FUSION PROTEINS WITH TOXIC ACTIVITY - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 250

successful to produce it as fusion protein secreted from
Pichia pastoris [32].
Thelectindomain(Bchain)ofricinbindstogalactoseorN-
acetylgalactosamine residues that can be found on the surface
of many eukaryotic cells. Since ricin is heavily glycosylated,
even attachment to mannose receptors on endothelial cells or
macrophages might occur; therefore, therapeutic variants are
preferably deglycosylated. Endocytosis by clathrin-coated
pits internalizes the surface bound ricin. From the early
endosome ricin travels in a retrograde transport via the Golgi
to the endoplasmic reticulum. There the disulfide bond is
reduced by protein disulfide isomerase, and the catalytic
domain (A chain) is translocated to the cytoplasm. The N-
glycosidase activity of the A chain modifies the 28S rRNA.
Therefore, the ribosome cannot interact with EF2 anymore,
which irreversibly interrupts protein synthesis [33].
The most recent application of ricin is its conjugation to
two antibodies targeting CD22 and CD19. This combination
therapy, called Combotox, is now in clinical Phase I [34].
Currently, no genetically fused version of ricin is under
development. Particularly when using ricin, a severe com-
plication, the vascular leak syndrome, is observed. Endo-
thelial cells are unspecifically affected by high toxin doses
and become leaky, releasing fluid into the tissues [35].
Overall plant toxins have been less successful than
microbial toxins, probably because of difficulties regarding
the association of toxin and ligand during internalization and
translocation [6].

intracellular signaling. The high affinity heterotrimeric
receptor consists of three subunits: a (CD25), b (CD122),
and g (CD132). However, an intermediate affinity variant
assembled only from bg subunits triggers a different path-
way. When designing an antibody-based immunocytokine
directed against the necrotic core of tumors, the IL-2 was
mutagenized to exhibit lower affinity to the bg receptor to
reduce unwanted toxicity. This fusion protein had full anti-
tumor activity against neuroblastoma and nonsmall cell lung
cancer metastases in mice, but was better tolerated [38].
HER2 positive tumors can be addressed with the dimer of an
anti-HER2 scFv linked to an Fc domain that was connected
to an IL-2 molecule. Owing to the presence of a functional
Fc part, this fusion protein is capable to induce antibody-
derived cellular cytotoxicity (ADCC) and IL-2-dependent
immune stimulation, leading to high antitumor activity [39].
In principle, IL-2 can be linked to any known antibody that
targets a tumor antigen, thus increasing the potency of
ADCC by additionally activating natural killer (NK) cells.
Furthermore, a fusion protein is more efficacious than
administering IL-2 and the antibody, since now a colocal-
ization of the antibody and the cytokine is achieved, increas-
ing the local concentration of the stimulating agent. This has
been demonstrated with the anti-GD2 disialoganglioside
antibody hu14.18, fused to IL-2 [40].

17.3.2 GM-CSF Fusion Proteins

The known properties of granulocyte-macrophage colony-
stimulating factor (GM-CSF) to enhance antigen presenta-
tion and co-stimulation lead to the concept of dual cytokines.
Fusion of GM-CSF to IL-2 resulted in highly active NK cells
that released more interferon- g (IFN- g ) than expected. But
this molecule, called GIFT2, did not enhance proliferation of
immune cells. Replacing IL-2 by IL-21 to generate GIFT21
differentiated dendritic cells and inhibited tumor growth in
vivo. A third tumor killing variant contains CCL2 as fusion
partner to GM-CSF. This chimera induced the upregulation
of Bax and ultimately apoptosis of CCR2 positive cells that
contribute to inflammation in an arthritis model. The utili-
zation of GM-CSF fused to different cytokines and the
sometimes-unexpected results show one dilemma of fusion
proteins. It is difficult to predict effects or synergy when
combining molecules of the same type. It is therefore more
advisable to combine proteins for targeting and toxicity [41].

17.3 IMMUNOCYTOKINES

The immune stimulatory activities of several cytokines are
well understood; therefore, it was logical to use them as
fusion partners. In contrast to directly acting toxins, cyto-
kines attract and activate immune cells that then eliminate
malignant cells through various mechanisms. Combining the
cytokines with a targeting motif also increases the size of the
resulting molecule, thus extending the usually short half-life
of cytokines. This approach is discussed in Chapter 19.
Initially a lot of work was done fusing cytokines to full
antibodies. Using the several N- or C-termini that are
available for coupling to an antibody, more than a single
cytokine molecule can be coupled. Alternatively, more than
one type of cytokine can be bound to an antibody thus
allowing synergistic activity [36]. Immunocytokines should
not be confused with targeted toxins where cytokines as
fusion partner take the role of targeting the toxin to cells
overexpressing the respective cytokine receptor [37].

17.3.3 Other Leukocyte Attracting Cytokines
and Chemokines

Another important target that has been evaluated with a wide
range of different immunocytokines is single stranded DNA,
which becomes exposed in degenerating cells. For this
purpose, the so-called tumor necrosis therapy (TNT) mono-
clonal antibody was generated and linked to IL-2, IFN- g ,

17.3.1 Interleukin 2 Fusion Proteins

The activity of interleukin 2 (IL-2) is transferred through its
binding to the respective receptor (IL-2R) and initiates