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

17.3 The Development of Recombinant DNA-based Immunotoxins
Recombinant immunotoxins contain antibody fragments as the targeting moiety.
These fragments can be produced in E. coli, and are the result of intensive research
and development in recombinant-antibody technologies [72±74]. Several antibody
fragments have been used to construct recombinant immunotoxins (Fig. 17.1). One
type contains Fab fragments in which the light chain and the heavy chain Fd fragment
(VH and CH1) are connected to each other via an interchain disulfide bond between
CL and CH1. The toxin moiety can be fused to the carboxyl end of either CL or
CH1. Fabs can be produced in E. coli, either by secretion, with co-expression of light
chains and Fd fragments or by expression of the chains in intracellular inclusion
bodies in separate cultures; in the latter case, they are reconstituted by a refolding reaction
using a redox-shuffling buffer system. Several immunotoxins with Fab fragments
have been constructed and produced in this way [1±4, 75].
The smallest functional modules of antibodies required for antigen binding are Fv
fragments. This makes them especially useful for clinical applications, not only for
generating recombinant immunotoxins, but also for tumor imaging, because their
small size improves tumor penetration. Fv fragments are heterodimers of the variable
heavy chain (VH) and the variable light chain (VL) domains. Unlike whole IgG
or Fab, in which the heterodimers are held together and stabilized by interchain disulfide
bonds, the VH and VL of Fvs are not covalently connected and are consequently
unstable; this instability can be overcome by making recombinant Fvs that
have the VH and VL covalently connected by a peptide linker that fuses the C terminus
of the V L or V H to the N-terminus of the other domain (Fig. 17.1). These molecules
are termed single-chain Fvs (scFvs) [76, 77], and many retain the specificity
and affinity of the original antibody. The cloning, construction and composition of
recombinant Fv fragments of antibodies and Fv±immunotoxins are described in
Fig. 17.3.
Many recombinant immunotoxins have been constructed using scFvs, in which molecules
the scFv gene is fused to PE38 to generate a potent cytotoxic agent with targeted
specificity [1±4, 78±84](Figs 17.1 and 17.3).
Until recently, the construction of scFvs was the only general method available to
make stable Fvs. However, many scFvs are unstable or have reduced affinity for the
antigen compared with the parent antibody or Fab fragment. This is because the linker
interferes with binding or because the linker does not sufficiently stabilize the Fv
structure, leading to aggregation and loss of activity. This is particularly true at physiological
temperatures (37 8C). To overcome these problems, an alternative strategy
has been developed that involves generating stable Fvs by connecting the VH and VL
domains by an interchain disulfide bond engineered between structurally conserved
framework residues of the Fv; these molecules are termed disulfide-stabilized Fvs
(dsFvs) [74, 85±87]. The positions at which the cysteine residues were to be placed
were identified by computer-based molecular modeling; as they are located in the
framework of each V H and V L, this location can be used as a general method to stabilize
almost all Fvs without the need for any structural information. Many dsFvs have
been constructed in the past 3 years (mainly as dsFv±immunotoxins, in which the
dsFv is fused to PE38) and they show several advantages over scFvs [74, 88±90]. In
addition to their increased stability (due to a decreased tendency to aggregate), they
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