Cancer Immune Therapy Edited by G. Stuhler and P. Walden ...

are often produced in higher yields than scFvs; in several cases, the binding affinity
of the dsFv was significantly improved over that of the scFv.
17.4
Construction and Production of Recombinant Immunotoxins
In the recombinant immunotoxins derived from PE, the recombinant antibody fragments
are fused to the N-terminus of the truncated derivative of PE (with the cellbinding
domain deleted, e.g. PE40 or PE38). This restores the original domain arrangement
of PE, which consists of an N-terminal-binding domain followed by the
translocation domain and the C-terminal ADP-ribosylation domain.
Only fusions of an antigen-binding domain (Fv) to the N-terminus of truncated PE
are active; C-terminal fusions are not active because the bulky antigen-binding domain
blocks translocation of the C-terminal fragment into the cytoplasm [1, 27].
DT immunotoxins are fusions of mutated DT with antigen-binding regions of a recombinant
antibody. However, in this case the antigen-binding domain must be
3 Fig. 17.3 Cloning, construction and composition
of scFv± and dsFv±immunotoxins. (A)Cloning
and construction of recombinant scFv± and
dsFv±immunotoxins. The genes encoding the
VH and VL variable domains are cloned usually
from hybridoma mRNA by reverse transcription,
cDNA synthesis and subsequent PCR amplification
using degenerate primers that are complementary
to the 5' or 3' end of the VH and VL
genes, or by primers which are designed according
to the N-terminal amino acid sequence of
the mAb to be cloned and conserved sequences
at the N-terminal of the heavy and light constant
regions. The variable genes can be also cloned
by constant domains primers and using the
RACE rapid amplification of cDNA ends
(method). Restriction sites for assembling the
peptide linker sequence which connects the VH
and V L domains, and for cloning into the expression
vector are also introduced by PCR. Construction
of dsFv involves the generation of two
expression plasmids which encode the two components
of the dsFv V H-Cys and V L-Cys. The cysteines
are introduced in position 44 in FR2 of
VH and position 100 of FR4 of VL or position 105
of FR4 in V H and position 43 of FR2 in V L (numbering
system of Kabat et al.)by site-directed
mutagenesis using as template a uracil-containing
single-stranded DNA of the scFv construct
from the F+ origin present in the expression
plasmid and co-transfection with M13 helper
17.4 Construction and Production of Recombinant Immunotoxins
phage. In addition to the cysteines, cloning sites,
ATG translation initiation codons and stop codons
are introduced at the 5'and 3' ends and of
the V H and V L genes as shown by site-directed
mutagenesis or PCR. The antibody variable
genes are subcloned into an expression vector
which contains the gene for a truncated form of
PE. This expression vector is controlled by the
T7 promoter and upon induction of the T7 RNA
polymerase, which is under the control of the lacUV5
promoter, in E. coli BL21 lDE3 by IPTG,
large amounts of recombinant protein are produced.
(B)Composition of recombinant immunotoxins.
In PE-derived recombinant Fv±immunotoxins,
the Fv region of the targeting antibody
is fused to the N-terminus of a truncated form of
PE which contains the translocation domain
(domain II)and enzymatically active ADP-ribosylation
domain (domain III]. The cell-binding
domain of whole PE (domain I)is replaced by
the Fv targeting moiety thus, preserving the relative
position of the binding domain function to
the other functional domains of PE. In the dsFv±
immunotoxins there are two components. In
one, the VH or VL domains are fused to the Nterminus
of the truncated PE and, in the other,
the variable domain is covalently linked by the
engineered disulfide bond. DT-derived immunotoxins
are fused to the C-terminus due to the inverse
arrangement of the functional modules of
PE and DT.
357