title: Phosphatidylserine-targeting antibodies augment the anti-tumorigenic activity of anti-PD-1 therapy by enhancing immune activation and downregulating pro-oncogenic factors induced by T-cell checkpoint inhibition in murine triple-negative breast cancers.
abstract: The purpose of this study was to investigate the potential of antibody-directed immunotherapy targeting the aminophospholipid phosphatidylserine, which promotes immunosuppression when exposed in the tumor microenvironment, alone and in combination with antibody treatment towards the T-cell checkpoint inhibitor PD-1 in breast carcinomas, including triple-negative breast cancers.
  
  
  
  Immune-competent mice bearing syngeneic EMT-6 or E0771 tumors were subjected to treatments comprising of a phosphatidylserine-targeting and an anti-PD-1 antibody either as single or combinational treatments. Anti-tumor effects were determined by tumor growth inhibition and changes in overall survival accompanying each treatment. The generation of a tumor-specific immune response in animals undergoing complete tumor regression was assessed by secondary tumor cell challenge and splenocyte-produced IFNγ in the presence or absence of irradiated tumor cells. Changes in the presence of tumor-infiltrating lymphocytes were assessed by flow cytometry, while mRNA-based immune profiling was determined using NanoString PanCancer Immune Profiling Panel analysis.
  
  
  
  Treatment by a phosphatidylserine-targeting antibody inhibits in-vivo growth and significantly enhances the anti-tumor activity of antibody-mediated PD-1 therapy, including providing a distinct survival advantage over treatment by either single agent. Animals in which complete tumor regression occurred with combination treatments were resistant to secondary tumor challenge and presented heightened expression levels of splenocyte-produced IFNγ. Combinational treatment by a phosphatidylserine-targeting antibody with anti-PD-1 therapy increased the number of tumor-infiltrating lymphocytes more than that observed with single-arm therapies. Finally, immunoprofiling analysis revealed that the combination of anti-phosphatidylserine targeting antibody and anti-PD-1 therapy enhanced tumor-infiltrating lymphocytes, and increased expression of pro-immunosurveillance-associated cytokines while significantly decreasing expression of pro-tumorigenic cytokines that were induced by single anti-PD-1 therapy.
  
  
  
  Our data suggest that antibody therapy targeting phosphatidylserine-associated immunosuppression, which has activity as a single agent, can significantly enhance immunotherapies targeting the PD-1 pathway in murine breast neoplasms, including triple-negative breast cancers.
authors.affiliation: Department of Preclinical Research, Peregrine Pharmaceuticals, Inc., Tustin, CA, USA. mgray@peregrineinc.com.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Gray
authors.affiliation: Department of Preclinical Research, Peregrine Pharmaceuticals, Inc., Tustin, CA, USA.
authors.forename: Jian
authors.initials: J
authors.lastname: Gong
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, USA.
authors.forename: Michaela M S
authors.initials: MM
authors.lastname: Hatch
authors.affiliation: Department of Preclinical Research, Peregrine Pharmaceuticals, Inc., Tustin, CA, USA.
authors.forename: Van
authors.initials: V
authors.lastname: Nguyen
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, USA.
authors.forename: Christopher C W
authors.initials: CC
authors.lastname: Hughes
authors.affiliation: Department of Preclinical Research, Peregrine Pharmaceuticals, Inc., Tustin, CA, USA.
authors.forename: Jeff T
authors.initials: JT
authors.lastname: Hutchins
authors.affiliation: Department of Preclinical Research, Peregrine Pharmaceuticals, Inc., Tustin, CA, USA.
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Freimark
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D060890:B7-H1 Antigen / Q000235:genetics / Q000378:metabolism; D014408:Biomarkers, Tumor; D045744:Cell Line, Tumor; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D005260:Female; D015870:Gene Expression; D020869:Gene Expression Profiling; D006801:Humans; D007167:Immunotherapy; D007371:Interferon-gamma / Q000096:biosynthesis; D053208:Kaplan-Meier Estimate; D016246:Lymphocytes, Tumor-Infiltrating / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D051379:Mice; D058990:Molecular Targeted Therapy; D010718:Phosphatidylserines / Q000037:antagonists & inhibitors; D061026:Programmed Cell Death 1 Receptor / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D013154:Spleen / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*; D064726:Triple Negative Breast Neoplasms / Q000188:drug therapy / Q000276:immunology* / Q000378:metabolism* / Q000473:pathology; D023041:Xenograft Model Antitumor Assays
chemical_list: D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D060890:B7-H1 Antigen; D014408:Biomarkers, Tumor; D016207:Cytokines; D010718:Phosphatidylserines; D061026:Programmed Cell Death 1 Receptor; D007371:Interferon-gamma
keywords: Breast cancer; Checkpoint inhibitor; Combination immunotherapy; Phosphatidylserine; Triple-negative breast cancer