title: Generation of a high-producing clone of a humanized anti-B-cell lymphoma monoclonal antibody (hLL2).
abstract: BACKGROUND
  LL2 is a murine immunoglobulin (Ig)G2a-kappa anti-B-cell monoclonal antibody with proven targeting and therapeutic efficacy in the management of non-Hodgkin's lymphoma (NHL). The authors had previously generated a humanized LL2 (hLL2) that demonstrated binding properties identical to those of LL2. Nevertheless, the productivity of the cell line was insufficient for large-scale production of the antibody for clinical studies. Therefore, the authors chose an amplifiable system for the generation of hLL2.
  
  
  METHODS
  The hLL2 sequences were ligated into the expression vector pdHL2, which has a dhfr amplifiable gene, and were incorporated into the SP2/0 cells by electroporation. A methotrexate (MTX) resistant clone producing hLL2 was identified. Stepwise increases in MTX concentrations, from 0.1 to 5 microM, and subcloning of the cells by limiting dilution were performed.
  
  
  RESULTS
  By amplifying the dhfr and hLL2 genes with stepwise increases in the MTX concentration, the antibody production was enhanced from its original 1.4 to 70 +/- 5 mg per liter of culture media. Subsequent subcloning further improved the productivity. Immunoreactivity of the antibody was conserved, as proven by enzyme-linked immunosorbent assay and cell-binding assays. By isoelectrofocusing, the isoelectric point (pI) of the antibody was measured at approximately 9.6. The productivity of the clone was not affected by culture conditions or storage of the cells in liquid nitrogen.
  
  
  CONCLUSIONS
  By means of gene amplification, the authors have generated a high-producing hLL2-IgG clone suitable for production of the quantity of antibody necessary for clinical diagnostic and therapeutic trials of NHL patients.
authors.affiliation: Immunomedics, Inc., Morris Plains, New Jersey 07950, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Losman
authors.forename: H J
authors.initials: HJ
authors.lastname: Hansen
authors.forename: H
authors.initials: H
authors.lastname: Dworak
authors.forename: I S
authors.initials: IS
authors.lastname: Krishnan
authors.forename: Z
authors.initials: Z
authors.lastname: Qu
authors.forename: L B
authors.initials: LB
authors.lastname: Shih
authors.forename: L
authors.initials: L
authors.lastname: Zeng
authors.forename: D M
authors.initials: DM
authors.lastname: Goldenberg
authors.forename: S O
authors.initials: SO
authors.lastname: Leung
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000912:Antibodies, Neoplasm / Q000096:biosynthesis*; D002460:Cell Line; D005784:Gene Amplification; D006801:Humans; D016393:Lymphoma, B-Cell / Q000276:immunology*; D008727:Methotrexate / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D008727:Methotrexate