Richard K. Yang

Quantification of Tetracycline Resistance Genes in Feedlot Lagoons by Real-Time PCR

ABSTRACT A new real-time PCR method is presented that detects and quantifies three tetracycline resistance (Tcr) genes [tet(O), tet(W), and tet(Q)] in mixed microbial communities resident in feedlot lagoon wastewater. Tcr gene real-time TaqMan primer-probe sets were developed and optimized to quantify the Tcr genes present in seven different cattle feedlot lagoons, to validate the method, and to assess whether resistance gene concentrations correlate with free-tetracycline levels in lagoon waters. The method proved to be sensitive across a wide range of gene concentrations and provided consistent and reproducible results from complex lagoon water samples. The log10 of the sum of the three resistance gene concentrations was correlated with free-tetracycline levels (r2 = 0.50, P < 0.001; n = 18), with the geometric means of individual resistance concentrations ranging from 4- to 8.3-fold greater in lagoon samples with above-median tetracycline levels (>1.95 μg/liter by enzyme-linked immunosorbent assay techniques) than in below-median lagoon samples. Of the three Tcr genes tested, tet(W) and tet(Q) were more commonly found in lagoon water samples. Successful development of this real-time PCR assay will permit other studies quantifying Tcr gene numbers in environmental and other samples.

Genotypes of NK cell KIR receptors, their ligands, and Fcγ receptors in the response of neuroblastoma patients to Hu14.18-IL2 immunotherapy.

Response to immunocytokine (IC) therapy is dependent on natural killer cells in murine neuroblastoma (NBL) models. Furthermore, killer immunoglobulin-like receptor (KIR)/KIR-ligand mismatch is associated with improved outcome to autologous stem cell transplant for NBL. Additionally, clinical antitumor response to monoclonal antibodies has been associated with specific polymorphic-FcγR alleles. Relapsed/refractory NBL patients received the hu14.18-IL2 IC (humanized anti-GD2 monoclonal antibody linked to human IL2) in a Childrens Oncology Group phase II trial. In this report, these patients were genotyped for KIR, HLA, and FcR alleles to determine whether KIR receptor-ligand mismatch or specific FcγR alleles were associated with antitumor response. DNA samples were available for 38 of 39 patients enrolled: 24 were found to have autologous KIR/KIR-ligand mismatch; 14 were matched. Of the 24 mismatched patients, 7 experienced either complete response or improvement of their disease after IC therapy. There was no response or comparable improvement of disease in patients who were matched. Thus KIR/KIR-ligand mismatch was associated with response/improvement to IC (P = 0.03). There was a trend toward patients with the FcγR2A 131-H/H genotype showing a higher response rate than other FcγR2A genotypes (P = 0.06). These analyses indicate that response or improvement of relapsed/refractory NBL patients after IC treatment is associated with autologous KIR/KIR-ligand mismatch, consistent with a role for natural killer cells in this clinical response.

Correlations between in situ denitrification activity and nir-gene abundances in pristine and impacted prairie streams.

Denitrification is a process that reduces nitrogen levels in headwaters and other streams. We compared nirS and nirK abundances with the absolute rate of denitrification, the longitudinal coefficient of denitrification (i.e., Kden, which represents optimal denitrification rates at given environmental conditions), and water quality in seven prairie streams to determine if nir-gene abundances explain denitrification activity. Previous work showed that absolute rates of denitrification correlate with nitrate levels; however, no correlation has been found for denitrification efficiency, which we hypothesise might be related to gene abundances. Water-column nitrate and soluble-reactive phosphorus levels significantly correlated with absolute rates of denitrification, but nir-gene abundances did not. However, nirS and nirK abundances significantly correlated with Kden, as well as phosphorus, although no correlation was found between Kden and nitrate. These data confirm that absolute denitrification rates are controlled by nitrate load, but intrinsic denitrification efficiency is linked to nirS and nirK gene abundances.

Intratumoral hu14.18–IL-2 (IC) Induces Local and Systemic Antitumor Effects That Involve Both Activated T and NK Cells As Well As Enhanced IC Retention

hu14.18–IL-2 (IC) is an immunocytokine consisting of human IL-2 linked to hu14.18 mAb, which recognizes the GD2 disialoganglioside. Phase 2 clinical trials of i.v. hu14.18–IL-2 (i.v.-IC) in neuroblastoma and melanoma are underway and have already demonstrated activity in neuroblastoma. We showed previously that intratumoral hu14.18–IL-2 (IT-IC) results in enhanced antitumor activity in mouse models compared with i.v.-IC. The studies presented in this article were designed to determine the mechanisms involved in this enhanced activity and to support the future clinical testing of intratumoral administration of immunocytokines. Improved survival and inhibition of growth of both local and distant tumors were observed in A/J mice bearing s.c. NXS2 neuroblastomas treated with IT-IC compared with those treated with i.v.-IC or control mice. The local and systemic antitumor effects of IT-IC were inhibited by depletion of NK cells or T cells. IT-IC resulted in increased NKG2D receptors on intratumoral NKG2A/C/E+ NKp46+ NK cells and NKG2A/C/E+ CD8+ T cells compared with control mice or mice treated with i.v.-IC. NKG2D levels were augmented more in tumor-infiltrating lymphocytes compared with splenocytes, supporting the localized nature of the intratumoral changes induced by IT-IC treatment. Prolonged retention of IC at the tumor site was seen with IT-IC compared with i.v.-IC. Overall, IT-IC resulted in increased numbers of activated T and NK cells within tumors, better IC retention in the tumor, enhanced inhibition of tumor growth, and improved survival compared with i.v.-IC.

In situ tumor vaccination by combining local radiation and tumor-specific antibody or immunocytokine treatments

Interest in combining radiotherapy and immune checkpoint therapy is growing rapidly. In this study, we explored a novel combination of this type to augment antitumor immune responses in preclinical murine models of melanoma, neuroblastoma, and head and neck squamous cell carcinoma. Cooperative effects were observed with local radiotherapy and intratumoral injection of tumor-specific antibodies, arising in part from enhanced antibody-dependent cell-mediated cytotoxicity (ADCC). We could improve this response by combining radiation with intratumoral injection of an IL2-linked tumor-specific antibody (termed here an immunocytokine), resulting in complete regression of established tumors in most animals associated with a tumor-specific memory T-cell response. Given the T-cell response elicited by combined local radiation and intratumoral immunocytokine, we tested the potential benefit of adding this treatment to immune checkpoint blockade. In mice bearing large primary tumors or disseminated metastases, the triple-combination of intratumoral immunocytokine, radiation, and systemic anti-CTLA-4 improved primary tumor response and animal survival compared with combinations of any two of these three interventions. Taken together, our results show how combining radiation and intratumoral immunocytokine in murine tumor models can eradicate large tumors and metastases, eliciting an in situ vaccination effect that can be leveraged further by T-cell checkpoint blockade, with immediate implications for clinical evaluation. Cancer Res; 76(13); 3929-41. ©2016 AACR.

Loss of the Polycomb mark from bivalent promoters leads to activation of cancer-promoting genes in colorectal tumors

In colon tumors, the transcription of many genes becomes deregulated by poorly defined epigenetic mechanisms that have been studied mainly in established cell lines. In this study, we used frozen human colon tissues to analyze patterns of histone modification and DNA cytosine methylation in cancer and matched normal mucosa specimens. DNA methylation is strongly targeted to bivalent H3K4me3- and H3K27me3-associated promoters, which lose both histone marks and acquire DNA methylation. However, we found that loss of the Polycomb mark H3K27me3 from bivalent promoters was accompanied often by activation of genes associated with cancer progression, including numerous stem cell regulators, oncogenes, and proliferation-associated genes. Indeed, we found many of these same genes were also activated in patients with ulcerative colitis where chronic inflammation predisposes them to colon cancer. Based on our findings, we propose that a loss of Polycomb repression at bivalent genes combined with an ensuing selection for tumor-driving events plays a major role in cancer progression.

Intratumoral delivery of low doses of anti-CD40 mAb combined with monophosphoryl lipid a induces local and systemic antitumor effects in immunocompetent and T cell-deficient mice.

In this study, an agonistic anti-CD40 monoclonal antibody was combined with monophosphoryl lipid A (MPL), a nontoxic derivative of lipopolysaccharide and agonist of toll-like receptor-4, to assess the immunomodulatory and antitumor synergy between the 2 agents in mice. Anti-CD40 was capable of priming macrophages to subsequent ex vivo activation by MPL in immunocompetent and T-cell–depleted mice. Intraperitoneal injections of anti-CD40+MPL induced additive to synergistic suppression of poorly immunogenic B16-F10 melanoma growing subcutaneously in syngeneic mice. When anti-CD40+MPL were injected directly into the subcutaneous tumor, the combination treatment was more effective, even with a 25-fold reduction in dose. Low-dose intratumoral treatment also slowed the growth of a secondary tumor growing simultaneously at a distant, untreated site. Antitumor effects were also induced in severe combined immunodeficiency mice and in T-cell–depleted C57BL/6 mice. Taken together, our results show that the antitumor effects of anti-CD40 are enhanced by subsequent treatment with MPL, even in T-cell–deficient hosts. These preclinical data suggest that an anti-CD40+MPL combined regimen is appropriate for clinical testing in human patients, including cancer patients who may be immunosuppressed from prior chemotherapy.

Soluble interleukin-2 receptor α activation in a Children's Oncology Group randomized trial of interleukin-2 Therapy for Pediatric Acute Myeloid Leukemia†‡

To assess associations of soluble IL‐2 receptor alpha (sIL‐2rα) concentration with outcomes in pediatric acute myeloid leukemia (AML) in a phase 3 trial of IL‐2 therapy.

Tumor-associated myeloid cells can be activated in vitro and in vivo to mediate antitumor effects

Tumor growth is often accompanied by the accumulation of myeloid cells in the tumors and lymphoid organs. These cells can suppress T cell immunity, thereby posing an obstacle to T cell-targeted cancer immunotherapy. In this study, we tested the possibility of activating tumor-associated myeloid cells to mediate antitumor effects. Using the peritoneal model of B16 melanoma, we show that peritoneal cells (PEC) in tumor-bearing mice (TBM) had reduced ability to secrete nitric oxide (NO) following in vitro stimulation with interferon gamma and lipopolysaccharide, as compared to PEC from control mice. This reduced function of PEC was accompanied by the influx of CD11b+ Gr-1+ myeloid cells to the peritoneal cavity. Nonadherent PEC were responsible for most of the NO production in TBM, whereas in naïve mice NO was mainly secreted by adherent CD11b+ F4/80+ macrophages. Sorted CD11b+ Gr-1− monocytic and CD11b+ Gr-1+ granulocytic PEC from TBM had a reduced ability to secrete NO following in vitro stimulation (compared to naïve PEC), but effectively suppressed proliferation of tumor cells in vitro. In vivo, treatment of mice bearing established peritoneal B16 tumors with anti-CD40 and CpG resulted in activation of tumor-associated PEC, reduction in local tumor burden and prolongation of mouse survival. Inhibition of NO did not abrogate the antitumor effects of stimulated myeloid cells. Taken together, the results indicate that in tumor-bearing hosts, tumor-associated myeloid cells can be activated to mediate antitumor effects.

Abstract 1538: Enhanced antitumor effects of intratumoral (IT) hu14.18-IL2 immunocytokine (IC) compared to intravenous (IV) hu14.18-IL2 are distinguished by increased activated tumor infiltrating lymphocytes (TILs) and IC retention

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Hu14.18-IL2 (APN301, Apeiron Biologics) is an immunocytokine (IC) consisting of human IL2 linked to each IgG heavy chain of the hu14.18 mAb, which recognizes the GD2 disialoganglioside. Phase 2 clinical trials of IV hu14.18-IL2 IC in neuroblastoma and melanoma are underway, with activity already demonstrated in neuroblastoma. We have previously shown that intratumoral IC treatment (IT-IC) results in enhanced anti-tumor activity in mouse models. These studies were designed to determine the mechanisms involved in the enhanced activity and to provide justification for future clinical testing of localized IT administration of this and other immunocytokines. Methods: We characterize tumor growth, survival outcomes, histology and phenotype of tumor infiltrating lymphocytes (TILs) by flow cytometry of IT-hu.14.18-IL2 treatment of A/J mice bearing subcutaneous NXS2 neuroblastomas. Looking at these parameters, IT-IC treated mice are compared to IV-IC treated, IT PBS treated and untreated mice. Data: Mice receiving IT-IC show significant increases [by immunohistochemistry (IHC) and by flow cytometry] of NKp46+ Natural Killer (NK) cells and CD8a+ cytotoxic T cells in TIL populations compared to control tumor-bearing mice. Improved survival and inhibition of tumor growth are observed in mice receiving IT-IC vs. untreated mice or mice receiving IV-IC. Comparisons within treatment groups, or independent of treatment groups, show that the number of NK or CD8 T cells in the tumor correlates inversely with change in tumor size. Analyses by IHC and flow-cytometry show greater IC detection in tumor after IT-IC vs. IV-IC. Moreover, IT-IC results in improved IC retention in tumors and increased NKG2D effector receptors on intratumoral NKG2A/C/E cells and on CD8 T cells when compared to control mice or mice receiving IV-IC. The augmented NKG2D seen in TILs was not seen in spleen cells, supporting the localized nature of the intratumoral changes induced by this treatment. Conclusions: In this murine neuroblastoma model, enhanced antitumor effects of IT hu14.18-IL2 compared to IV hu14.18-IL2 are distinguished by increased activated TILs and IC retention, improved survival and inhibition of tumor growth. These observations suggest that localized administration of immunocytokines in human patients may show analogous advantages over IV administration. In accordance, we have designed a phase I/II clinical trial protocol investigating the maximum tolerated dose (MTD) and efficacy endpoints of intratumorally administered hu14.18-IL2 in stage III/IV melanoma patients with recurrent or refractory disease, and are working towards approval for its activation. Supported by R01-CA-32685-27. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1538. doi:1538-7445.AM2012-1538