Antibodies are highly specific, naturally evolved molecules that recognize and eliminate pathogens and tumour associated antigens. The organisation into distinct structural and functional domains facilitates their engineering. Driven by a variety of antibody engineering methods and technological developments, chimeric and humanized antibodies are commonly used as therapeutics for cancer, infectious and inflammatory diseases, for imaging or in targeted delivery systems. This chapter describes the cloning and expression of chimeric antibodies using Lonza’s “double-gene vectors” (Lonza Biologics’ Glutamine Synthetase (GS) gene expression system) in mammalian cells.