Ann E. Sluder

Spiroindolines identify the vesicular acetylcholine transporter as a novel target for insecticide action.

The efficacy of all major insecticide classes continues to be eroded by the development of resistance mediated, in part, by selection of alleles encoding insecticide insensitive target proteins. The discovery of new insecticide classes acting at novel protein binding sites is therefore important for the continued protection of the food supply from insect predators, and of human and animal health from insect borne disease. Here we describe a novel class of insecticides (Spiroindolines) encompassing molecules that combine excellent activity against major agricultural pest species with low mammalian toxicity. We confidently assign the vesicular acetylcholine transporter as the molecular target of Spiroindolines through the combination of molecular genetics in model organisms with a pharmacological approach in insect tissues. The vesicular acetylcholine transporter can now be added to the list of validated insecticide targets in the acetylcholine signalling pathway and we anticipate that this will lead to the discovery of novel molecules useful in sustaining agriculture. In addition to their potential as insecticides and nematocides, Spiroindolines represent the only other class of chemical ligands for the vesicular acetylcholine transporter since those based on the discovery of vesamicol over 40 years ago, and as such, have potential to provide more selective tools for PET imaging in the diagnosis of neurodegenerative disease. They also provide novel biochemical tools for studies of the function of this protein family.

Mature B cells accelerate wound healing after acute and chronic diabetic skin lesions

Chronic wounds affect 12–15% of patients with diabetes and are associated with a drastic decrease in their quality of life. Here, we demonstrate that purified mature naive B220+/CD19+/IgM+/IgD+ B cells improve healing of acute and diabetic murine wounds after a single topical application. B cell treatment significantly accelerated acute wound closure by 2–3 days in wild‐type mice and 5–6 days in obese diabetic mice. The treatment led to full closure in 43% of chronic diabetic wounds, as compared to only 5% in saline‐treated controls. Applying equivalent numbers of T cells or disrupted B cells failed to reproduce these effects, indicating that live B cells mediated pro‐healing responses. Topically applied B cell treatment was associated with significantly reduced scar size, increased collagen deposition and maturation, enhanced angiogenesis, and increased nerve growth into and under the healing wound. β‐III tubulin+ nerve endings in scars of wounds treated acutely with B cells showed increased relative expression of growth‐associated protein 43. The improved healing associated with B cell treatment was supported by significantly increased fibroblast proliferation and decreased apoptosis in the wound bed and edges, altered kinetics of neutrophil infiltration, as well as an increase in TGF‐β and a significant reduction in MMP2 expression in wound granulation tissue. Our findings indicate that the timeline and efficacy of wound healing can be experimentally manipulated through the direct application of mature, naive B cells, which effectively modify the balance of mature immune cell populations within the wound microenvironment and accelerate the healing process.

AMD3100 Augments the Efficacy of Mesothelin-Targeted, Immune-Activating VIC-008 in Mesothelioma by Modulating Intratumoral Immunosuppression

CXCR4 antagonist AMD3100 promoted conversion of Tregs to helper-like cells and reduced PD-1 expression on CD8+ T cells in mesothelioma. The combination of AMD31000 with a mesothelin-targeting immune stimulator, VIC-008, controlled tumors and prolonged survival of tumor-bearing animals. AMD3100 (plerixafor), a CXCR4 antagonist, has been demonstrated to suppress tumor growth and modulate intratumoral T-cell trafficking. However, the effect of AMD3100 on immunomodulation remains elusive. Here, we explored immunomodulation and antitumor efficacy of AMD3100 in combination with a previously developed mesothelin-targeted, immune-activating fusion protein, VIC-008, in two syngeneic, orthotopic models of malignant mesothelioma in immunocompetent mice. We showed that combination therapy significantly suppressed tumor growth and prolonged animal survival in two mouse models. Tumor control and survival benefit were associated with enhanced antitumor immunity. VIC-008 augmented mesothelin-specific CD8+ T-cell responses in the spleen and lymph nodes and facilitated intratumoral lymphocytic infiltration. However, VIC-008 treatment was associated with increased programmed cell death protein-1 (PD-1) expression on intratumoral CD8+ T cells, likely due to high CXCL12 in the tumor microenvironment. AMD3100 alone and in combination with VIC-008 modulated immunosuppression in tumors and the immune system through suppression of PD-1 expression on CD8+ T cells and conversion of regulatory T cells (Tregs) into CD4+CD25–Foxp3+IL2+CD40L+ helper-like cells. In mechanistic studies, we demonstrated that AMD3100-driven Treg reprogramming required T cell receptor (TCR) activation and was associated with loss of PTEN due to oxidative inactivation. The combination of VIC-008 augmentation of tumor-specific CD8+ T-cell responses with AMD3100 abrogation of immunosuppression conferred significant benefits for tumor control and animal survival. These data provide new mechanistic insight into AMD3100-mediated immunomodulation and highlight the enhanced antitumor effect of AMD3100 in combination with a tumor antigen–targeted therapy in mouse malignant mesothelioma, which could be clinically relevant to patients with this difficult-to-treat disease. Cancer Immunol Res; 6(5); 539–51. ©2018 AACR.

Near-infrared 1064 nm laser modulates migratory dendritic cells to augment the immune response to intradermal influenza vaccine

Brief exposure of skin to near-infrared (NIR) laser light has been shown to augment the immune response to intradermal vaccination and thus act as an immunologic adjuvant. Although evidence indicates that the NIR laser adjuvant has the capacity to activate innate subsets including dendritic cells (DCs) in skin as conventional adjuvants do, the precise immunological mechanism by which the NIR laser adjuvant acts is largely unknown. In this study we sought to identify the cellular target of the NIR laser adjuvant by using an established mouse model of intradermal influenza vaccination and examining the alteration of responses resulting from genetic ablation of specific DC populations. We found that a continuous wave (CW) NIR laser adjuvant broadly modulates migratory DC (migDC) populations, specifically increasing and activating the Lang+ and CD11b−Lang− subsets in skin, and that the Ab responses augmented by the CW NIR laser are dependent on DC subsets expressing CCR2 and Langerin. In comparison, a pulsed wave NIR laser adjuvant showed limited effects on the migDC subsets. Our vaccination study demonstrated that the efficacy of the CW NIR laser is significantly better than that of the pulsed wave laser, indicating that the CW NIR laser offers a desirable immunostimulatory microenvironment for migDCs. These results demonstrate the unique ability of the NIR laser adjuvant to selectively target specific migDC populations in skin depending on its parameters, and highlight the importance of optimization of laser parameters for desirable immune protection induced by an NIR laser–adjuvanted vaccine.

Q-vaxcelerate: A distributed development approach for a new Coxiella burnetii vaccine

ABSTRACT Development of vaccines that are both safe and effective remains a costly and time-consuming challenge. To accelerate the pace of development and improve the efficacy and safety of candidate vaccines for both existing and emerging infectious agents, we have used a distributed development approach. This features the managed integration of individual expert groups having the requisite vaccine platforms, pre-clinical models, assays, skills and knowledge pertinent to a specific pathogen into a single, end-to-end development team capable of producing a new vaccine tailored to that particular agent. Distributed development focuses on integrating existing effort across multiple institutions rather than developing new capabilities or consolidating resources within an individual organization. Previously we have used the distributed development strategy to generate vaccine candidates for emerging viral diseases. Coxiella burnetii is a highly infectious and resilient bacterium and the causative agent of Q fever. Treatment for Q fever can require months of antibiotics. The current vaccine for Q-fever is only approved in Australia and requires prescreening due to the potential for severe reactogenicity in previously exposed individuals. Here we discuss Q-VaxCelerate, a distributed development consortium for the development of a new vaccine to prevent Q fever.

Standardized guinea pig model for Q fever vaccine reactogenicity

Historically, vaccination with Coxiella burnetii whole cell vaccines has induced hypersensitivity reactions in humans and animals that have had prior exposure to the pathogen as a result of infection or vaccination. Intradermal skin testing is routinely used to evaluate exposure in humans, and guinea pig hypersensitivity models have been developed to characterize the potential for reactogenicity in vaccine candidates. Here we describe a refinement of the guinea pig model using an alternate vaccine for positive controls. An initial comparative study used viable C. burnetii to compare the routes of sensitizing exposure of guinea pigs (intranasal vs intraperitoneal), evaluation of two time points for antigen challenge (21 and 42 days) and an assessment of two routes (intradermal and subcutaneous) of challenge using the ruminant vaccine Coxevac as the antigenic control. Animals sensitized by intraperitoneal exposure exhibited slightly larger gross reactions than did those sensitized by intranasal exposure, and reactions were more pronounced when skin challenge was performed at 42 days compared to 21 days post-sensitization. The intradermal route proved to be the optimal route of reactogenicity challenge. Histopathological changes at injection sites were similar to those previously reported and a scoring system was developed to compare reactions between groups receiving vaccine by intradermal versus subcutaneous routes. Based on the comparative study, a standardized protocol for assessment of vaccine reactogenicity in intranasally-sensitized animals was tested in a larger confirmatory study. Results suggest that screens utilizing a group size of n = 3 would achieve 90% power for detecting exposure-related reactogenic responses of the magnitude induced by Coxevac using either of two outcome measures.

Abstract 1633: Immunotherapy for malignant mesothelioma that combines a mesothelia-targeted immune-activating protein and CXCL12/CXCR4 blockade

Background and Purpose: There is a significant unmet need for new treatment strategies for malignant mesothelioma (MM). Despite relevant advances in many cancer treatment areas, including improvements in diagnosis, staging, and the clinical course of treated patients, MM remains a highly lethal disease. The purpose of this study is to develop a combination immunotherapy for MM, which involves a fusion protein to target and evoke a cellualr immune response to mesothelin (MSLN) and the blockade of CXCL12/CXCR4 pathway to mobilize cytotoxic effector cells into tumors. Experimental Procedures: The efficacy of the MSLN targeted immune activating fusion protein (scFv-MtbHsp70), FDA-approved small molecule CXCR4 antagonist AMD3100 (plerixafor), and the combination were evaluated in two syngeneic and orthotopic murine models of MM in immune competent C57BL/6 mice. Mice received 4 intraperitoneal (i.p.) treatments from 7 days post i.p. injection of luciferase-expressing 40L and AE17 cells. Tumor growth was monitored by in vivo imaging of luciferase activity with an IVIS Spectrum. Survival time was calculated as life span from the day of tumor inoculation. In immunological studies, mice were sacrificed 4 weeks after tumor cell inoculation. Immune cells from spleens and tumors were labeled with antibodies against CD3, CD4, CD8, CD25 and Foxp3 antibodies, and examined by flow cytometry. Splenocytes were stimulated with MSLN and assessed for intracellular IFN-γ production by flow cytometry. Results: In both murine mesothelioma models, the fusion protein scFv-MtbHsp70 alone delayed tumor growth and prolonged mouse survival, which was associated with increased tumor infiltration by CD3+CD8+ T cells. Treatment enhanced the cytotoxic function of tumor-specific CD3+CD8+ T cells by evoking dendritic cell activation as well as antigen presentation and cross presentation. AMD3100 alone reduced the proportion of CD4+CD25+Foxp3+ Treg cells in tumors and decreased PD-1 expression on CD3+CD8+ T cells. The combination of the fusion protein and AMD3100 further significantly slowed tumor growth and enhanced mouse survival while augmenting tumor-specific CD8+ T-cell immune responses and abrogating intratumoral immunosuppression. Conclusion: Our findings demonstrated for the first time the synergistic effect of combination of MSLN-targeted immune-activating fusion protein scFv-MtbHsp70 and AMD3100 in treatment of MM in mice. This is a new therapeutic strategy which may significantly prolong survival of patients with this disease. Citation Format: Huabiao Chen, Binghao Li, Yang Zeng, Patrick Reeves, Qiuyan Liu, Ann Sluder, Jeffrey Gelfand, Timothy Brauns, Mark Poznansky. Immunotherapy for malignant mesothelioma that combines a mesothelia-targeted immune-activating protein and CXCL12/CXCR4 blockade [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 1633. doi:10.1158/1538-7445.AM2017-1633

Abstract 3649: Combination of mesothelin-targeted immune-activating fusion protein and anti-PD-L1 augments antitumor immunity and prolongs survival in murine model of ovarian cancer

Background and Purpose: Although immunotherapy as an adjuvant to surgery and chemotherapy has been investigated in ovarian cancer (OC) as a means of reducing tumor recurrence and improving survival, there remains a significant unmet need for combinatorial strategies to enhance the antitumor immune response. The purpose of this study was to develop a novel combination immunotherapy for OC, utilizing our novel fusion protein to target and generate a cellular immune response to mesothelin (MSLN) in conjunction with blockade of the PD-1/PD-L1 checkpoint to restore the function of cytotoxic T cells in order to enhance cancer control and prolong survival. Experimental Procedures: Luciferase-expressing ID8 cells were employed to establish an intraperitoneal ovarian tumor model in immunocompetent C57BL/6 mice. The efficacies of the MSLN-targeted immune-activating fusion protein (VIC-008), αPD-L1, and the combination were evaluated. Mice received 4 intraperitoneal (i.p.) treatments of VIC-008 from day 7 post tumor inoculation weekly, and 6 treatments of αPD-L1 i.p. every other day from 4 weeks post inoculation. Tumor growth was monitored by in vivo imaging of luciferase activity. Survival time was calculated as life span from the day of tumor inoculation. In immunological studies, mice were sacrificed 7 weeks after tumor cell inoculation. Immune cells from lymph nodes, ascites and tumors were stained with antibodies against multiple immune cell markers and profiled by flow cytometry. Results: VIC-008, αPD-L1 or combination treatment delayed tumor growth. The combination treatment resulted in the greatest prolongation in survival, followed by αPD-L1 treatment and then VIC-008 treatment. Improved survival was associated with increased levels of intratumoral CD3+CD8+ T cells (P Conclusion: Our results suggest that, through activating dendritic cells and enhancing antigen presentation and cross-presentation, VIC-008 augments antitumor CD8+ T cell responses and facilitates generation of memory T cells when combined with PD-1/PD-L1 blockade, providing long-term antitumor effects. Our findings demonstrate for the first time a mechanistic rationale for combining VIC-008 and αPD-L1 in treatment of OC in mice, positioning this combination therapy as a potential promising new immunotherapeutic approach for OC. Citation Format: Yang Zeng, Binghao Li, Qiuyan Liu, Patrick Reeves, Ann Sluder, Jeffrey Gelfand, Timothy Brauns, Mark Poznansky, Huabiao Chen. Combination of mesothelin-targeted immune-activating fusion protein and anti-PD-L1 augments antitumor immunity and prolongs survival in murine model of ovarian cancer [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 3649. doi:10.1158/1538-7445.AM2017-3649