Richard Walsh

STUDIES OF THE CYTOCHEMISTRY OF NUCLEOPROTEINS II. IMPROVED STAINING METHODS WITH TOLUIDINE BLUE AND AMMONIUM MOLYBDATE

1 . Stainimig of the nucleoproteins by toluidine blue-molybdate depends upon graded imiactivation of proteimi-bound amino groups or alteration in the concentrations and time of reaction of the dye and molvbdate to allow the formation of a nucleic acid-toluidimie blue complex that is capable of reacting with polymolybdates to produce a metachromatic l)olymner. Toluidine blue sampIes of high )urity differed from older, less pure dyes iii the miature of dye binding and in the rate of formation of the nucleic acid-dye complex that reacts with molvbdate. 2. The staining procedure has l)eeli comisiderably modified and improved amid certain technical l)rot)lems have l)eeli elucidated. Five procedures are reqUire(1 to produce optimal stainimig of deoxyribonucleoprotein and one or more of nine types of ribonucleoprotein in the cell. 3. The oliaracteristics of the nine types of ribonucleoprotein demonstrable by these methods are reviewed. The results suggest that, as the ribonucleic acid which is synthesized in the chromatin and chromosomes diffuses to the perichromosomal and interchromosomal regions, it is functionally and molecularly changed by virtue of association with amino-acids and protein.

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 A08: Distribution, cutoff, and functional significance of a potential biomarker for Imprime PGG, an experimental cancer immunotherapeutic, in a healthy subject population

Imprime PGG (Imprime) is a yeast-derived beta-1,3/1,6 glucan that binds complement receptor 3 (CR3) on innate immune cells and enables these cells to exert anti-tumor activity against tumor cells that have been opsonized by iC3b following targeting by anti-tumor antibodies. Following incubation with whole blood (WB) from healthy subjects, Imprime was shown to bind to neutrophils and monocytes via CR3 and modulate complement receptor expression, activation marker expression, and interleukin-8 (IL-8) production. These effects, however, differed widely among subjects. It was subsequently observed that Imprime-induced functional activities occurred predominantly in individuals with higher levels of endogenous immunoglobulin G (IgG) or IgM anti-beta-glucan antibodies (ABA) and that ABA are a prerequisite for opsonizing Imprime to allow its binding to CR3. A small pilot study (n=32) with healthy subjects using prototype enzyme-linked immunosorbent assays (ELISAs) to measure IgG and IgM ABA suggested correlations between ABA levels, Imprime binding to neutrophils in WB, and consequent induction of functional responses. The objectives of this study were to further optimize and qualify the prototype ELISAs and, in a larger cohort of healthy subjects (n=143), to confirm the significance of elevated ABA levels with respect to binding of Imprime to neutrophils and monocytes and Imprime–induced functional changes. Qualification of the IgG and IgM ABA ELISAs yielded good inter-assay precision (coefficients of variation 5% of cells) in samples from 52% of the subjects. Samples from these same subjects also exhibited high Imprime binding to monocytes. ABA levels in the high binding individuals were significantly greater than those in the low binding individuals (p Citation Format: Diane McMurray, Ben Harrison, Katie Ertelt, Richard Walsh, Lindsay Wurst, Steven Leonardo, Nadine Ottoson, Adria Bykowski Jonas, Xiaohong Qiu, Nandita Bose, Peter Maimonis. Distribution, cutoff, and functional significance of a potential biomarker for Imprime PGG, an experimental cancer immunotherapeutic, in a healthy subject population. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A08.

Abstract B65: Immune cell priming and potentiation of anti-tumor effects by Imprime PGG®

Biothera is developing Imprime PGG® (Imprime PGG) for cancer that is synergistic with multiple anti-tumor monoclonal antibodies, demonstrating the potential to improve patient outcomes in a wide range of cancer indications. Although, the antitumor potential of Imprime PGG has now been well documented in several preclinical models, its immunomodulatory potential has not been fully elucidated. Here, we studied the in vivo effects of Imprime PGG, a pharmaceutical grade soluble yeast-derived β-1,3/1,6 glucan, on immune cell binding and function, using both tumor bearing and non-tumor bearing mice. Immunodeficient nude mice, with or without tumor (i.e. MiaPaCa-2 human pancreatic tumor), were administered a single dose of Imprime PGG (i.v.) and 3 days later, immune cells in peripheral blood, spleen, and/or tumor were isolated and evaluated for Imprime PGG binding and function. Using flow cytometric methods, Imprime PGG was found to bind B cells, macrophages, and neutrophils in whole blood and in addition, bound to NK cells in spleen. Notably, in mice bearing tumor, Imprime PGG administration lead to a relatively high percentage of Imprime PGG-bound macrophages and neutrophils in the tumor microenvironment. Furthermore, for the first time, we were able to demonstrate that Imprime PGG administered in vivo had the ability to “prime” immune cells and enhance their antitumor function. Administration of Imprime PGG increased the cytotoxic ability of immune cells, such as macrophages and NK cells, and lead to higher antibody mediated cytotoxicity and increased sensitivity to non-specific stimuli. Likewise, immune cells primed in vivo with Imprime PGG demonstrated increased cytokine responses (such as KC; a mouse isoform of human IL-8, IL-6, G-CSF, and MIP-1α and preliminary cytokine data on tumor infiltrating effector cells support an antitumor profile. Collectively, these results indicate that Imprime PGG when administered in vivo “primes” immune effector cells for enhanced antitumor activity. These observations further validate the potential benefit of combining this unique immunomodulatory drug with antitumor monoclonal antibodies as a positive treatment strategy in cancer. Citation Format: Yumi Yokoyama, Mariana I. Nelson, Nadine C. Ottoson, Mark N. Kirstein, Richard Walsh, Kyle S. Michel, Michael E. Danielson, Mary A. Antonysamy. Immune cell priming and potentiation of anti-tumor effects by Imprime PGG®. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B65.