Nevala, Wendy K.

Skin cancer affects more individuals in the USA than any other malignancy and malignant melanoma is particularly deadly because of its metastatic potential. Melanoma has been recognized as one of the most immunogenic malignancies; therefore, understanding the mechanisms of tumor–immune interaction is key for developing more efficient treatments. As the tumor microenvironment shows an immunosuppressive action, immunotherapeutic agents promoting endogenous immune response to cancer have been tested (interleukin-2, anticytotoxic-T-lymphocyte-associated antigen 4, and antiprogrammed cell death protein 1 monoclonal antibodies) as well as combinations of cytotoxic chemotherapy agents and inhibitors of angiogenesis (taxol/carboplatin/avastin). However, clinical outcomes are variable, with only a minority of patients achieving durable complete responses. The variability of immune homeostasis, which may be more active or more tolerant at any given time, in cancer patients and the interaction of the immune system with the tumor could explain the inconsistency in clinical outcomes among these patients. Recently, the role of the lymphocyte-to-monocyte-ratio (LMR) in the peripheral blood has been investigated and has been proven to be an independent predictor of survival in different hematological malignancies and in solid tumors. In melanoma, our group has validated the significance of LMR as a predictor of relapse after resection of advanced melanoma. In this study, we examined the dynamics in the immune system of patients with advanced melanoma by performing serial multiday concentration measurements of cytokines and immune cell subsets in the peripheral blood. The analysis of outcomes of chemotherapy administration as related to LMR on the day of treatment initiation showed that progression-free survival was improved in the patients who received chemotherapy on the day when LMR was elevated.

Galectin-9, a β-galactoside-binding protein, is defined as a negative regulator of T helper 1 (Th1) immune responses, favoring Th2 bias. Systemic immunity in patients with metastatic melanoma is predominantly Th2 biased. We hypothesized that galectin-9 can modulate systemic immunity toward Th2 polarization in patients with advanced melanoma. The presence or concentration of galectin-9 was assessed in tumors and plasma, in patients with metastatic melanoma. The immunomodulatory function of galectin-9 was determined by exposing human peripheral blood mononuclear cells to galectin-9 in vitro. Galectin-9 was expressed in 57% of tumors and was significantly (3.6-fold) increased in the plasma of patients with advanced melanoma compared with healthy controls (P<0.001). High plasma galectin-9 concentration was associated with systemic Th2 polarization and reduced 2-year survival compared with low/no galectin-9 expression. In-vitro, galectin-9 reduced proliferation of healthy peripheral blood mononuclear cells, and promoted Th1 cell apoptosis, Th2-biased cell phenotypes, and cytokine secretion. Galectin-9 also stimulated monocyte differentiation toward an M2 macrophage phenotype, as assessed by chemokine/cytokine secretion and CD206 expression, observed both in vitro as well as in patients with metastatic melanoma. Elevated galectin-9 in patient plasma correlated with Th2 systemic bias and less favorable clinical outcomes for patients with metastatic melanoma. This Th2 bias appears to be not only a feature of the known mechanisms of Th1 apoptosis by T-cell immunoglobulin and mucin-domain containing-3 binding, but also mediated by myeloid cell differentiation toward an M2 phenotype, that favors tumor progression. These data support galectin-9 as a novel therapeutic target for patients with metastatic melanoma.

Immune checkpoint therapy with PD-1 blockade has emerged as an effective therapy for many advanced cancers; however, only a small fraction of patients achieve durable responses. To date, there is no validated blood-based means of predicting the response to PD-1 blockade. We report that Bim is a downstream signaling molecule of the PD-1 pathway, and its detection in T cells is significantly associated with expression of PD-1 and effector T cell markers. High levels of Bim in circulating tumor-reactive (PD-1 + CD11a hi CD8 + ) T cells were prognostic of poor survival in patients with metastatic melanoma who did not receive anti–PD-1 therapy and were also predictive of clinical benefit in patients with metastatic melanoma who were treated with anti–PD-1 therapy. Moreover, this circulating tumor-reactive T cell population significantly decreased after successful anti–PD-1 therapy. Our study supports a crucial role of Bim in both T cell activation and apoptosis as regulated by PD-1 and PD-L1 interactions in effector CD8 + T cells. Measurement of Bim levels in circulating T cells of patients with cancer may provide a less invasive strategy to predict and monitor responses to anti–PD-1 therapy, although future prospective analyses are needed to validate its utility.

Abstract Antibody-directed chemotherapy (ADC) offers an advantage over conventional chemotherapy because it provides antibody-directed targeting, with resultant improvement in therapeutic efficacy and reduced toxicity. Despite extensive research, with notable exceptions, broad clinical application of ADC remains elusive; major hurdles include the instability of antibody–chemotherapy linkers and reduced tumor toxicity of the chemotherapy when bound to the antibody. To address these challenges, we have developed a platform technology that utilizes the nab-paclitaxel formulation of paclitaxel, Abraxane, in which hydrophobic paclitaxel is suspended in 130-nm albumin nanoparticles and thus made water-soluble. We have developed a method to noncovalently coat the Abraxane nanoparticle with recombinant mAbs (anti-VEGF, bevacizumab) and guide Abraxane delivery into tumors in a preclinical model of human A375 melanoma. Here, we define the binding characteristics of bevacizumab and Abraxane, demonstrate that the chemotherapy agent retains its cytotoxic effect, while the antibody maintains the ability to bind its ligand when the two are present in a single nanoparticle (AB160), and show that the nanoparticle yields improved antitumor efficacy in a preclinical human melanoma xenograft model. Further data suggest that numerous therapeutic monoclonal IgG1 antibodies may be utilized in this platform, which has implications for many solid and hematologic malignancies. Cancer Res; 76(13); 3954–64. ©2016 AACR.

Abstract Background: Phase 3 clinical results demonstrate that nab-paclitaxel (ABX) exhibits single agent activity in patients with metastatic melanoma relative to dacarbazine. Additionally, phase 2 clinical data suggest that ABX combined with bevacizumab (BEV) with/without carboplatin exhibits greater clinical efficacy than ABX alone. We hypothesized that the latter may be due to a pharmacokinetic, non-covalent interaction between bevacizumab (BEV) and nab-paclitaxel (ABX) allowing for greater localization of ABX to sites of VEGF overproduction (metastases). Herein we present pre-clinical data describing the utility of experimentally created ABX/BEV complex nanoparticles used to deliver ABX to VEGF producing cancer targets. Methods and Results: To test whether BEV binds to ABX we employed flow cytometry and discovered that the BEV does bind to ABX while preserving the ability of BEV to bind its ligand, VEGF. To measure binding affinity we used Biolayer Interferometry (BLItz) to measure association and dissociation constants. Results of BLItz experiments demonstrate that the binding affinity of BEV to ABX is pH and temperature dependent. The size and stability of the nanoparticle formed by binding BEV and ABX was determined using light refraction (Mastersizer) and Brownian motion (Nanosight). Results of these experiments suggest that a range of nanoparticles can be produced and stability of the BEV particle is increased relative to ABX particles alone. Finally the tumor efficacy of the BEV/ABX complex relative to ABX and BEV as well as sequential administration of the two drugs was tested in a xenograft model of A375 human melanoma cell line. Pharmacokinetic studies in these mice suggest that more ABX is deposited in the tumor site when treated with the complexes relative to ABX alone. Conclusions: The therapeutic antibody BEV has the capability to bind to the chemotherapy drug ABX while still binding its ligand, VEGF. The resulting nanoparticle is more stable than the original ABX particle. In a preclinical model of human A375 xenografts in nude mice, AB160 treatment offers significantly improved antitumor activity beyond that of ABX, BEV or ABX+BEV with no additional toxicity. Initial results indicate that the improved efficacy of the ABX/BEV nanoparticle relative to ABX alone is primarily due to the ability of the anti-VEGF antibody (BEV) to target the chemotherapy agent to the tumor where local VEGF concentration is very high. Clinical translation of the safety of a 160nm AB particle (AB160) in patients with metastatic melanoma is in progress. Citation Format: Wendy K. Nevala, Svetomir N. Markovic. Targeted nano-immune conjugates to melanoma: Preclinical testing of bevacizumab targeted nab-paclitaxel. [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 B77.

Abstract Background: The metastatic advancement of melanoma is largely dependent on its ability to evade immunity and gain access to the lymphatics. Characterization of tumor-draining, sentinel lymph nodes in patients has identified the nodes to express an immunosuppressed, Th2-dominant immune profile. Importantly, this profile is observed in sentinel lymph nodes even prior to evidence of metastasis supporting a model in which melanoma-derived factors prime tumor-draining, sentinel lymph nodes for tumor progression. Recent work in our lab has characterized the cellular interactions and the contribution of soluble factors in facilitating this immunosuppressive immune profile in fresh sentinel lymph nodes of patients; however, an appreciation for the role of subcellular counterparts remains to be explored. In this study, we considered a novel role for melanoma-derived, membrane-bound nanoparticles as drivers in polarizing immunity towards an immunosuppressive, tumor-promoting state. Methods and Results: Utilizing recent technology advances in Nanoparticle Tracking Analysis (NTA), characterization of melanoma derived vesicles from human melanoma cell lines showed a size distribution of particles ranging from 30nm to 1um, representing both exosomes and microvesicles. Following hypoxic culture, vesicle production was amplified, suggesting the increasingly hypoxic tumor microenvironment may trigger vesicle secretion. To evaluate the immune regulating potential of melanoma-derived vesicles, proteomic analysis of vesicle cargo by mass spectrometry identified over 100 proteins including known immune regulators Galectin 3, MHC I, ICAM1 and MIF. Functional assessment revealed that dendritic cells matured in vitro in the presence of melanoma-derived vesicles have significantly reduced surface marker expression of co-stimulatory markers CD80 and CD86 compared to controls. In addition to an activation defect, microarray analysis of dendritic cells matured in the presence of melanoma vesicles displayed a unique RNA expression profile. Pathway analysis of the expression arrays identified numerous overlapping signaling pathways involving proteins present in the melanoma-derived vesicles by mass spectrometry suggesting tumor-derived vesicle cargo has the ability to modulate dendritic cell function. Conclusion: Taken together, this study demonstrates a critical function for melanoma-derived vesicles in driving immunosuppression by characterizing their generation, protein cargo and immune regulating properties. The enrichment of melanoma-derived vesicles for immune mediators and ability of the vesicles to suppress dendritic cell function suggests a mechanism by which regional immunosuppression precedes nodal involvement and may foster an environment for metastasis to occur. Moving forward, we are currently translating these findings into fresh sentinel lymph nodes obtained from patients in order to elucidate the role of melanoma-derived vesicles in metastasis. Understanding this mechanism may yield valuable insight as we seek to develop efficacious immunotherapies targeting nodal metastasis. Citation Format: Rachel Goldenstein, Wendy Nevala, Svetomir N. Markovic. Melanoma-derived nanovesicles as drivers of immunosuppression. [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 A70.

Abstract Introduction: Galectin-9 (Gal-9) is a carbohydrate-binding protein that interacts with T-cell immunoglobulin mucin 3 (TIM3) to modulate immune homeostasis. The Gal-9/TIM3 interaction down regulates activated T cells which promotes tumor growth, much like program death 1 (PD-1) and its ligand (PD-L1). Still, the effect Gal-9 has on the immune environment of melanoma patients is weakly understood. This study focuses on the role of Gal-9 in immunoregulation of healthy persons and metastatic melanoma patients. Methods: Soluble Gal-9 levels were determined from plasma collected from patients and healthy persons using standard ELISA methods. Proliferation and apoptosis of cells in the presence and absence of Gal-9 was determined by mixed lymphocyte reactions and Tunnel staining. Supernatants from these reactions were collected and run using a cytokine bead assay to determine changes in immune parameters with and without Gal-9. Results: Soluble Gal-9 was significantly higher in metastatic melanoma patients compared to healthy controls (3929 vs. 877 pg/mL, p=0.0003 respectively). In the presence of Gal-9, the proliferative effect of an allostimulus could be overridden until the level of responder cells to stimulator cells was 4:1. Using an apoptosis assay, Gal-9 was found to increase the level of cellular apoptosis; however, apoptosis was decreased as the allostimulus increased. Healthy cells incubated with Gal-9 were found to increase secretion of growth factors (EGF and FGF-2), chemokines (CXCL1, CXCL8, CCL2 and CCL3), T helper 1 cytokines (IFN-g and IL-7) which have shown tumor promoting affects and T helper 2 cytokines (IL-13 and IL-4). Conclusions: Gal-9 may potentially play a role in suppressing anti-tumor immunity. This suggests the use of a Gal-9 neutralizing antibody for therapeutic benefit in melanoma. Citation Format: Elizabeth Ann L. Enninga, Wendy K. Nevala, Svetomir N. Markovic. Galectin-9 has tumor promoting properties that may be targetable in metastatic melanoma. [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 A41.

Several recent studies have shown differences in the maternal immune milieu at different phases of pregnancy, but most studies have been cross-sectional or of relatively few time points. Levels of 42 cytokines were determined using a multiplex bead-based assay on archived serum from a cohort of pregnant womenN=16at median of 18 time points tested, from the first trimester through to parturition, per woman. Unconditional growth modeling was then used to determine time-dependent changes in levels of these cytokines. Macrophage-derived chemokine (MDC, aka CCL22) decreases as pregnancy progresses. IL-1β, IL-6, IL-8, IL-12p70, IL-13, IL-15, IP-10, and FLT3-ligand increase as a function of gestational weeks, and IFNα2, IL-1ra, IL-3, IL-9, IL-12p40, and soluble CD40 ligand increase as a function of trimester. As pregnancy normally progresses, a maternal shift away from a type 2-biased immune response and toward an inflammatory/counterregulatory response is observed.

ProblemSeveral pregnancy complications have disparities based on the sex of the fetus. It is unknown whether the sex of the fetus differentially alters the maternal immune milieu, potentially contributing to the observed differences.Method of studyUsing maternal plasma collected during 38 uncomplicated pregnancies (19 males, 19 females), we compared levels of cytokines, sex hormones, and angiogenic factors throughout gestation and postpartum.ResultsMale fetal sex was associated with higher levels of proinflammatory cytokines (G‐CSF, IL‐12p70, IL‐21, and IL‐33) and angiogenic factors (PlGF and VEGF‐A) compared with female fetal sex at multiple timepoints. Female fetal sex was associated with higher levels of regulatory cytokines (IL‐5, IL‐9, IL‐17, and IL‐25). IL‐27 increased throughout pregnancy regardless of fetal sex. There was no fetal sex‐based difference in analyte concentrations at the postpartum measurement.ConclusionWomen carrying a male fetus exhibit a more proinflammatory/proangiogenic immune milieu than women carrying a female fetus.

Abstract Monocytes may contribute to tumor progression in part by mediating tumor-induced immunosuppression. Alterations to the monocyte populations and functions in untreated patients with late-stage melanoma are not fully understood. To characterize these alterations, we compared the frequency, phenotype, and functional capacity of peripheral blood monocytes and other myeloid cells in untreated, newly diagnosed stage IV melanoma patients (n = 18) with those in healthy volunteers. Stage IV untreated melanoma patients exhibited a sizeable decrease in the percentage of monocytes (P &lt; 0.0001) that included a drop in the percentage of the CD14+CD16− classical monocyte pool (P = 0.006). Although there was not a significant difference in the CD14+HLA-DRlow/− monocyte population between the patients with melanoma and the healthy volunteers, the HLA-DR levels were considerably lower in the patients' CD14+CD16+ intermediate (P &lt; 0.0001) and CD14lowCD16+ nonclassical monocyte populations (P = 0.001). Decreased surface expression of CD86 (P = 0.0006) and TNFRII (P = 0.0001) and increased expression of tissue factor and PD-L1 (P = 0.003) were identified on monocytes from patients with melanoma. Furthermore, these monocytes had decreased ability to upregulate CD80 expression and cytokine production following stimulation with agonist of Toll-like receptor 3 (TLR3). Peripheral blood dendritic cell subsets were decreased in untreated stage IV melanoma patients. Our study demonstrates that untreated late-stage melanoma patients exhibit monocytopenia in addition to phenotypic and functional deficiencies that may negatively affect their immune function. These findings open new avenues into examining the role of monocyte populations in melanoma development. Cancer Immunol Res; 2(3); 241–8. ©2013 AACR.