A general method for chimerization of monoclonal antibodies by inverse polymerase chain reaction which conserves authentic N-terminal sequences.
Chimerization of antibodies (Ab) by cloning the V (variable) regions encoding the light and heavy chains with degenerate oligodeoxyribonucleotide primers matching to framework region 1 and to the joining regions, leads to Ab with altered amino acids at the N-terminus compared to those of the parental Ab. This is due to N-terminal framework 1 sequences in the expression vectors [Larrick et al., Bio/Technology 7 (1989) 937-938; Le Boeuf et al., Gene (1989) 371-377; Orlandi et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837]. This might lead to Ab with altered affinity to the antigen due to interaction of framework sequences with complementarity determining regions. Moreover, some V regions may be refractory to cloning by this procedure. Here, we describe a method to circumvent these potential problems. The V regions for both chains of the Ab are cloned by inverse polymerase chain reaction (PCR) with primers matching the known constant region sequences of the Ab. After sequencing, PCR fragments corresponding to the V regions of both chains are inserted in-frame into appropriate expression vectors leading to Ab with unaltered N-terminal sequences after expression in mammalian cells. The procedure is illustrated with an Ab directed against the beta chain of the human interleukin-2 receptor.
Kaluza
B
B
Boehringer Mannheim GmbH, Department of Biotechnology, Penzberg, Germany.
Betzl
G
G
Shao
H
H
Diamantstein
T
T
Weidle
U H
UH
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA; D005822:Genetic Vectors; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D015375:Receptors, Interleukin-2 / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D012333:RNA, Messenger; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins; D004247:DNA