A functional recombinant human 4-1BB ligand for immune costimulatory therapy of cancer

J Immunother. 2011 Mar;34(2):175-82. doi: 10.1097/CJI.0b013e318206dac1.


Costimulatory factors hold great promise for development into novel anticancer biotherapeutics. An agonist to 4-1BB is ranked number 8 by National Cancer Institute on the list of 20 agents with high potential for use in treating cancer. We earlier reported on a recombinant murine 4-1BB ligand fusion protein that binds 4-1BB receptor on murine T cells and stimulates their proliferation in tumor-bearing mice. To facilitate clinical translation,we constructed a corresponding recombinant human 4-1BB ligand fusion protein (hIg-h4-1BBLs) and showed its ability to activate human T cells in vitro. Using Chinese hamster ovary cells transformed with a plasmid coexpressing hIg-h4-1BBLs and rat glutamine synthetase, we generated a high-producing clone by sequential selection with methionine sulfoximine. The hIg-h4-1BBLs was partially purified by protein A column chromatography and characterized biochemically and functionally, using human 4-1BB binding and human T-cell proliferation assays, in vitro.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western Blot confirmed that the hIg-h4-1BBLs is expressed predominantly as a functionally active multimeric protein with the ability to specifically bind to cells expressing human 4-1BB receptor and induce significant T-cell proliferation in vitro using both human and monkey peripheral blood mononuclear cells. The hIg-h4-1BBLs can be produced in large quantities from the high producer clone and developed as a novel immune costimulatory biotherapeutic to treat, alone and in combination with other modalities, various malignant diseases in patients through T-cell activation. Process development of this clinical agent has been discussed with the Food and Drug Administration in a pre-Investigational New Drug meeting and presented to the Office of Biotechnology Activities in a public hearing.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • 4-1BB Ligand / genetics*
  • 4-1BB Ligand / immunology*
  • Adjuvants, Immunologic / pharmacology*
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • CHO Cells
  • Cell Proliferation / drug effects
  • Cricetinae
  • Cricetulus
  • Gene Order
  • Glutamate-Ammonia Ligase / immunology
  • Glutamate-Ammonia Ligase / isolation & purification
  • Glutamate-Ammonia Ligase / metabolism
  • Haplorhini
  • Humans
  • Immunotherapy*
  • Leukocytes, Mononuclear / drug effects*
  • Leukocytes, Mononuclear / immunology
  • Lymphocyte Activation / drug effects
  • Mice
  • Molecular Sequence Data
  • Neoplasms / therapy*
  • Rats
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / pharmacology*
  • T-Lymphocytes / immunology
  • T-Lymphocytes / metabolism


  • 4-1BB Ligand
  • Adjuvants, Immunologic
  • Recombinant Fusion Proteins
  • Glutamate-Ammonia Ligase