Ross B. Fulton

Abstract 5034: Imprime PGG decreases regulatory T cell suppression and enhances T cell proliferation and differentiation revealing additional mechanisms for its anti-tumor activity

Imprime PGG is a soluble yeast-derived β-glucan immunomodulator being developed for treatment of cancer in combination with anti-tumor antibodies (Abs). Imprime PGG binds to complement receptor (CR)3 on innate immune cells (neutrophils and monocytes), and primes them to exert anti-tumor activity against Ab-targeted, complement opsonized tumor cells. Numerous studies in syngeneic and human xenogeneic tumor models in mice have demonstrated that administration of Imprime PGG in combination with anti-tumor Ab treatment reduces tumor growth and prolongs survival beyond that observed with either agent alone. In clinical trials, Imprime PGG in combination with tumor-targeting Abs has also been associated with improvements in objective tumor response and survival. Imprime PGG9s mechanism of action has been extensively studied in the context of the innate immune system. Whether Imprime PGG may also play a role in the modulation of adaptive immune responses and the cross-talk that exists between the innate immune system and T-cell responses was unclear. Recent work has revealed that Imprime PGG is capable of activating not only the innate immune system but also has the potential of inhibiting the polarization of macrophages into the immunosuppressive M2 phenotype. Since considerable cross-talk exists between the innate and adaptive immune cells in the immunosuppressive tumor microenvironment, we investigated whether Imprime PGG may also play a role in direct or indirect modulation of one of the adaptive immunosuppressive cell types, regulatory T cells (Tregs). We demonstrated that plasma from Imprime PGG treated whole blood (WB) was able to decrease the suppressive capability of Tregs on conventional CD4+ T cells. We also determined that plasma from Imprime PGG treated WB was able to enhance the proliferation of both CD4+ and CD8+ T cells in response to CD3/28 beads and to peptide stimulation in vitro. This enhanced proliferation was accompanied by upregulation of the transcription factor Tbet and increased IFN-γ production suggesting Imprime PGG may drive polarization of T cells to a Th1, anti-tumor phenotype. Interestingly, these effects were greatest when Imprime PGG was added to WB and not to isolated plasma, indicating that the effects of Imprime PGG on the T cell compartment are indirect and may involve cross-talk from the innate immune system. These data are the first to show that Imprime PGG may exert anti-tumor activity not only by re-directing the innate immune system against opsonized tumor cells, but also by re-orienting the adaptive immune compartment toward a Th1, anti-tumor phenotype. Citation Format: Steven M. Leonardo, Keith Gorden, Ross Fulton, Lindsay Wurst. Imprime PGG decreases regulatory T cell suppression and enhances T cell proliferation and differentiation revealing additional mechanisms for its anti-tumor activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5034. doi:10.1158/1538-7445.AM2015-5034

Abstract B019: Imprime PGG, a soluble β-glucan, binds to and activates dendritic cells resulting in enhanced T cell priming, expansion, and cytokine production

Imprime PGG (Imprime), a soluble yeast 1,3/1,6 β-glucan, is being developed as a novel cancer immunotherapy in conjunction with anti-tumor antibodies in several cancers. Randomized Phase 2 clinical trials of Imprime in the 1st-line treatment of stage IV non-small cell lung cancer have shown promising efficacy in both objective tumor response and survival. Mechanistic work has revealed that Imprime acts to stimulate a coordinated, anti-cancer immune response in conjunction with antibody therapy. Imprime represents a pathogen associated molecular pattern (PAMP) and, as such, can be efficiently and effectively recognized by cells of the innate immune system, triggering macrophage repolarization, neutrophil activation, monocyte-derived dendritic cell maturation, NK cell activation and, via cross-talk with the adaptive immune system, expansion of CD4 and CD8 T cells. As PAMPs have been shown to stimulate dendritic cells (DCs), which are critical for generating robust and durable anti-tumor responses, we sought to better understand the effects of Imprime on DCs. Here we demonstrate that Imprime binds to various DC subsets, stimulating the critical antigen presenting functional activity of these DC subsets. Following i.v. administration in mice, Imprime bound both classical splenic DCs, including CD8α+ cross-presenting DCs, and migratory DC subsets within peripheral lymph nodes. In vitro treatment of human whole blood showed binding in the inflammatory DC subset (Lin-HLA-DR+CD11c+CD16+) as well as the classical (Lin-HLA-DR+CD11c+CD1c+) DC subset. Binding of Imprime to both mouse and human DCs resulted in the upregulation of MHC class II and the co-stimulatory molecules CD80/86 that are critical for antigen presentation and T cell activation. Furthermore, in vivo treatment of mice with Imprime in combination with H-2Kb-restricted OVA257-264 peptide resulted in enhanced expansion of adoptively transferred OVA-specific OT-I CD8 T cells. In contrast to mice immunized with OVA peptide alone, which did not generate a functional CD8 T cell response, Imprime co-administration resulted in functional OT-I capable of ex vivo degranulation and production of the cytokines IFN-γ and IL-2. These data suggest that, in addition to previous studies showing Imprime primes monocytes, macrophages, and neutrophils, Imprime also enhances T cell activation and expansion by directly stimulating dendritic cell maturation and efficient antigen presentation. These data demonstrate that Imprime PGG treatment may enhance the adaptive immune response necessary for durable tumor control and, together with previously published data, indicate that Imprime PGG treatment triggers an orchestrated anti-cancer immune response involving both the innate and adaptive immune systems. Citation Format: Ross B. Fulton, Steven M. Leonardo, Kyle S. Michel, Michael E. Danielson, Keith B. Gorden, Jeremy R. Graff. Imprime PGG, a soluble β-glucan, binds to and activates dendritic cells resulting in enhanced T cell priming, expansion, and cytokine production. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B019.

Abstract A160: Imprime PGG binds to neutrophils through complement, Fc, and dectin-1 receptors, priming these cells for enhanced ROS production and tumor cell cytotoxicity

Imprime PGG (Imprime), a soluble yeast 1,3/1,6 β-glucan, is being developed as a novel cancer immunotherapy in conjunction with anti-tumor antibodies in several cancers. In clinical studies, including randomized Phase 2 clinical trials in the 1st-line treatment of stage IV non-small cell lung cancer with bevacizumab, Imprime treatment has shown promising efficacy in both objective tumor response rates and survival. In numerous syngeneic and xenogeneic mouse tumor models, Imprime treatment in combination with an anti-tumor antibody reduced tumor growth and prolonged survival beyond that observed with antibody alone. Mechanistic studies have now demonstrated that, with endogenous anti-β-glucan antibodies (ABA), Imprime forms an immune complex. This Imprime-ABA complex then activates the classical complement pathway and is subsequently opsonized by iC3b. This immune complex then binds to and primes innate immune cells, including macrophages, monocytes and neutrophils, triggering a coordinated immune attack against antibody-targeted cancer cells. In this study, we sought to explore more fully the functional consequences of Imprime-ABA immune complex binding to, and priming of, neutrophils. Neutrophils are the first line of defense against fungal infections. Neutrophils detect fungal infections through recognition of β-glucans on the surface of yeast and fungi. These β-glucans serve as fungal pathogen associated molecular patterns (PAMPs) that are efficiently and effectively recognized by receptors on innate immune cells, initiating a coordinated immune response to infection. Here we demonstrate that Imprime, in individuals with high ABA levels, is recognized as a PAMP, directly interacting with neutrophils via multiple receptors, priming these cells and ultimately leading to enhanced effector responses including reactive oxygen species (ROS) generation and tumor cell killing. Our data now show that Imprime elicits enhanced neutrophil survival as measured by cell viability dye exclusion using flow cytometry. Imprime treatment also enhances the responsiveness of neutrophils to Fc receptor activation. As measured by luminol-based read-outs, ROS production in response to anti-Fc receptor antibody-coated beads is profoundly enhanced by Imprime treatment. Moreover, Imprime treated neutrophils show a remarkable surge in ROS production compared to vehicle–treated neutrophils when exposed to B cell lymphoma cells decorated with anti-CD20 monoclonal antibodies (i.e. rituximab). These Imprime-treated neutrophils also exhibit a robust increase in cytotoxicity against these B cell lymphoma cells coated with rituximab using in vitro Calcein AM dye release assays. We next sought to delineate which receptors might be most critical for Imprime binding and priming of neutrophils. As demonstrated by antibody blocking studies, the Imprime-ABA complex can bind to both complement receptors (notably CR3) and Fc receptors. Imprime can also block binding of a dectin-1 agonizing antibody to dectin-1 and can bind directly to HL-60 and HEK cells lines engineered to overexpress dectin-1 by retroviral transduction. Collectively, these data show for the first time that Imprime directly primes neutrophils to recognize and kill antibody-decorated tumor cells and interacts with the Fc, complement and dectin-1 receptors on the surface of neutrophils. Citation Format: Steven M. Leonardo, Ross B. Fulton, Lindsay R. Wurst, Keith B. Gorden, Adria Bykowski Jonas, Xiaohong Qui, Anissa SH Chan, Jeremy R. Graff. Imprime PGG binds to neutrophils through complement, Fc, and dectin-1 receptors, priming these cells for enhanced ROS production and tumor cell cytotoxicity. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A160.

Abstract LB-080: Imprime PGG, a β-glucan PAMP (pathogen-associated molecular pattern) activates the direct killing functions of innate immune cells in concert with tumor targeting antibodies

Imprime PGG (Imprime) is a soluble yeast 1,3/1,6 β-glucan PAMP (pathogen-associated molecular pattern). As a PAMP, Imprime triggers innate immune function, activating the direct killing functions of innate immune cells, facilitating MDSC differentiation and macrophage repolarization as well as enabling dendritic cell maturation and antigen presentation, driving T cell expansion and activation. In the clinic, Imprime is administered intravenously and is well-tolerated. In multiple clinical trials (> 400 subjects), including randomized phase 2 studies in NSCLC, Imprime has consistently shown promising increases in both objective tumor response and patient survival. To date, the clinical experience with Imprime has centered on combinations with tumor targeting monoclonal antibodies (Mabs). For instance, Imprime combined with rituximab and alemtuzumab in CLL patients yielded a 65% complete response rate (vs 37% historical CR rate for alemtuzumab plus rituximab). We sought to better characterize the effect of Imprime in concert with tumor-targeting mAbs. We show that Imprime enhances the effector functions of multiple innate immune cell lineages. We first evaluated the generation of Reactive Oxygen Species (ROS) in neutrophils isolated from human healthy volunteer whole blood. These neutrophils, but not those from vehicle treated whole blood, specifically recognized B cell lymphomas (Raji) only after opsonization with anti-CD20 Mabs (rituximab, ofatumumab, obinatuzumab), generating a substantial ROS burst that coincided with enhanced tumor cell cytotoxicity. Similarly, increased antibody dependent cellular phagocytosis (ADCP) mediated by monocyte-derived macrophages was evident against antibody-opsonized lymphomas (Z138 B cell lymphomas with obinutuzumab) and solid tumor cells (SKBr3 breast cancer cells with trastuzumab) from Imprime-treated whole blood. Likewise, increased Natural Killer (NK) cell-mediated antibody dependent cellular cytotoxicity (ADCC) was evident only after Imprime treatment against antibody-opsonized cancer cells (SKBR3 with trastuzumab, K562 erythroleukemia cells with anti-glycophorin-A). In vivo, we now show that Imprime administered intravenously significantly enhances the anti-tumor efficacy of trastuzumab in a patient derived xenograft model of breast cancer, reducing mean tumor volume to ∼ 50% of that achieved by trastuzumab alone. In the B16 lung experimental metastasis model, the addition of Imprime to the anti-TRP1 tumor targeting antibody TA-99 significantly reduces both the number and size of B16 lung metastases. Together, these data show that Imprime stimulates the innate immune system, augmenting the anti-tumor efficacy of a diverse array of tumor targeting antibodies in multiple tumor types. Citation Format: Steven M. Leonardo, Ross B. Fulton, Keith B. Gorden, Katy Fraser, Ben Harrison, Takashi Kangas, Adria Jonas, Yumi Yokoyama, Nadine Ottoson, Nandita Bose, Jeremy R. Graff. Imprime PGG, a β-glucan PAMP (pathogen-associated molecular pattern) activates the direct killing functions of innate immune cells in concert with tumor targeting antibodies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-080.

Abstract 3767: Imprime PGG, a soluble yeast b-glucan PAMP, enhancement of anti-tumor responses in combination with tumor targeting antibody is highly dependent on NK cell killing

Cancer therapy has been reshaped by checkpoint inhibitors (CPIs), making it possible for durable responses against cancers with traditionally low cure rates. Current efforts are focused on combination therapies in the hopes of evading resistance to CPIs and improving overall response. One escape mechanism attributed to acquired resistance to CPIs includes defective antigen presentation, namely a loss in MHC class I expression. This leads to loss of CD8 T cell-mediated tumor killing and disease relapse. This recent revelation has stimulated a need for therapies that activate other cytotoxic effector cells such as NK cells to kill tumors. Imprime PGG (Imprime) is a soluble, systemically delivered yeast 1,3/1,6 β-glucan PAMP (pathogen-associated molecular pattern) capable of triggering innate immune cell function leading to a cascade of immune activation and enhanced tumor killing. Imprime activates the innate immune system via dectin-1, eliciting production of a variety of chemokines and cytokines, including type I IFN, leading to the mobilization and stimulation of innate cell types including dendritic cells and monocytes. Unlike other PAMPs which systemic administration often leads to toxic side effects, Imprime has been administered safely by intravenous infusion to >400 human subjects. Currently, Imprime PGG is being evaluated in combination with αPD1 therapy in multiple clinical trials. Previously we have shown that combination therapy of anti-Trp1 antibody and Imprime leads to a significant reduction in both number and size of lung metastases in the B16F10 metastatic melanoma tumor model over anti-Trp1 antibody alone. This reduction of metastases is highly dependent on NK cells but not CD8 T cells. To explore the impact of Imprime on NK-mediated cytotoxicity, we further evaluated in vivo killing of MHC class I deficient TapKO cells after intravenous administration of Imprime. In these experiments Imprime was able to enhance the NK cytotoxic killing of the targets. All NK cell killing observed was dependent on type I IFN, phagocytic cells and dectin 1. Imprime treatment increased cytokines that drive enhanced NK activation and effector phenotype. Significant increases were observed in the cytokines IL15/IL15rα, IL18, IL12p70 in lymph node lysates as well as increases in the effector molecules CD69, GrB, and CD107a on splenic NK cells. The upregulation of all of these molecules during Imprime treatment was dependent on dectin 1. Additionally, IL15/IL15rα production was also dependent on type I IFN, and phagocytic cells. Interestingly, Ly6c hi monocytes, which are increased after Imprime treatment, also show enhancement of IL15rα expression. Collectively, these data demonstrate that Imprime contributes to enhanced NK functionality and killing which may provide a unique immunotherapeutic approach to complement existing therapies. Citation Format: Kathryn A. Fraser, Takashi Kangas, Ross B. Fulton, Steven M. Leonardo, Ben Harrison, Yumi Yokoyama, Nandita Bose, Jeremy R. Graff, Mark Uhlik, Keith B. Gorden. Imprime PGG, a soluble yeast b-glucan PAMP, enhancement of anti-tumor responses in combination with tumor targeting antibody is highly dependent on NK cell killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3767.

Abstract 1703: A novel tumor vaccine platform: direct conjugation of antigens to the β glucan PAMP Imprime PGG enhances antigen presentation and T cell priming

Significant clinical and pre-clinical research has shown that PAMPs- Pathogen Associated Molecular Patterns- can trigger an integrated anti-cancer immune response involving both innate and adaptive immunity. Imprime PGG is a soluble yeast β-1,3/1,6 glucan currently in multiple phase 2 clinical studies in combination with the immune checkpoint inhibitor (CPI), pembrolizumab. Preclinical mechanistic research has shown that Imprime PGG can re-polarize immunosuppressive myeloid cells in the tumor microenvironment and activate antigen presenting cells to prime antigen-specific CD8 T cells thereby boosting effector T cell function and expansion. Based on this ability to activate dendritic cells and induce type I interferon, we sought to explore the use of Imprime as an immune activating/antigen-directing scaffold onto which we could attach tumor antigen to drive an antigen-specific T cell based immune response. Imprime PGG has been safely administered intravenously to more than 400 human subjects and thus represents a safe starting point for the development of a cancer vaccine. Imprime’s 1,3/1,6 β-glucan polymeric structure allows for the straightforward attachment of peptides and proteins via three different conjugation routes. To explore this function we covalently linked the chicken ovalbumin (OVA) protein to Imprime to generate a β-glucan-protein conjugate (Imprime-OVA). Using T cell receptor transgenic OT-I CD8 and OT-II CD4 T cells to track responses to OVA, we treated mice with Imprime-OVA intravenously and examined the expansion and functional quality of the T cell response 7 days later at the peak of expansion. Following Imprime-OVA treatment, both OVA-specific CD8 and CD4 T cells underwent vigorous expansion. OT-I CD8 T cells upregulated the transcription factor Tbet, which is central to developing effector functions, and were highly multifunctional in their ability to produce IFN-γ, TNF-α and IL-2. OT-I T cells responded similarly to vaccination with the minimal H-2Kb/OVA257-264 peptide covalently attached to Imprime. By comparison, vaccination with unconjugated OVA (protein or peptide) and Imprime was much less effective in driving T cell expansion and differentiation. The CD8 T cell response required Batf3-dependent cross-presenting DCs whereas the CD4 T cell response did not, and both CD4 and CD8 responses required the C-type lectin receptor Dectin-1. These data show that an Imprime PGG-protein conjugate can effectively elicit the expansion and functional activation of cytotoxic T cells and may have utility as a potential cancer vaccine platform. These data provide evidence that Imprime PGG not only serves as a combination therapy for CPIs, anti-angiogenics, and tumor targeting antibodies but may also be a robust platform for multiple tumor vaccine strategies. Citation Format: Kyle S. Michel, Ross B. Fulton, Steven M. Leonardo, Keith B. Gorden, Jeremy R. Graff, Michael E. Danielson. A novel tumor vaccine platform: direct conjugation of antigens to the β glucan PAMP Imprime PGG enhances antigen presentation and T cell priming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1703. doi:10.1158/1538-7445.AM2017-1703

Abstract 3688: Imprime PGG, a novel innate immune therapeutic in phase 2 clinical development, induces mobilization of monocytes and focalized recruitment of innate immune cells to tumor sites

Immune checkpoint inhibitors (CPI) have shown compelling clinical efficacy in multiple tumor types, though only in a minority of treated patients. Significant research and clinical development are focused on expanding CPI efficacy. Imprime PGG is a novel, IV administered 1,3/1,6 β-glucan PAMP (pathogen-associated molecular pattern) that activates innate immune effector cells to enhance tumor killing, to repolarize the suppressive myeloid cells of the tumor microenvironment and to activate the antigen presentation capability of dendritic cells, macrophages and monocytes. In multiple preclinical models, Imprime enhances the anti-tumor efficacy of CPIs. Imprime is now in multiple phase 2 clinical studies in combination with the CPI, pembrolizumab. We sought to understand more precisely how Imprime activates the innate immune system to enable a concerted innate and adaptive anti-cancer immune response. Using multispectral fluorescence IHC we now show that Imprime induces focalized recruitment of innate immune cells to tumor bearing tissue. In the B16F10 experimental metastasis model, Imprime dosed in combination with the tumor-targeting antibody TA-99 can nearly completely repress the outgrowth of pulmonary metastases across a 19 day time course. At 24h post-Imprime treatment, the presence of Ly6G+ neutrophils was evident throughout the lung tissue. At later time points (72h and beyond) the formation of immune cell clusters was readily evident in lungs from Imprime treated mice and rarer in control mice or mice treated only with TA-99. These immune cell clusters were predominately localized to arterioles near B16 tumor sites and comprised of multiple immune cell subtypes including macs, B cells, T cells as well as a monocyte population that are CD11b+, Ly6G- and F4/80- and strongly positive for MHCII. Consistent with these preclinical findings, IV administration of Imprime to healthy human volunteers increased neutrophil and monocyte mobilization into peripheral blood 2-3 fold 4h post infusion. Imprime treatment also resulted in a significantly increased subset of CD16+ monocytes that are known to have higher antigen presentation capability and express higher levels of the activation markers CD86, PD-L1, and HLA-DR (MHCII). Furthermore, RNA expression profiling of whole blood from Imprime-treated volunteers shows increased expression of the CCL3, CCL4, IL-1β and TNF-α, functional mediators produced by these monocyte populations. Together, these data show that Imprime drives the concerted activation of multiple innate immune subtypes and promotes the appearance of unique monocyte populations that may be critical for an Imprime-induced anti-cancer immune response. Citation Format: Steven Leonardo, Nadine Ottosson, Keith Gorden, Takashi Kangas, Xiaohong Qiu, Ross Fulton, Benjamin Harrison, Adria Jonas, Richard Walsh, Katie Ertelt, Jamie Lowe, Richard Huhn, Jeremy Graff, Nandita Bose, Mark T. Uhlik. Imprime PGG, a novel innate immune therapeutic in phase 2 clinical development, induces mobilization of monocytes and focalized recruitment of innate immune cells to tumor sites [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3688. doi:10.1158/1538-7445.AM2017-3688

Abstract LB-199: Imprime PGG modulates immunosuppressive myeloid components of the tumor microenvironment and drives enhanced antitumor efficacy in combination with checkpoint inhibitor therapies

Checkpoint inhibitor therapies (CPI) have shown great promise, however in a limited percentage of patients. One of the key mechanisms behind the limited efficacy of CPI therapy is immune resistance mediated by immunosuppressive myeloid cells at the tumor microenvironment (TME), namely M2 macrophages and myeloid-derived suppressor cells (MDSC). Multiple therapeutic interventions are being developed to target these cell types with the intention of reshaping the TME and enhancing the effector functions of the infiltrating cytotoxic T cells.Molecules containing pathogen associated molecular patterns (PAMPS) are one of the unique combination partners that can sensitize tumors to respond to CPI. Imprime PGG (Imprime), an intravenously administered soluble yeast β-1,3/1,6 glucan PAMP, is being clinically developed in combination with tumor-targeting antibodies, anti-angiogenics, and CPI. Imprime has shown promising results in two randomized phase 2 studies in non-small cell lung cancer. Mechanistic studies have shown Imprime to repolarize M2 macrophages and MDSC in in vitro human systems as well as multiple xenograft models. The objective of this study was to evaluate Imprime’s ability to counteract immunosuppression and thereby influence the effector functions of T cells in a syngeneic tumor model. To this end, we first evaluated the anti-tumor efficacy of Imprime in combination with anti-PD-1 or anti-PD-L1 antibody in the MC-38 colon cancer model and found that both combinations repressed tumor growth more effectively than either single agent. Flow cytometric evaluation of single cell suspensions of spleen and tumor tissue after one week of Imprime dosing revealed that the tumor associated macrophages showed a shift to an M1-like phenotype with increased expression of PD-L1, CD86, inducible nitric oxide synthase, MHC class II and downmodulation of Arginase-1. qRT-PCR analyses also demonstrated an increase in transcripts for M1 markers (Il12b p35, Ifng, Tnfa) with a coincident decrease in transcripts for M2 markers (VEGF, Fizz1, CCL17). Adaptive immune resistance, increased PD-L1 expression on the tumor cells as a result of immune activation was observed. In the spleen, the monocytic MDSC also showed increased expression of M1 markers. Furthermore, increased number of CD8 cells in the spleen were of effector memory phenotype with enhanced expression of PD-1, granzyme B, and Ki-67. At the tumor site, the CD8 cells from Imprime treated mice demonstrated increased proliferative and cytokine producing capabilities (IL-2, IFNg, and TNFa) in response to CD3/CD28 stimulation. Collectively, these data show that Imprime’s ability to remold the TME such that the myeloid cells are less suppressive and the cytotoxic T cells are more functionally active can have a tremendous impact on overcoming resistance to CPI therapy. Citation Format: Adria B. Jonas, Anissa SH Chan, Xiaohong Qiu, Kathryn Fraser, Nadine Ottoson, Takashi Kangas, Richard Walsh, Steven M. Leonardo, Ross Fulton, Keith Gorden, Mark Uhlik, Jeremy Graff, Nandita Bose. Imprime PGG modulates immunosuppressive myeloid components of the tumor microenvironment and drives enhanced antitumor efficacy in combination with checkpoint inhibitor therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-199. doi:10.1158/1538-7445.AM2017-LB-199

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 LB-089: Imprime PGG, a β-glucan PAMP (pathogen-associated molecular pattern), effectively elicits in vivo maturation of antigen presenting cells in mice and humans, suggesting potential synergy with checkpoint inhibitor therapy

Recognition of PAMPs via pattern recognition receptors is central to immune recognition of foreign threats and to the generation of a coordinated innate and adaptive immune response. Cancers lack PAMPs and are poorly immunogenic. Consequently, the immune system often fails to mount an effective, coordinated anti-cancer immune attack. Though immunotherapies (e.g. checkpoint inhibitors) are effective in some cancer patients, the majority of patients fail to achieve substantial therapeutic benefit. To fully realize the potential of immune checkpoint inhibitors, there is substantial interest in developing therapeutically-viable PAMPs capable of enabling the maturation and function of professional antigen presenting cells (e.g. dendritic cells, DCs). Bacterial and viral PAMPs (i.e. TLR and STING agonists) can elicit DC maturation but are poorly tolerated systemically and are thereby limited to intra-tumoral delivery. The soluble yeast β-1,3/1,6 glucan, Imprime PGG (Imprime), is a PAMP that has been successfully administered intravenously (IV), is well-tolerated and shows promising efficacy in a series of clinical trials in > 400 total subjects. We sought to determine whether Imprime could drive maturation and enhanced function of antigen presenting cells in vivo. We now show that Imprime binds in vivo to various dendritic cell (DC) subsets in both human and mouse. In mice dosed IV, Imprime also elicits DC maturation as indicated by CD86 upregulation and successfully induces a type I interferon transcriptional profile. In C57Bl/6 mice immunized with the OVA257-264 model antigen, Imprime treatment elicits the specific expansion of adoptively transferred OT-I CD8 T cells (transgenic T cells engineered to recognize the OVA antigen). These OT-I T cells are functional effector cells, showing enhanced degranulation and increased capacity to produce IFN-γ and IL-2 when compared to OT-I cells isolated from mice challenged with OVA peptide alone. In ex vivo human whole blood, Imprime also enables DC maturation (enhanced expression of CD86, CD83, MHC class II), T cell expansion and production of the potent anti-tumor cytokine IFN-γ. Importantly, we now show preliminary data that peripheral blood monocytes and classical DCs from Imprime-treated cancer patients show elevated CD86 expression. Collectively, these data show that Imprime, a novel, systemically-administered, well-tolerated PAMP, can effectively elicit the maturation and function of antigen presenting cells in vivo and may thereby enhance T cell priming and anti-tumor immune response elicited by checkpoint inhibitors. Citation Format: Ross B. Fulton, Steven M. Leonardo, Adria B. Jonas, Kathryn A. Fraser, Anissa S.H. Chan, Nadine R. Ottoson, Michael E. Danielson, Nandita Bose, Jeremy R. Graff, Keith Gorden. Imprime PGG, a β-glucan PAMP (pathogen-associated molecular pattern), effectively elicits in vivo maturation of antigen presenting cells in mice and humans, suggesting potential synergy with checkpoint inhibitor therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-089.