title: Targeting TLR-4 with a novel pharmaceutical grade plant derived agonist, Immunomax®, as a therapeutic strategy for metastatic breast cancer.
abstract: BACKGROUND
  Previously 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.
  
  
  METHODS
  The 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.
  
  
  RESULTS
  Immunomax® 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.
  
  
  CONCLUSIONS
  This 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.
authors.affiliation: Department of Molecular Immunology, Institute for Molecular Medicine, 16371 Gothard Street, Suite H, Huntington Beach, CA, 92647, USA. aghochikyan@immed.org.
authors.forename: Anahit
authors.initials: A
authors.lastname: Ghochikyan
authors.affiliation: The Institute of Immunology, Federal Medical-Biological Agency, Moscow, 115478, Russia. pichalvas@gmail.com.
authors.forename: Alexey
authors.initials: A
authors.lastname: Pichugin
authors.affiliation: The Institute of Immunology, Federal Medical-Biological Agency, Moscow, 115478, Russia. Bavmofc@mail.ru.
authors.forename: Alexander
authors.initials: A
authors.lastname: Bagaev
authors.affiliation: Department of Molecular Immunology, Institute for Molecular Medicine, 16371 Gothard Street, Suite H, Huntington Beach, CA, 92647, USA. adav@immed.org.
authors.forename: Arpine
authors.initials: A
authors.lastname: Davtyan
authors.affiliation: Department of Molecular Immunology, Institute for Molecular Medicine, 16371 Gothard Street, Suite H, Huntington Beach, CA, 92647, USA. ahov@immed.org.
authors.forename: Armine
authors.initials: A
authors.lastname: Hovakimyan
authors.affiliation: Gamaleya Research Institute for Epidemiology and Microbiology, Russian Ministry of Health Moscow, Moscow, 123098, Russia. amir_tuhvatulin@yahoo.com.
authors.forename: Amir
authors.initials: A
authors.lastname: Tukhvatulin
authors.affiliation: Department of Molecular Immunology, Institute for Molecular Medicine, 16371 Gothard Street, Suite H, Huntington Beach, CA, 92647, USA. hayk.davtyan@immed.org.
authors.forename: Hayk
authors.initials: H
authors.lastname: Davtyan
authors.affiliation: Gamaleya Research Institute for Epidemiology and Microbiology, Russian Ministry of Health Moscow, Moscow, 123098, Russia. sdmitryv@yahoo.com.
authors.forename: Dmitry
authors.initials: D
authors.lastname: Shcheblyakov
authors.affiliation: Gamaleya Research Institute for Epidemiology and Microbiology, Russian Ministry of Health Moscow, Moscow, 123098, Russia. ldenisy@yahoo.com.
authors.forename: Denis
authors.initials: D
authors.lastname: Logunov
authors.affiliation: The Institute of Immunology, Federal Medical-Biological Agency, Moscow, 115478, Russia. chulkina@gmail.com.
authors.forename: Marina
authors.initials: M
authors.lastname: Chulkina
authors.affiliation: The Institute of Immunology, Federal Medical-Biological Agency, Moscow, 115478, Russia. bushueva@gmail.com.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Savilova
authors.affiliation: The Institute of Immunology, Federal Medical-Biological Agency, Moscow, 115478, Russia. d.trofimov@dna-technology.ru.
authors.forename: Dmitry
authors.initials: D
authors.lastname: Trofimov
authors.affiliation: Department of Molecular Biology & Biochemistry, University of California, Irvine, CA, 92697, USA. enelson@uci.edu.|Department of Medicine, Division of Hematology and Oncology University of California, Irvine, CA, 92697, USA. enelson@uci.edu.
authors.forename: Edward L
authors.initials: EL
authors.lastname: Nelson
authors.affiliation: Department of Molecular Immunology, Institute for Molecular Medicine, 16371 Gothard Street, Suite H, Huntington Beach, CA, 92647, USA. magadjanyan@immed.org.|The Institute for Memory Impairments and Neurological Disorders, University of California, Irvine, CA, 92697, USA. magadjanyan@immed.org.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Agadjanyan
authors.affiliation: The Institute of Immunology, Federal Medical-Biological Agency, Moscow, 115478, Russia. ravshan.ataullakhanov@gmail.com.
authors.forename: Ravshan I
authors.initials: RI
authors.lastname: Ataullakhanov
mesh_terms: D000818:Animals; D005260:Female; D008214:Lymphocytes / Q000276:immunology; D008325:Mammary Neoplasms, Experimental / Q000276:immunology / Q000473:pathology / Q000628:therapy*; D051379:Mice; D008807:Mice, Inbred BALB C; D009362:Neoplasm Metastasis; D010936:Plant Extracts / Q000494:pharmacology*; D051197:Toll-Like Receptor 4 / Q000187:drug effects*
chemical_list: D010936:Plant Extracts; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4