Daniel E. Cramer

Yeast β-Glucan Amplifies Phagocyte Killing of iC3b-Opsonized Tumor Cells via Complement Receptor 3-Syk-Phosphatidylinositol 3-Kinase Pathway

Anti-tumor mAbs hold promise for cancer therapy, but are relatively inefficient. Therefore, there is a need for agents that might amplify the effectiveness of these mAbs. One such agent is β-glucan, a polysaccharide produced by fungi, yeast, and grains, but not mammalian cells. β-Glucans are bound by C receptor 3 (CR3) and, in concert with target-associated complement fragment iC3b, elicit phagocytosis and killing of yeast. β-Glucans may also promote killing of iC3b-opsonized tumor cells engendered by administration of anti-tumor mAbs. In this study, we report that tumor-bearing mice treated with a combination of β-glucan and an anti-tumor mAb show almost complete cessation of tumor growth. This activity evidently derives from a 25-kDa fragment of β-glucan released by macrophage processing of the parent polysaccharide. This fragment, but not parent β-glucan, binds to neutrophil CR3, induces CBRM 1/5 neoepitope expression, and elicits CR3-dependent cytotoxicity. These events require phosphorylation of the tyrosine kinase, Syk, and consequent PI3K activation because β-glucan-mediated CR3-dependent cytotoxicity is greatly decreased by inhibition of these signaling molecules. Thus, β-glucan enhances tumor killing through a cascade of events, including in vivo macrophage cleavage of the polysaccharide, dual CR3 ligation, and CR3-Syk-PI3K signaling. These results are important inasmuch as β-glucan, an agent without evident toxicity, may be used to amplify tumor cell killing and may open new opportunities in the immunotherapy of cancer.

Combined yeast {beta}-glucan and antitumor monoclonal antibody therapy requires C5a-mediated neutrophil chemotaxis via regulation of decay-accelerating factor CD55.

Administration of a combination of yeast-derived beta-glucan with antitumor monoclonal antibodies (mAb) has significant therapeutic efficacy in a variety of syngeneic murine tumor models. We have now tested this strategy using human carcinomas implanted in immunocompromised severe combined immunodeficient mice. Combined immunotherapy was therapeutically effective in vivo against NCI-H23 human non-small-cell lung carcinomas, but this modality was surprisingly ineffective against SKOV-3 human ovarian carcinomas. Whereas NCI-H23 tumors responded to this combination therapy with increased intratumoral neutrophil infiltration and C5a production, these responses were lacking in treated SKOV-3 tumors. Further results suggested that SKOV-3 tumors were protected by up-regulation of the membrane complement regulatory protein CD55 (decay-accelerating factor). Blockade of CD55 in vitro led to enhanced deposition of C activation product C3b and increased cytotoxicity mediated by beta-glucan-primed neutrophils. In vivo, administration of anti-CD55 mAb along with beta-glucan and anti-Her-2/neu mAb caused tumor regression and greatly improved long-term survival in animals bearing the previously resistant SKOV-3 tumors. This was accompanied by increased intratumoral neutrophil accumulation and C5a production. We conclude that CD55 suppresses tumor killing by antitumor mAb plus beta-glucan therapy (and, perhaps, in other circumstances). These results suggest a critical role for CD55 to regulate iC3b and C5a release and in turn to influence the recruitment of beta-glucan-primed neutrophils eliciting killing activity.

Therapeutic potential of various β-glucan sources in conjunction with anti-tumor monoclonal antibody in cancer therapy

Combined β-glucan with anti-tumor mAb therapy has demonstrated therapeutic efficacy in murine tumor models. The current study was designed to compare the therapeutic efficacy of various sources of β-glucans. Our studies demonstrated that yeast β-glucan, in combination with anti-tumor mAb, resulted in significantly smaller tumor burdens and achieved enhanced long-term survival compared to mAb alone or β-glucan extracts from mushrooms. Further studies indicated that yeast β-glucan particle was superior to mushroom extracts in inducing cytokine secretion, particularly IL-12 production in dendritic cells (DCs). In addition, results showed that cytokine production was markedly decreased in MyD88-deficient macrophages and DCs but not in complement receptor 3 (CR3)-deficient mice. Our data suggest that yeast β-glucan demonstrates much stronger adjuvant activity compared to mushroom β-glucan extracts in tumor therapy. This effect of yeast β-glucan may be in part ascribed to the cytokine secretion by DCs and macrophages and bioavailability of active β-glucan moiety.

Yeast-Derived β-Glucan Augments the Therapeutic Efficacy Mediated by Anti–Vascular Endothelial Growth Factor Monoclonal Antibody in Human Carcinoma Xenograft Models

Purpose: Bevacizumab is a recombinant IgG1 humanized monoclonal antibody against vascular endothelial growth factor (VEGF). Its proposed mechanism of action is independent of immune effector functions. Many human carcinomas not only secrete VEGF but also express membrane-bound VEGF. In addition, VEGF receptors are expressed on tumor cells. It is hypothesized that bevacizumab could bind membrane-bound VEGF or VEGF-VEGF receptor complexes on tumors, thereby initiating potential immunologic consequences. We previously showed that yeast-derived β-glucan functions with antitumor antibodies that activate complement to recruit complement receptor 3–expressing leukocytes capable of mediating complement receptor 3–dependent cellular cytotoxicity of tumors opsonized with iC3b. In the current study, the therapeutic efficacy mediated by combining bevacizumab with yeast-derived β-glucan was studied in human carcinoma xenograft models. Experimental Design: Human tumor cell lines were screened for membrane-bound VEGF expression both in vitro and in vivo. Complement activation mediated by bevacizumab was examined. Tumor cell lines positive or negative for membrane-bound VEGF expression were implanted in severe combined immunodeficient mice to establish xenograft models. Tumor-bearing mice were treated with different regimens. Tumor regression and long-term survival were recorded. Results: Human ovarian carcinoma SKOV-3 cells expressed membrane-bound VEGF both in vitro and in vivo. Bevacizumab was bound to membrane-bound VEGF, activated complement, and synergized with β-glucan to elicit cellular cytotoxicity in vitro. In vivo study showed that β-glucan could significantly augment the therapeutic efficacy mediated by bevacizumab. Conclusions: Yeast-derived β-glucan can synergize with anti-VEGF monoclonal antibody bevacizumab for the treatment of cancer with membrane-bound VEGF expression.

CD8+, αβ-TCR+, and γδ-TCR+ Cells in the Recipient Hematopoietic Environment Mediate Resistance to Engraftment of Allogeneic Donor Bone Marrow

Historically, conditioning for engraftment of hematopoietic stem cells has been nonspecific. In the present study, we characterized which cells in the recipient hematopoietic microenvironment prevent allogeneic marrow engraftment. Mice defective in production of αβ-TCR+, γδ-TCR+, αβ- plus γδ-TCR+, CD8+, or CD4+ cells were transplanted with MHC-disparate allogeneic bone marrow. Conditioning with 500 cGy total body irradiation (TBI) plus a single dose of cyclophosphamide (CyP) on day +2 establishes chimerism in normal recipients. When mice were conditioned with 300 cGy TBI plus a single dose of CyP on day +2, all engrafted, except wild-type controls and those defective in production of CD4+ T cells. Mice lacking both αβ- and γδ-TCR+ cells engrafted without conditioning, suggesting that both αβ- and γδ-TCR T cells in the host play critical and nonredundant roles in preventing engraftment of allogeneic bone marrow. CD8 knockout (KO) mice engrafted without TBI, but only if they received CyP on day +2 relative to the marrow infusion, showing that a CD8− cell was targeted by the CyP conditioning. The CD8+ cell effector function is mechanistically different from that for conventional T cells, and independent of CD4+ T helper cells because CD4 KO mice require substantially higher levels of conditioning than the other KO phenotypes. These results suggest that a number of cell populations with different mechanisms of action mediate resistance to engraftment of allogeneic marrow. Targeting of specific recipient cellular populations may permit conditioning approaches to allow mixed chimerism with minimal morbidity and could potentially avoid the requirement for myelotoxic agents altogether.

Mobilization of Hematopoietic Progenitor Cells by Yeast-Derived β-Glucan Requires Activation of Matrix Metalloproteinase-9

Poly‐(1,6)‐β‐d‐glucopyranosyl‐(1,3)‐β‐d‐glucopyranose (PGG) β‐glucan is a soluble yeast‐derived polysaccharide that has previously been shown to induce hematopoietic progenitor cell (HPC) mobilization. However, the mobilizing mechanism of action remains unknown. Here, we confirmed that PGG β‐glucan alone or in combination with granulocyte colony‐stimulating factor (G‐CSF) mobilizes HPC into the periphery. Optimal mobilizing effects were seen 24–48 hours after PGG β‐glucan doses of 4.8–9.6 mg/kg. Animals treated with G‐CSF and PGG β‐glucan showed a collaborative effect in HPC mobilization compared with G‐CSF treatment alone. Additional studies demonstrated that neither complement 3 nor complement receptor 3 played a role in this effect and that PGG β‐glucan treatment did not induce proinflammatory cytokine secretion. However, bone marrow cells from PGG β‐glucan‐treated mice secreted abundant matrix metalloproteinase‐9 (MMP‐9), and PGG β‐glucan‐induced HPC mobilization was abrogated in MMP‐9 knockout mice. Moreover, we demonstrated that both hematopoietic and nonhematopoietic cells contributed to MMP‐9 secretion upon PGG β‐glucan treatment. In addition, HPCs mobilized by PGG β‐glucan had similar levels of engraftment in host and lineage differentiation capability compared with those mobilized by G‐CSF. Thus, PGG β‐glucan is an agent that enhances HPC mobilization and may improve the outcome of clinical stem cell transplantation.

The role of alphabeta- and gammadelta-T cells in allogenic donor marrow on engraftment, chimerism, and graft-versus-host disease.

BACKGROUND We previously characterized a facilitating cell (FC) in mouse marrow that enables engraftment of allogeneic hematopoietic stem cells (HSCs) without causing graft-versus-host disease (GVHD). The FC shares some cell surface molecules with T cells (Thy1+, CD3epsilon+, CD8+, CD5+, and CD2+) but is T-cell receptor (TCR) negative. Historically, depletion of CD3+ or CD8+ cells from rat marrow was associated with an increased rate of failure of engraftment. In this study, we evaluated whether depletion of alphabeta- and gammadelta-TCR(+) T cells from donor marrow would retain engraftment potential yet avoid GVHD. METHODS Wistar-Furth rats were conditioned with 950 cGy of total body irradiation and transplanted with ACI bone marrow processed to remove either alphabeta-TCR(+), gammadelta-TCR(+), or alphabeta- plus gammadelta-TCR(+) T cells. Recipients were typed for chimerism at 28 days and monthly thereafter. RESULTS Recipients of marrow depleted of alphabeta- (group A), gammadelta- (group B), or alphabeta- and gammadelta-TCR(+) T cells (group C) engrafted and had an average chimerism level of 73.0+/-8.3%, 92.3+/-9.2%, and 46.3+/-32.8%, respectively. Aggressive T-cell depletion did not remove the FC population (CD8+/CD3+/TCR(-)). Group A and group B both developed GVHD, with a higher incidence of GVHD in group B compared to group A. None of the recipients in group C developed GVHD. CONCLUSIONS These data demonstrate that depletion of T cells from rat marrow does not impair engraftment of HSCs, indirectly supporting the existence of FCs in rat marrow. Moreover, donor alphabeta- and gammadelta-TCR(+) T cells contribute to GVHD in a nonredundant fashion, although alphabeta-TCR(+) T cells are more potent as the effector cells. Finally, the level of donor chimerism is influenced by the composition of the graft, because recipients of marrow that contain alphabeta-TCR(+) T cells exhibited significantly higher donor chimerism compared to recipients of marrow depleted of both alphabeta- and gammadelta-TCR(+) T cells.

Discovery of a novel compound with anti-venezuelan equine encephalitis virus activity that targets the nonstructural protein 2.

Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC50 = 0.84 µM), for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection.

Discovery of a Broad-Spectrum Antiviral Compound That Inhibits Pyrimidine Biosynthesis and Establishes a Type 1 Interferon-Independent Antiviral State

ABSTRACT Viral emergence and reemergence underscore the importance of developing efficacious, broad-spectrum antivirals. Here, we report the discovery of tetrahydrobenzothiazole-based compound 1, a novel, broad-spectrum antiviral lead that was optimized from a hit compound derived from a cytopathic effect (CPE)-based antiviral screen using Venezuelan equine encephalitis virus. Compound 1 showed antiviral activity against a broad range of RNA viruses, including alphaviruses, flaviviruses, influenza virus, and ebolavirus. Mechanism-of-action studies with metabolomics and molecular approaches revealed that the compound inhibits host pyrimidine synthesis and establishes an antiviral state by inducing a variety of interferon-stimulated genes (ISGs). Notably, the induction of the ISGs by compound 1 was independent of the production of type 1 interferons. The antiviral activity of compound 1 was cell type dependent with a robust effect observed in human cell lines and no observed antiviral effect in mouse cell lines. Herein, we disclose tetrahydrobenzothiazole compound 1 as a novel lead for the development of a broad-spectrum, antiviral therapeutic and as a molecular probe to study the mechanism of the induction of ISGs that are independent of type 1 interferons.

Hematopoietic stem cells from the marrow of mice treated with Flt3 ligand are significantly expanded but exhibit reduced engraftment potential.

BACKGROUND Hematopoietic stem cells (HSC) can be significantly expanded by hematopoietic growth factors. Flt3 ligand (FL) is a hematopoietic growth factor that induces proliferation and mobilization of HSC into the peripheral blood. We previously reported that FL-mobilized HSC exhibit superior engraftment potential. The engraftment potential of FL-expanded HSC in the bone marrow compartment has not been evaluated. In this study, we investigated the effect of in vivo administration of FL on the engraftment potential of HSC expanded in the marrow. METHODS B10.BR (H-2k) donor mice were treated for 10 days with 10 microg of FL per day. Partially conditioned allogeneic B10 (H-2b) recipients received whole bone marrow. Purified HSC (c-Kit+/Sca1+/lin-) from the marrow were also transplanted in ablated syngeneic B10.BR recipients. RESULTS FL treatment significantly expanded HSC in the marrow compartment. The absolute number of T cells and granulocytes were unchanged whereas dendritic cells, facilitating cells, and HSC were significantly increased in the bone marrow of donor mice treated with FL compared with untreated mice. Mice conditioned with 700 cGy and transplanted with FL-treated allogeneic bone marrow showed a significantly lower rate of engraftment (14%) compared with recipients of bone marrow from untreated mice (100%). Syngeneic recipients transplanted with 500, 1000, 2000, or 3000 purified HSC from FL-treated donors also showed reduced long-term survival compared with mice transplanted with HSC from untreated donors. Cell cycle analysis revealed that significantly more bone marrow HSC were in cycle after FL treatment compared with unmanipulated controls. CONCLUSION These data show that FL treatment for 10 days induces proliferation of HSC but reduces the engraftment potential of HSC harvested from the marrow. The reduced syngeneic engraftment of HSC indicates that FL treatment induces intrinsic changes in HSC, resulting in failure of long-term engraftment or self-renewal despite no change in characteristic phenotype of HSC.