Modular Cloning of MUC1 Recombinant Antibodies by Assembly of Synthetic Domain Genes

Abstract

Antibody immunotherapy has emerged as a constructive way to fight cancer. New immunotherapies require the identification of a tumor‐specific antigen. The protein mucin 1 (MUC1) has long been recognized as an important specific antigen and a potential immunotherapy target. In healthy human tissues, MUC1 is responsible for protecting the epithelial lining. However, in cancer cells, MUC1 displays truncated glycosylation, revealing tumor‐specific epitopes. Recombinant antibodies are preferred for therapeutic development, and in some cases, it is desirable to produce different antibody formats, including intact IgG or Fab fragments. Using the MUC1 specific antibody 4H5 as a model, we have developed a modular cloning approach that can rapidly produce different antibody modalities to facilitate the rapid production of both IgG and Fab antibody formats. The 4H5 variable domains were PCR amplified to contain 20 base‐pair (bp) overlaps with the pcDNA3.1 vector on the 5’ end, and 20 bp overlaps to either the human CH1 or Ck domains on the 3’end of the genes. To produce the Fab, a His‐tag and a 20 base‐pair (bp) overlap to the vector was added to the 3’ end of a human CH1 domain gene sequence. For IgG production, human CH1‐CH2‐CH3 domains were PCR amplified, and the same vector overlap sequence was added to the 3’ end of the sequence. Finally, a 20 bp overlap to the vector was added to the 3’ end of a human Ck domain. The PCR products were purified, and the constructs were assembled using the NEBuilder®HiFi DNA Assembly Cloning Kit. DNA sequencing confirmed the successful assembly of the coding sequence, and the 4H5 Fab fragments and IgG were produced by transient transfection in CHO cells. The antibodies (Fab and IgG) were purified by affinity chromatography and binding to recombinant MUC1 antigens confirmed by ELISA. The cloning method presented here simultaneously produces multiple antibody formats. This approach will permit rapid assembly and screening of different antibody formats and isotypes for therapeutic antibody discovery and analysis.