Targeting TLR-4 with a novel pharmaceutical grade plant derived agonist, Immunomax®, as a therapeutic strategy for metastatic breast cancer.

Anahit Ghochikyan,Alexander Bagaev,Edward L Nelson,Armine Hovakimyan,Hayk Davtyan,Arpine Davtyan,Anastasia Savilova,Alexey Pichugin,Marina Chulkina,Denis Logunov,Michael G Agadjanyan,Amir Tukhvatulin,Dmitry Shcheblyakov,Ravshan I Ataullakhanov,Dmitry Trofimov

doi:10.1186/s12967-014-0322-y

Abstract

BackgroundPreviously we demonstrated that the resection of primary 4T1 tumors only slightly prolongs mouse survival, but importantly, creates a “window of opportunity” with attenuated suppressor cell and increased activated T cell populations. This suggests that additional activation of the immune system by immunostimulatory agents during this period may enhance anti-tumor immunity and potentially eradicate micro-metastatic disease in this stringent model.We hypothesized that the immunostimulator Immunomax®, which is comprised of a plant-derived polysaccharide, is non-toxic in humans and stimulates immune defense during the infectious diseases treatment, may have also anti-tumor activity and be beneficial in the adjuvant setting when endogenous anti-tumor responses are present and during the “window of opportunity” in post-resection metastatic breast cancer model. Here we provide the initial report that Immunomax® demonstrates the capacity to eliminate micro-metastatic disease in the post-resection, 4T1 mouse model of breast cancer.MethodsThe efficacy of Immunomax® was evaluated by analyzing survival rate and the number of spontaneous clonogenic tumor cells in the lung homogenates of mice. The frequencies of activated NK, CD4+ and CD8+ cells as well as myeloid-derived suppressor cells and Treg cells were evaluated using flow cytometry. Highly purified mouse and human dendritic and NK cells were sorted and the effect of Immunomax® on activation status of these cells was assessed by flow cytometry. The property of Immunomax® as TLR-4 agonist was determined by NF-κB/SEAP reporter gene assay, WB, RT-PCR.ResultsImmunomax® injections significantly prolonged overall survival and cured 31% of mice. This immunostimulator activates DCs via the TLR-4, which in turn stimulates tumoricidal NK cells and in vitro, completely inhibits growth of 4T1 cells. Incubation of PBMC from healthy donors with Immunomax® activates NK cells via activation of plasmacytoid DC leading significantly higher efficacy in killing of human NK-target cells K562 compared with non-treated cells.ConclusionThis is the first demonstration that Immunomax® is a TLR-4 agonist and the first report of a documented role for this pharmaceutical grade immunostimulator in augmenting anti-tumor activity, suggesting that incorporation of Immunomax® into developing breast cancer therapeutic strategies may be beneficial and with less potential toxicity than checkpoint inhibitors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-014-0322-y) contains supplementary material, which is available to authorized users.

Highlights

Breast cancer is the most commonly diagnosed cancer worldwide and it is the second leading cause of death in women [1]
We demonstrated that inhibition of TLR-4 signal transduction and its downstream signaling by CLI-095 inhibited killing of 4T1 tumor cells by the mixture of purified BM-DC and NK cells activated by Immunomax® in vitro, reversing it to the level exhibited in NK/BM-DC co-culture before the addition of Immunomax® (Figure 5E)
The capacity to limit the growth and development of metastatic tumor in a stringent 4T1 breast cancer model makes this novel TLR-4 agonist attractive. This is the first demonstration of anti-tumor activity of Immunomax® against occult micrometastatic tumor and the first in vitro demonstration that this immunostimulator is a TLR-4 agonist directly activating DC and NK cells co-operation

Summary

Introduction

Breast cancer is the most commonly diagnosed cancer worldwide and it is the second leading cause of death in women [1]. Primary treatments (surgery, radiation therapy, and chemotherapy) are beneficial and lead to increased disease free and overall survival, there is a continuous relapse rate that leads to a substantial proportion of breast cancer patients developing recurrent and/or metastatic disease This indicates persistence of occult microscopic disease that leads to significant morbidity and mortality (~40,000 deaths in 2011 in the US) despite improved primary and adjuvant treatment. We demonstrated that the resection of primary 4T1 tumors only slightly prolongs mouse survival, but importantly, creates a “window of opportunity” with attenuated suppressor cell and increased activated T cell populations This suggests that additional activation of the immune system by immunostimulatory agents during this period may enhance anti-tumor immunity and potentially eradicate micro-metastatic disease in this stringent model. We provide the initial report that Immunomax® demonstrates the capacity to eliminate micro-metastatic disease in the post-resection, 4T1 mouse model of breast cancer

Methods

Results

Discussion

Conclusion