title: Process validation and clinical evaluation of a protocol to generate gene-modified T lymphocytes for imunogene therapy for metastatic renal cell carcinoma: GMP-controlled transduction and expansion of patient's T lymphocytes using a carboxy anhydrase IX-specific scFv transgene.
abstract: BACKGROUND
  Adoptive transfer of autologous T cells that are gene-transduced to express Ag-specific receptors represents an experimental strategy to provide tumor-specific immunity to cancer patients. We studied this concept in patients with metastatic renal cell cancer (RCC) using retroviral transduction of T cells with a single-chain Ab-G250 chimeric receptor [scFv(G250)]. We describe the validation of our clinical protocol for gene transduction and expansion of human T lymphocytes.
  
  
  METHODS
  A batch of scFv(G250) transgene-containing retrovirus was produced under conditions of good manufacturing practice (GMP). In addition to quality control and safety testing of the virus batch, extensive potency testing was performed, i.e. assessment of its functional transduction efficiency in primary human T cells. Subsequently, the clinical gene transduction and cell-expansion protocol was subjected to a series of process validations and a clinical evaluation using T cells obtained from healthy donors and three RCC patients.
  
  
  RESULTS
  The clinical batch of scFv(G250) transgene-containing retrovirus met the quality and safety control criteria. Small-scale transductions yielded 62-92% scFv(G250)+ T cells and, at a clinical scale, 50-84% transduction efficiencies were obtained. Patient and healthy donor T cells showed similar expansion potencies, and also yielded similar levels of scFv(G250)-mediated immune functions, i.e. specific cytolysis of G250-ligand expressing RCC cells and production of IFN-gamma upon stimulation with such cells. All T cell cultures were free of replication competent retroviruses.
  
  
  CONCLUSIONS
  We have shown that the validated batch of scFv(G250) transgene-containing retrovirus in combination with our GMP T-cell transduction and expansion protocol successfully generates clinically relevant numbers of functional scFv(G250) gene-modified T cells for patient treatment.
authors.affiliation: Unit Clinical and Tumor Immunology, Department of Medical Oncology, Erasmus University Medical Center-Daniel den Hoed Cancer Center, Rotterdam, the Netherlands. c.lamers@erasmusmc.nl
authors.forename: C H J
authors.initials: CH
authors.lastname: Lamers
authors.forename: P
authors.initials: P
authors.lastname: van Elzakker
authors.forename: S C L
authors.initials: SC
authors.lastname: Langeveld
authors.forename: S
authors.initials: S
authors.lastname: Sleijfer
authors.forename: J W
authors.initials: JW
authors.lastname: Gratama
mesh_terms: D019264:Adoptive Transfer / Q000379:methods*; D002256:Carbonic Anhydrases / Q000378:metabolism; D002292:Carcinoma, Renal Cell / Q000473:pathology / Q000628:therapy*; D049109:Cell Proliferation; D002985:Clinical Protocols / Q000592:standards*; D018628:Gene Dosage; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006157:Guanosine Monophosphate / Q000494:pharmacology; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007680:Kidney Neoplasms / Q000473:pathology / Q000628:therapy; D008213:Lymphocyte Activation; D009362:Neoplasm Metastasis; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics; D057127:Single-Chain Antibodies; D013601:T-Lymphocytes / Q000276:immunology* / Q000821:virology; D014161:Transduction, Genetic / Q000379:methods; D019076:Transgenes
chemical_list: D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C410726:scFV green fluorescent protein, recombinant; D049452:Green Fluorescent Proteins; D006157:Guanosine Monophosphate; D002256:Carbonic Anhydrases