CLASSIC IMMUNOTOXINS WITH PLANT OR MICROBIAL TOXINS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 265

cytosol. The cytosolic C-terminal fragment then catalyzes the
ADP-ribosylation of EF2, resulting in cell death similar toDT.
PE-based immunotoxins have been made with various
truncated forms of PE and full-length PE by either chemical
conjugation or protein engineering (Figure 18.1). The full-
length PE is conjugated to the OVB3 monoclonal antibody
that has affinity to ovarian cancer cell lines and colon cancer
cell lines. PE40 is a 40-kDa truncated form of PE in which
residues 1-250 of domain Ia have been removed. Recombi-
nant NLysPE40, a modified form of PE40 having an N-
terminal lysine residue for chemical conjugation, is conju-
gated to a monoclonal antibody. The truncated PE40 is used
to synthesize TP-40 consisting of transforming growth
factor a (TGF a ) as the ligand. PE38 is a 38-kDa truncated
form of PE in which residues 1-250 of domain Ia and
residues 365-384 of domain Ib have been removed. PE38K-
DEL, PE38QQR, and PE38-8X are modified forms of PE38.
PE38KDEL has C-terminal KDEL residues as a endoplas-
mic reticulum (ER) retention signal that enhances internal
routing to ER and subsequently improves potency. To
minimize conjugation of heterobifunctional cross-linking
reagents to the C terminal catalytic domain of PE38, lysines
590, 606, and 613 of PE38 are replaced with glutamines and
arginine (PE38QQR) [50]. PE38-8X is a deimmunized
version of PE38 with eight point mutations on seven major
B-cell epitopes of PE38. The deimmunized version is less
immunogenic in animals [51]. PE25-LR is lysosomal prote-
ase-resistant and the smallest truncated form of PE contain-
ing residues 274-284 and residues 395-613 [52].

ligand with a sulfhydryl group, which provides a reactive
group for chemical conjugation with an antibody [61]. The
deglycosylated ricin A has less nonspecific toxicity than the
blocked ricin and the ricin A chain. Deglycosylation of ricin
A chain is required to reduce nonspecific normal tissue
binding because mannose receptors on liver cells recognize
terminal oligosaccharides of ricin A chain [62,63]. Degly-
cosylated ricin A chain is obtained by chemical treatment
with a mixture of sodium metaperiodate and sodium cya-
noborohydride [64] or expression in Escherichia coli [65].

18.2.4 Gelonin and Saporin

Gelonin, PAP, and saporin are potent single-chain ribosome-
inactivating proteins (28-30 kDa) from plants of Gelonium
multiflorum, Phytolacca americana, and Saponaria offici-
nalis, respectively. These toxins and ricin A chain
have similar protein structure (Figure 18.1). They are all
N-glycosidases which remove the same adenine base from
the 28S ribosomal RNA of the mammalian ribosomes [66].
Unlike ricin, these toxins lack any cell-binding or cyto-
plasmic delivery domains. For immunotoxins, these toxins
need to escape from the endosomes following internalization
by ligand-directed endocytosis in order to intoxicate a cell.
However, the endosomal escape is an inefficient rate-limit-
ing step [67]. A conserved C-terminal hydrophobic motif of
ribosome-inactivating proteins [68] may play an important
role in the endosomeal escape.

18.3 IMMUNOTOXIN DESIGN AND SYNTHESIS

18.2.3 Ricin

In the past three decades, over 1000 immunotoxins have
been engineered. The first generation immunotoxins are
chemical conjugates of toxins and tumor-selective ligands.
Large amounts of toxin and ligand molecules are needed to
synthesize chemical conjugates because of low conjugation
yield. Chemical conjugation yields a heterogeneous product
due to the random reaction of derivatizing reagents with
multiple amino acid residues in the ligand and/or toxin.
Serum half-lives of chemical conjugates are relatively
longer (9-24 h), compared to recombinant immunotoxins
(1-5 h). Many chemical conjugates have reached clinical
testing. The second-generation immunotoxins are recombi-
nant fusion proteins. Toxin structure information has been
used in immunotoxin design to minimize unwanted toxin
effect and maximize specific cytotoxicity to target cells.
They have only one tumor-selective ligand and a truncated
toxin in a single chain protein. Owing to a relatively simple
protein structure, these molecules are produced in E. coli
with reasonable yields. One of good examples is the first
approved immunotoxin, DAB 389 IL2 (Ontak 1 , denileukin
diftiftox) for the treatment of cutaneous T-cell lymphoma
(CTCL) in 1999 by the FDA. Most recombinant immuno-
toxins belong to this class.

Ricin is a two-chain toxin extracted from castor beans
(Ricinus communis). The A chain of 267 residues (RTA)
is linked to the B chain of 262 residues (RTB) by a disulfide
bond (Figure 18.1). RTA has N-glycosidase activity that
specifically depurinates an invariant adenine residue of the
28S ribosomal subunit. RTB has three lectin binding sites,
which bind galactosyl residues on mammalian cell surface
glycoproteins [53,54]. After cell binding, ricin internalizes
into endosomes using both clathrin-dependent and clathrin-
independent pathways [55] and reaches the trans-reticular
Golgi compartment [56]. In the Golgi, the toxin is retro-
grade transported to the ER via COP-I-independent, Rab6-
regulated vesicles [57,58]. In the ER, the toxin is reduced, the
A chain unfolds with the help of chaperones, and the A chain
translocates to the cytosol using the Sec61p transposon [59].
The A chain depurinates the adenine base of the 28S
ribosomal RNA, resulting in irreversible blocking of protein
elongation [60].
A blocked ricin, a ricin A chain and a deglycosylated
ricin A chain have been used in preparation of ricin immu-
notoxin. The blocked ricin has the A and B chains. The B
chain is blocked by covalent attachment of a glycopeptide