IMMUNORNASE FUSIONS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 331

through the (G4S3) 3 spacer. The construct was expressed in
transiently transfected Chinese hamster ovary (CHO) cells.
It was highly active against CD22 þ Daudi cells (Table 22.1)
but not toward Jurkat cells which do not overexpress this
antigen. A high-level concentration (5-10 mg/L) of this
fusion protein was recently attained from supernatants of
stably transfected myeloma (NSO) cells from the same
laboratory [88].
Hetzel et al. [89] constructed a fusion protein comprising
scFv of h22 humanized antibody and angiogenin. The anti-
body binds Fc g R (CD64) receptor, which is frequently over-
expressed by acute myeloid leukemia cells [90]. The fusion
protein was produced in E. coli BL21 Star (DE3) cells using a
periplasmic space secretion system under osmotic shock [91].
Interestingly, insertion of the membrane transfer peptide
followed by the cytosolic-cleavable peptide [92,93] between
h22 scFv and angiogenin [89] substantially increased fusion
protein activity against CD64 þ U937 cells (Table 22.1). The
use of “cleavable adaptors” for the construction of immu-
noRNases is discussed later in this chapter (Section 3.3).

immune targeting to cancer cells. This is attributed to its
high catalytic activity and expected low immunogenic
potential as a human protein. Zewe et al. [79] designed a
fusion protein of this enzyme and a single-chain antibody to
transferrin receptor (CD71). It was expressed in E. coli and
recovered from inclusion bodies by denaturation and rena-
turation. This fusion protein was only slightly less active
against three different human cancer cell lines compared to
the analogous RNase 2 construct described in the same paper
and discussed in Section 2.1 (Table 22.1). As mentioned
earlier, the anti-CD71 antibody is no longer considered a
candidate for construction of fusion proteins because it is at
risk of crossing the blood brain barrier.
ErbB2, a transmembrane tyrosine kinase receptor (also
known as HER2 or CD340) emerged as a promising target
antigen because of its preferential expression in tumors [99].
It is highly expressed in breast, ovary, and lung carcinomas
[100,101] and only marginally in normal tissues [102]. As
mentioned in Section 1.3, Lorenzo et al. [60] were first to
employ an anti ErbB2 scFv for RNase targeting. This fusion
protein comprised a murine scFv with (G 4 S) 3 linker, a
GSPEFM spacer, and RNase 1. It was expressed in E.
coli, isolated from cell lysate by metal chelate chromatog-
raphy (His 6 tag located at the N-terminus) and re-natured.
The product (immunoRNase) was toxic to ErbB2 þ SKBR3
and MDA-MB453 breast carcinoma cells (Table 22.1) but
not to ErbB2 negative cells. Two years later, the same group
reported the construction of a similar fusion protein with a
human instead of a murine immune moiety [103]. In their
earlier work [104], the authors successfully isolated a novel,
fully human ErbB2-specific scFv (Erbicin) from a phage
display library [105]. Erbicin was then fused to RNase 1 to
obtain the first fully human immunoRNase [103]. The novel
fusion protein contained a modified V H -V L linker, SS
(G 4 S 2 ) 3 GGS as well as a longer spacer between the immune
and effector moieties (AAASGGPEGGS). The His 6 tag
located at the C-terminal position. It was expressed in E.
coli as a soluble protein (molecular mass 46 kDa) in the
periplasmic space. The imunoRNase was cytotoxic to four
different ErbB2-positive cell lines with IC 50 s 12.5, 47, 52,
and 60 nM, respectively (Table 22.1). It also strongly inhib-
ited the growth of tumor in mice, which were inoculated
with ErbB2-positive TUBO cells. Studies of the mechanism
of action of this novel fusion protein [106] revealed expected
cytotoxicity dependence on the fusion protein's catalytic
activity. It was shown that inactivation of the RNase moiety
by alkylation abolished cell killing activity. The authors also
demonstrated that the construct translocates directly from
endosomes to the cytosol, where it interacts with ribonucle-
ase inhibitor protein. However, because of high cellular
uptake, the immunoRNase neutralizes the inhibitor and
elicits apoptotic cell death.
A dimeric immunoRNase was then designed [107] based
on Erbicin and a previously engineered cytotoxic dimer of

22.2.2 Immuno-Fusion Proteins of Bovine Seminal
Plasma RNase or Human Pancreatic RNase

Bovine seminal plasma RNase (BsRNase) is another member
of pancreatic ribonucleaseA superfamily [29,30]. This RNase
was recently reviewed along with other inherently cytotoxic
RNases [17]. BsRNase is the only enzyme in the superfamily
with quaternary structure. It is a natural homodimer consisting
of two identical 124 residue subunits linked by two disulfide
bonds and noncovalent interactions. The subunits are 80%
identical to bovine pancreatic RNase A. Deonarain and
Epenetos [94,95] fused a monomer subunit of BsRNase to
the C-terminus of single-chain fragment of murine H17E2
antibody. It was specific to the human placental-like alkaline
phosphatase antigen (PLAP) that is expressed in ovarian,
testicular, bladder, and head and neck cancers [96,97]. V H
andV L of this scFvwere connected by the (G 4 S) 3 linker. In the
original construct, the scFv was directly fused to the
N-terminus of the BsRNase subunit. Insertion of the APAAS-
PADA spacer and/or a diphtheria toxin disulfide loop between
the immune and the effector moieties increased its activity.
Also, C-terminal extension of the constructs with the KDEL
sequence segment (endoplasmic reticulum retention signal)
lowered the IC 50 values against PLAP positive human oral
epidermal carcinoma KB cells. Themost active fusion protein
comprised the scFv, the disulfide loop followed by the spacer,
and the RNase extended with KDEL (Table 22.1). The fusion
proteins were

10 4 fold more active than native
BsRNase. All variants were expressed in E. coli, renatured
and purified by metal chelate chromatography through the
N-terminally located His 6 tag.
Along with angiogenin and RNase 2, human pancreatic
RNase (RNase 1) [98] was an attractive candidate for

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