude that IRMs can modulate NK-cell function both in vitro and in vivo and that distinct indirect pathways control human NK-cell activation by TLR-7 and TLR-8 agonists.

Synthetic TLR agonists reveal functional differences between human TLR7 and TLR8.

Although TLR7 and TLR8 are phylogenetically and structurally related, their relative functions are largely unknown. The role of TLR7 has been established using TLR7-deficient mice and small molecule TLR7 agonists. The absence of TLR8-selective agonists has hampered our understanding of the role of TLR8. In this study TLR agonists selective for TLR7 or TLR8 were used to determine the repertoire of human innate immune cells that are activated through these TLRs. We found that TLR7 agonists directly activated purified plasmacytoid dendritic cells and, to a lesser extent, monocytes. Conversely, TLR8 agonists directly activated purified myeloid dendritic cells, monocytes, and monocyte-derived dendritic cells (GM-CSF/IL-4/TGF-beta). Accordingly, TLR7-selective agonists were more effective than TLR8-selective agonists at inducing IFN-alpha- and IFN-regulated chemokines such as IFN-inducible protein and IFN-inducible T cell alpha chemoattractant from human PBMC. In contrast, TLR8 agonists were more effective than TLR7 agonists at inducing proinflammatory cytokines and chemokines, such as TNF-alpha, IL-12, and MIP-1alpha. Thus, this study demonstrated that TLR7 and TLR8 agonists differ in their target cell selectivity and cytokine induction profile.

Plasmacytoid dendritic cells produce cytokines and mature in response to the TLR7 agonists, imiquimod and resiquimod.

The immune response modifiers, imiquimod and resiquimod, are TLR7 agonists that induce type I interferon in numerous species, including humans. Recently, it was shown that plasmacytoid dendritic cells (pDC) are the primary interferon-producing cells in the blood in response to viral infections. Here, we characterize the activation of human pDC with the TLR7 agonists imiquimod and resiquimod. Results indicate that imiquimod and resiquimod induce IFN-alpha and IFN-omega from purified pDC, and pDC are the principle IFN-producing cells in the blood. Resiquimod-stimulated pDC also produce a number of other cytokines including TNF-alpha and IP-10. Resiquimod enhances co-stimulatory marker expression, CCR7 expression, and pDC viability. Resiquimod was compared throughout the study to the pDC survival factors, IL-3 and IFN-alpha; resiquimod more effectively matures pDC than either IL-3 or IFN-alpha alone. These results demonstrate that imidazoquinoline molecules directly induce pDC maturation as determined by cytokine induction, CCR7 and co-stimulatory marker expression and prolonging viability.