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Generation of a high‐producing clone of a humanized anti‐B‐cell lymphoma monoclonal antibody (hLL2)
Author(s) -
Losman Michele J.,
Hansen Hans J.,
Dworak Heather,
Krishnan Indira S.,
Qu Zhengxing,
Shih Lisa B.,
Zeng Li,
Goldenberg David M.,
Leung Shuion
Publication year - 1997
Publication title -
cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.052
H-Index - 304
eISSN - 1097-0142
pISSN - 0008-543X
DOI - 10.1002/(sici)1097-0142(19971215)80:12+<2660::aid-cncr43>3.0.co;2-7
Subject(s) - clone (java method) , antibody , subcloning , monoclonal antibody , microbiology and biotechnology , immunotoxin , cell culture , biology , gene , recombinant dna , immunology , biochemistry , genetics
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 μM, 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. Cancer 1997; 80:2660‐6. © 1997 American Cancer Society.