336
Antibody Molecules, Genetic Engineering of
problems would be overcome by produc-
ing human monoclonal antibodies using
human B-cells from immunized human
donors. However, human monoclonal an-
tibodies are difFcult to produce using the
hybridoma technology originally designed
for the production of murine antibodies:
the cell lines are unstable and frequently
produce antibodies of the IgM isotype,
which have low afFnity for the antigen and
are difFcult to purify and handle. In addi-
tion, there are ethical and safety problems
in obtaining humans immunized with cer-
tain antigens.
2.2
Chimeric Antibodies
To overcome the problems associated with
the administration of murine monoclonal
antibodies to humans, protein engineer-
ing has been used to convert murine
monoclonal antibodies to mouse/human
chimeric antibodies by genetically fusing
the mouse variable regions to the human
constant regions (±ig. 4), a procedure that
is facilitated by the structure of antibodies.
Initially, variable region genes were ob-
tained from genomic or cDNA libraries
produced using DNA or mRNA from
antibody-producing cells. More recently,
cloning of genes encoding for the antibody
variable region has been greatly facilitated
by polymerase chain reaction (PCR)-based
procedures. The variable region consists of
hypervariable complementarity determin-
ing regions CDRs that are responsible for
antibody speciFcity supported by relatively
conserved framework regions. A limited
number of different hydrophobic leader
sequences are also found associated with
the different variable regions. Therefore, it
is possible to design sets of oligonucleotide
primers on the basis of either the frame-
work or the leader regions that will bind to
virtually all mouse variable regions. A lim-
ited number of primers are required for all
constant regions. Using upstream consen-
sus primers for framework or leader re-
gions in light- and heavy-chain variable re-
gions and downstream primers for the con-
stant regions, PCR can be used to amplify
cDNAs generated by reverse-transcription
(RT-PCR) directly from hybridoma mRNA.
Mouse
monoclonal
antibody
Human
monoclonal
antibody
Mouse–human
chimeric
antibody
Humanized
antibody
Fig. 4
Schematic representation of a murine monoclonal antibody, a human
monoclonal antibody, a mouse–human chimeric antibody, and a CDR-grafted or
humanized antibody. Chimeric antibodies have murine derived variable regions and
binding speciFcities joined to human constant regions with their corresponding effector
functions. Humanized antibodies are composed mostly of human sequences except for
the areas in contact with the antigen (CDR regions), which are derived from mouse
sequences.
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