IMMUNORNASE FUSIONS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 335

example, the previously mentioned fusion protein, which
comprises an anti-CD30 single-chain Fv fragment of murine
Ber-H2 antibody and RNase 1, evaded the inhibitor [115]. It
was apparently due to a steric hindrance generated by the
scFv, preventing the enzyme-inhibitor interaction. On
the other hand, another fusion protein created with the
same enzyme, specific to ErbB2-positive solid tumors,
strongly interacted with the inhibitor but, simultaneously
effectively killed target cells [103]. Evidently, the immu-
noRNase reached the cytosol in large enough amount to
overwhelm the inhibitor (usually present in cytosol in
roughly 4 m M concentration [32]) and elicit apoptosis in
tumor cells [103]. It seems, therefore, that lack of cytotoxic
activity of some other RNase 1 immunofusions [167] could
result from their inadequate internalization and/or intra-
cellular routing and therefore, low uptake to the cytosol.
Consequently, a molar excess of RNase inhibitor over the
enzyme is created. Most immunofusions with mammalian
RNases as effector moieties were active against target cells.
It seems, however, that some fusion proteins could have been
more effective if RNase variants with engineered resistance
to the inhibitor [32] were used instead. Generally though, the
inhibitor cannot compromise the effects of immunoRNases
with high cytosol uptake.

toxicity [93,172]. They were peptide segments containing
sequences cleavable by proteolytic enzymes present either in
cytosol or endosomes. The “cytosolic cleavable unit,” YVH-
DEVDRGP, contained cleavable sites for caspases 1 and 3.
They were -DE- and -DR-, respectively. The 13 amino acid
“endosomal cleavable unit” had two sites available for furin
(-RG- and -RS-) [93]. Insertion of both cleavable units
between the anti-CD64 scFv and angiogenin enhanced
cytotoxicity of this immunoRNase by a factor of 20 but
simultaneously significantly reduced stability of this con-
struct in serum [89]. This could be due to degradation of the
endosomal cleavable unit by proteases with trypsin speci-
ficity present in the circulatory system. The fusion protein
with cytosolic cleavable unit alone was stable and retained
its acquired increased activity against target cells [89]. Chen
et al. [173] recently presented a novel approach toward
cleavable fusion proteins. The authors designed a dithiocy-
clopeptide based on the amino acid sequence of somatotro-
pin. It was used as a cleavable (reducible) linker/spacer
between functional components of a fusion protein that
contained colony stimulating factor and transferrin.
The amino acid sequence stretch of somatotropin,
AGCKNFFWKTFTSC (C3-C14 disulfide bond), was modi-
fied by replacing the WKT segment with PRS (the thrombin
specific site). The peptide was inserted between the
C-terminus of colony stimulating factor and the N-terminus
of transferrin through short Leu-Glu linkers. The fusion
protein expressed in HEK293 cells, contained a circular
linker/spacer with the original disulfide bond of somatotro-
pin. In vitro treatment with thrombin cleaved the circular
spacer but the components of the fusion protein remained
connected by the disulfide bond. Pretreatment with thrombin
improved activity of this fusion protein but the disulfide bond
was not very stable in the mouse circulatory system. This
may be a limiting factor of this method when used for the
construction of other fusion proteins such as immunoRNases.

22.3.3 Cleavable ImmunoRNases
In chemical conjugates of Onconase 1 or RNase A with
antibodies, the functional components are connected either
by disulfide or thioether linkages [58,59,168-170]. The
former bond is cleavable in the reducing environment of
the cell cytosol while the latter remains intact. The cleava-
ble, disulfide conjugates were found more active against
target cells than stable thioether conjugates [167-170].
Similar observations were also made in our laboratory
(Saxena SK, Ardelt W, unpublished). Most probably, sepa-
ration of the reducible conjugates in cytosol augmented the
enzymatic activity of the released RNases. It is possible that
large antibody molecules attached to the enzymes could
have obstructed their interaction with intracellular substrates
(steric hindrance). In fusion proteins, stable peptide seg-
ments (spacers) [171] do not allow for separation of the
functional components inside the cell. In this respect, there-
fore, fusion proteins are similar to the thioether-linked
chemical conjugates. Component inseparability creates
even more limitations in fusion proteins than in chemical
conjugates. Steric hindrance may not only directly affect
catalytic activity but also impair the process of protein
folding. This was observed in an antibody-barnase fusion
protein [151]. Thus, development of cleavable immunoR-
Nases may be in some cases advantageous. Hetzel et al. [89]
first worked in this direction. “Cleavable adapters” were
initially inserted between the binding and toxic moieties of
classic immunotoxins in an attempt to reduce nonspecific

22.3.4 High-Level Production of ImmunoRNases

Numerous classic immunotoxins and immunoRNases
were expressed in E. coli where they accumulated in
inclusion bodies as miss-folded, nonfunctional aggregates
[60,77,79,80,94,95,127]. Their recovery required in vitro
unfolding by denaturation and reduction of miss-formed
disulfide bonds followed by refolding at native conformation
and purification. Buchner et al. [174] designed the original
protocol for this process. It is a tedious, complicated, and
costly procedure with usually low product yield. It is there-
fore, not suitable for large-scale manufacturing and another
methodology clearly needed to be developed.
Periplasmic space expression in E. coli, with the use of
the pET22b(

) expression vector leads to soluble and
properly folded proteins. This approach was used for the
preparation of immunoRNases [86,103,107,129,152,155]

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