IMMUNORNASE FUSIONS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 328

engaged in the machinery of protein biosynthesis would
eventually inhibit translation and cause cell death. However,
most known RNases do not exhibit innate cytotoxic or
cytostatic functions although some were found cytotoxic
when microinjected to Xenopus oocytes [14,15]. Therefore,
it seems that the cell entry is a prerequisite for RNase
cytotoxicity [16]. A number of RNases demonstrate inherent
cytostatic and cytotoxic activities and have been considered
as potential drugs for anticancer therapy. Their structures
and functions, as well as mechanisms of toxicity and thera-
peutic potentials, were exhaustively reviewed [17-28].
Cytotoxic RNases are small basic proteins of 10-28 kDa
molecular mass. They were found among members of both,
RNase A (reviewed in References 29 and 30) and T1
(reviewed in References 31) superfamilies. They bind to
cancer cell membranes by ionic interactions, enter cells by
endocytosis, avoid degradation by proteinases inside the
endosomes [19] (that is largely due to their high confor-
mational stability), then translocate to the cytosol, where
they evade mammalian protein ribonuclease inhibitor [32]
and degrade various species of RNA that leads to apoptotic
cell death.
The most studied enzyme of this group is Onconase 1
(ranpirnase or, formerly, P30-protein) discovered by
Alfacell Corporation (presently Tamir Biotechnology,
Monmouth Junction, NJ, USA) in oocytes of a frog Rana
pipiens [33]. Onconase 1 (recently reviewed [17,18]) is the
smallest known member of pancreatic RNase A superfamily
[33]. It consists of 104 amino acid residues and has a
molecular weight 11,820. Onconase 1 is an unusually stable
protein and is remarkably resistant to proteolysis. Its very
high isoelectric point (9.7) facilitates binding to highly
negative [34] cancer cell surfaces. The enzyme is then
internalized by energy-dependent endocytosis [35] based
on clathrin adaptors and the GTPase dynamin (clathrin/Ap-2
mediated endocytic pathway) [36,37]. From endosomes,
Onconase 1 is delivered to the cell cytosol through the
receptor recycling compartment, apparently bypassing the
Golgi and endoplasmic reticulum [36,37] (intracellular
routing of other cytotoxic RNases is discussed in Reference
37). In the cytosol, the enzyme resists proteolysis and
does not interact with mammalian protein RNase inhibitor
[38-40]. This allows for the degradation of RNA, preferen-
tially tRNA. This in turn, leads to inhibition of protein
synthesis, induction of G 1 arrest and apoptosis (see Refer-
ences 17 and 18 for more discussion). Inhibition of protein
synthesis did not, however, explain certain effects of Onco-
nase 1 . For example, it was found that the enzyme was able
to suppress the expression of certain proteins and simulta-
neously upregulate other proteins [41] (similar results were
recently published [42]). Therefore it was postulated that
Onconase 1 , in addition to tRNAs [43,44], may also target
other noncodingRNAs-microRNAs involved in the regulation
of gene expression through RNA interference (RNAi) [45].

Indeed, it was recently found that the silencing of the gene
encoding glyceraldehyde-3-phosphate dehydrogenase in
human lung adenocarcinoma A549 cells by a siRNA was
effectively prevented by this RNase [46].
Onconase 1 is the only RNase that has advanced to
clinical trials and has been tested in the United States and
Europe. It has been tested as a single anticancer agent or in
combination with other drugs [47-51]. Even though Onco-
nase 1 is not a human protein, no serious immunogenicity
complications were noticed in the clinic. Approximately, a
total of 1000 of patients have been treated to date and there
have been few problems with repeated administering. This
may be due to a high degree similarity between the 3-D
structure of Onconase 1 and human RNases. The lack of
complications may also manifest from the enzyme's ability
to specifically enhance activation-induced apoptosis of
peripheral blood lymphocytes [52]. Thus, Onconase 1 has
been well tolerated by cancer patients; the main side effect,
renal toxicity at high doses was found reversible and less
pronounced or prevented by patient hydration.
Onconase 1 and other naturally cytotoxic RNases dem-
onstrate substantial specificity to rapidly proliferating cells,
for example, cancer cells. Endocytic internalization and
translocation to the cell's cytosol are limiting factors for
the cytotoxicity of these enzymes as well as of RNases with
engineered cytotoxicity [16,37]. Therefore, linking of
RNases by chemical conjugation or recombinant fusion to
cell binding, internalizing molecules has been used to
improve the specificity and cellular uptake of these enzymes.

22.1.3 Ribonuclease Targeting to Cancer Cells

The first evidence that RNases could be specifically targeted
to cancer cells was generated nearly two decades ago.
Bovine pancreatic RNase A [53,54] and Onconase 1 [55]
were chemically conjugated to transferrin or to antibodies
binding transferrin receptor. All these early conjugates were
found highly cytotoxic to cancer cells and demonstrated
IC 50 values below 1 m M. RNases were also chemically
conjugated to a number of other targeting molecules like
growth factors, cytokines, monoclonal antibodies, or their
fragments (see References 8,56, and 57 for reviews).
Chemical immunoconjugates of RNases are discussed later
in this chapter.
Two groups of authors provided excellent proofs of
concept for immune targeting of RNases to cancer cells.
Rybak et al. [58,59] described a chemical conjugate of LL2
anti-CD22 antibody with Onconase 1 . Both, immune and
effector molecules were modified prior to conjugation.
Sulfhydryl groups were introduced to the antibody in the
reaction with 2-iminothiolane and a heterobifunctional
crosslinker was incorporated to Onconase 1 through its
amino-reactive arm. Then, the sulfhydryl-reactive arm of
the incorporated crosslinker formed a disulfide bond linking