title: Different cytokine and stimulation conditions influence the expansion and immune phenotype of third-generation chimeric antigen receptor T cells specific for tumor antigen GD2.
abstract: OBJECTIVE
  Chimeric antigen receptor (CAR) T cells are a novel immunotherapy for cancer. To achieve anti-tumor efficacy, these cells must survive, expand, and persist after infusion into patients, functions that are reportedly best achieved by cells with a stem or central-memory rather than effector-memory phenotype. We have developed third-generation CAR T cells specific for the tumor-associated antigen GD2 for use in a phase I clinical trial. We investigated the optimal cell culture conditions for CAR T-cell production, and here we describe the relative effects of 3 activation and cytokine conditions on CAR T-cell expansion, effector function and phenotype.
  
  
  METHODS
  Peripheral blood mononuclear cells were activated by anti-CD3 and anti-CD28 or anti-CD3 and Retronectin. Activated cells were transduced with the CAR-encoding retroviral vector and expanded in either interleukin (IL)-2 or IL-7 and IL-15. Immune phenotype and expansion were tracked throughout the culture, and transduction efficiency, and subsequent GD2-specific effector functions were evaluated by flow cytometry and cytotoxic T lymphocytes assay.
  
  
  RESULTS
  CD3/Retronectin stimulation with IL-2 resulted in poorer activation, expansion and Th1 cytokine secretion of CAR T cells than CD3/CD28 stimulation with either IL-2 or IL-7 and IL-15. However, CAR T cells cultured in CD3/CD28/IL7/IL-15 and CD3/Retronectin/IL-2 had superior cytotoxic T lymphocyte activity and a more stem-like phenotype.
  
  
  CONCLUSIONS
  The combination of CD3 and CD28 with IL-7 and IL-15 gave the best balance of CAR T-cell expansion and potent GD2-specific effector functions while retaining a stem/memory phenotype, and these growth conditions will therefore be used to manufacture CAR T cells for our phase I clinical trial.
authors.affiliation: Translational Oncology, Centre for Cancer Biology, SA Pathology, and University of South Australia, Adelaide, Australia. Electronic address: Tessa.Gargett@health.sa.gov.au.
authors.forename: Tessa
authors.initials: T
authors.lastname: Gargett
authors.affiliation: Translational Oncology, Centre for Cancer Biology, SA Pathology, and University of South Australia, Adelaide, Australia; Cancer Clinical Trials Unit, Royal Adelaide Hospital, Adelaide, Australia; Discipline of Medicine, University of Adelaide, Adelaide, Australia.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Brown
mesh_terms: D019264:Adoptive Transfer; D000951:Antigens, Neoplasm / Q000276:immunology*; D018106:CD28 Antigens / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D005353:Fibronectins / Q000494:pharmacology; D006801:Humans; D019409:Interleukin-15 / Q000494:pharmacology; D007376:Interleukin-2 / Q000494:pharmacology; D015851:Interleukin-7 / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008213:Lymphocyte Activation / Q000276:immunology*; D010641:Phenotype; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D011994:Recombinant Proteins / Q000494:pharmacology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology* / Q000637:transplantation; D018417:Th1 Cells / Q000276:immunology
chemical_list: D000951:Antigens, Neoplasm; D018106:CD28 Antigens; D017252:CD3 Complex; D005353:Fibronectins; C569274:IL15 protein, human; C508594:IL2 protein, human; C508605:IL7 protein, human; D019409:Interleukin-15; D007376:Interleukin-2; D015851:Interleukin-7; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C545354:retronectin
keywords: IL-15; IL-2; IL-7; Retronectin; anti-CD3 and anti-CD28 stimulation; chimeric antigen receptor T cells; effector function; immune phenotype