title: Combined immunostimulatory monoclonal antibodies extend survival in an aggressive transgenic hepatocellular carcinoma mouse model.
abstract: OBJECTIVE
  Immunostimulatory monoclonal antibodies (ISmAb) that unleash antitumor immune responses are showing efficacy in cancer clinical trials. Anti-B7-H1 (PD-L1) monoclonal antibodies (mAb) block a critical inhibitory pathway in T cells, whereas anti-CD137 and OX40 mAbs provide T-cell costimulation. A combination of these ISmAbs (anti-CD137 + anti-OX40 + anti-B7-H1) was tested using a transgenic mouse model of multifocal and rapidly progressing hepatocellular carcinoma, in which c-myc drives transformation and cytosolic ovalbumin (OVA) is expressed in tumor cells as a model antigen.
  
  
  METHODS
  Flow-cytometry and immunohistochemistry were used to quantify tumor-infiltrating lymphocytes (TIL) elicited by treatment and assess their activation status and cytolytic potential. Tolerance induction and its prevention/reversal by treatment with the combination of ISmAbs were revealed by in vivo killing assays.
  
  
  RESULTS
  The triple combination of ISmAbs extended survival of mice bearing hepatocellular carcinomas in a CD8-dependent fashion and synergized with adoptive T-cell therapy using activated OVA-specific TCR-transgenic OT-1 and OT-2 lymphocytes. Mice undergoing therapy showed clear increases in tumor infiltration by activated and blastic CD8(+) and CD4(+) T lymphocytes containing perforin/granzyme B and expressing the ISmAb-targeted receptors on their surface. The triple combination of ISmAbs did not result in enhanced OVA-specific cytotoxic T lymphocyte (CTL) activity but other antigens expressed by cell lines derived from such hepatocellular carcinomas were recognized by endogenous TILs. Adoptively transferred OVA-specific OT-1 lymphocytes into tumor-bearing mice were rendered tolerant, unless given the triple mAb therapy.
  
  
  CONCLUSIONS
  Extension of survival and dense T-cell infiltrates emphasize the translational potential of combinational immunotherapy strategies for hepatocellular carcinoma. Clin Cancer Res; 19(22); 6151-62. ©2013 AACR.
authors.affiliation: Authors' Affiliations: Centro de Investigación Médica Aplicada (CIMA), Universidad de Navarra; Department of Oncology, Clinica Universidad de Navarra; Liver Unit, Clínica Universidad de Navarra and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Pamplona, Spain; Oncology Drug Discovery division, Bristol-Myers Squibb, Lawrenceville, New Jersey; and Department of Pathology, University of Bonn, Bonn, Germany.
authors.forename: Aizea
authors.initials: A
authors.lastname: Morales-Kastresana
authors.forename: Miguel F
authors.initials: MF
authors.lastname: Sanmamed
authors.forename: Inmaculada
authors.initials: I
authors.lastname: Rodriguez
authors.forename: Asis
authors.initials: A
authors.lastname: Palazon
authors.forename: Ivan
authors.initials: I
authors.lastname: Martinez-Forero
authors.forename: Sara
authors.initials: S
authors.lastname: Labiano
authors.forename: Sandra
authors.initials: S
authors.lastname: Hervas-Stubbs
authors.forename: Bruno
authors.initials: B
authors.lastname: Sangro
authors.forename: Carmen
authors.initials: C
authors.lastname: Ochoa
authors.forename: Ana
authors.initials: A
authors.lastname: Rouzaut
authors.forename: Arantza
authors.initials: A
authors.lastname: Azpilikueta
authors.forename: Elixabet
authors.initials: E
authors.lastname: Bolaños
authors.forename: Maria
authors.initials: M
authors.lastname: Jure-Kunkel
authors.forename: Ines
authors.initials: I
authors.lastname: Gütgemann
authors.forename: Ignacio
authors.initials: I
authors.lastname: Melero
mesh_terms: D019264:Adoptive Transfer; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000627:therapeutic use*; D060890:B7-H1 Antigen / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000276:immunology* / Q000401:mortality; D045744:Cell Line, Tumor; D003602:Cytotoxicity, Immunologic / Q000276:immunology; D004195:Disease Models, Animal; D007167:Immunotherapy / Q000379:methods*; D008113:Liver Neoplasms / Q000188:drug therapy / Q000276:immunology* / Q000401:mortality; D008213:Lymphocyte Activation / Q000276:immunology; D018655:Lymphocyte Count; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology; D008562:Membrane Glycoproteins / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D053278:OX40 Ligand; D010047:Ovalbumin / Q000096:biosynthesis / Q000276:immunology; D013534:Survival; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9 / Q000276:immunology; D048069:Tumor Necrosis Factors / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D060890:B7-H1 Antigen; C498919:Cd274 protein, mouse; D008562:Membrane Glycoproteins; D053278:OX40 Ligand; C504586:Tnfsf4 protein, mouse; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9; D048069:Tumor Necrosis Factors; D010047:Ovalbumin