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

354 17 Immunotoxins and Recombinant Immunotoxins in Cancer Therapy
toxin and after a variety of other steps including a pH-induced conformational
change [60±62], proteolytic cleavage at a specific site in domain II [29], and a reductive
step that separates the amino and carboxyl fragments. Ultimately, the C-terminal
portion of PE is translocated from the endoplasmic reticulum into the cytosol.
Despite a similar mode of action of PE and DT, which is ADP-ribosylation, and a similar
initial pathway of cell entry (internalization via coated pits and endocytic vesicles)
and of processing (proteolytic cleavage and a reductive step), they share almost
no sequence homology. The only similarity is the spatial arrangement of key residues
in their active sites that are arranged around residue Glu553 in PE and
Glu145 in DT [63±66].
When the whole toxin is used to make an immunotoxin, non-specific toxicity occurs
mainly due to binding of the toxin portion to cells, mediated by the binding domain.
Consequently, the goal of making improved derivatives of PE-based immunotoxins
has been to inactivate or remove the binding domain. Molecules in which the binding
domain has been retained but inactivated by mutations were made [67]; however,
a better alternative to inactivating the cell-binding domain by mutations is to remove
it from PE. The prototype molecule with this sort of deletion is PE40 (amino acids
253±613, MW 40 kDa]. Because PE40 and its derivatives described below lack the
binding domain (amino acids 1±252), they have very low non-specific toxicity, but
make very active and specific immunotoxins when fused to recombinant antibodies
[68, 69]. Currently, almost all PE-derived recombinant immunotoxins are constructed
with PE38 (MW 38 kDa), a PE40 derivative that has, in addition to the deletion of domain
la, a second deletion encompassing a portion of domain Ib (amino acids 365±
379) [58]. Another useful mutation is to change the C-terminal sequence of PE from
REDLK to KDEL. This improves the cytotoxicity of PE and its derivatives, presumably
by increasing their delivery to the endoplasmic reticulum where translocation
takes place [70, 71].
17.3.2
The Targeting Moiety ± Recombinant Antibody Fragments
The antibody moiety of the recombinant immunotoxin is responsible for specifically
directing the immunotoxin to the target cell, thus the usefulness of the immunotoxin
depends on the specificity of the antibody or antibody fragment that is connected
to the toxin. Consequently, for the construction of recombinant immunotoxins,
the only antibodies that should be used are those that recognize antigens that
are expressed on target cancer cells and are not present on normal cells, present at
very low levels or are only present on less-essential cells (Tab. 17.1). Receptors for
growth factors like EGF, IL-2, IL-4, IL-6 or erbB2 are common targets for targeted
cancer therapy because they are highly expressed on many cancer cells. Other carcinoma-related
antigens include developmental antigens such as complex carbohydrates,
which are often highly abundant on the surface of cancer cells.
The use of antibodies for immunotoxin production also requires that the antibody±
antigen complex be internalized, because the mechanism of PE-toxin killing requires
endocytosis as a first step in the entry of the toxin into the cell.