IMMUNORNASE FUSIONS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 333

E. coli, recovered from inclusion bodies by denaturation and
re-naturation and finally purified by cation exchange chro-
matography. The novel fusion protein was cytotoxic to the
MCF7 breast cancer cell line (Table 22.1).
The same group of authors designed a series of human-
ized scFv fragments [128] derived from murine RFB4 anti-
body, which recognizes the CD22 antigen (discussed in
Section 2.1). Some of the fragments were later employed
for the construction of monomeric and dimeric fusion
proteins with RapLR [129]. Shortening of the V L -V H
(G 4 S) 3 linker to the five residue form (G 4 S) based on the
work of other authors [130] allowed for generation of a
diabody and, consequently a dimeric fusion protein with
RapLR [129]. The scFvs to be fused to the enzyme were
further engineered by replacing the V L Leu 36 with Tyr in
order to improve their solubility and stability. Both mono-
and dimeric fusion proteins were expressed in E. coli (strain
TG1) periplasmic space and purified by immobilized metal
chromatography and size exclusion. Dimerization caused
substantial increase of cytotoxicity to CD22 positive cell
lines, Daudi, Raji, and CA46 cells (Table 22.1).
Onconase 1 was immunotargeted to CD74, a type II
transmembrane glycoprotein that is highly expressed in
hematologic malignances like non-Hodgkin lymphoma
and multiple myeloma [131,132]. Humanized murine LL1
anti-CD74 internalizing antibody was first engineered [134]
by changing its constant region from g 1to g 4 and introduc-
ing an S228 to P replacement in the hinge region (to prevent
half molecule formation [134]). Then, its' (V L ) N-terminus
was genetically fused to the C-terminus of Onconase 1
through the (G 4 S) 3 spacer [133]. The construct, 2L-Onc-
hLL1-V4P, was expressed in transfected Sp2/0-Ag14 mye-
loma cells and purified by protein A affinity chromatogra-
phy. The molecular weight of the product corresponded to
one IgG fused to two enzyme molecules. This first recom-
binant RNase-IgG fusion protein was highly cytotoxic to
CD74 positive non-Hodgkin lymphoma (Daudi and Rai) or
multiple myeloma (MC/Car) cells with IC 50 values 0.63, 1.3,
and 0.63 nM, respectively. CD74 negative, small cell lung
cancer cell line was insensitive.
The same group recently succeeded in the construction
of a similar immunoRNase [135] targeting Onconase 1 to
the Trop-2 (EGP-1or TACSTD2) antigen, a tumor-associ-
ated calcium signal transducer [136] highly expressed by
human carcinomas but not by most normal tissues ([135] and
references therein). The humanized murine RS7 anti-Trop-2
antibody [137,138] was used for generation of a fusion
protein with Onconase 1 . The single putative N-glycosyla-
tion site in the enzyme was disrupted (by the replacement of
Asn69 with Gln) to prevent glycan heterogeneity of the
fusion protein when expressed in mammalian cells. Two
molecules of this Onconase 1 variant were then recombi-
nantly fused to the antibody [135]. Each was connected to
the N-terminus of one L chain in a similar way that used in

the previous paper [133]. The fusion protein was expressed
in stably transfected Sp2/0 myeloma cells and purified from
the supernatant on a Protein A column. The product was
apparently homogenous based on size exclusion high per-
formance liquid chromatography and demonstrated two
bands in reducing SDS-PAGE. The bands were of molecular
mass 37 kDa and, respectively, corresponded to the

heavy chain of the antibody and the light chain fused to
Onconase 1 . It was very active against cultured breast cancer
cellsMDA-MB-468 andT-47D (IC 50 s 3.8 and 2.0 nM, respec-
tively). It was also active toward nonsmall cell lung cancer
Calu-3 (IC50 8.5 nM) and cervical cancer ME-180 (IC50
1.5 nM) cells. The immunoRNase also significantly inhibited
the growth of Calu-3 xenographs on athymic nu/nu mice.
Very recently, a development of Onconase 1 fusion pro-
teins with humanized anti-CD22 antibody was announced
[139].

50 and

22.2.4 Barnase Immuno-Fusion Proteins

So far, barnase is the only microbial RNase targeted to cancer
cells. It is a small (110 amino acid residues, 12.4 kDa)
extracellular enzyme secreted by Bacillus amyloliquefaciens.
It was first described in 1959 [140] and has been well
characterized since then [141-143]. Unlike most known
RNases it contains no disulfide bonds. Barnase and its
specific protein inhibitor barstar are reviewed [144,145].
Deyev et al. [146] designed fusion proteins composed of a
single-chain variable antibody fragment specific to the
ErbB2 (HER2) surface receptor (discussed in Section 2.2)
and barnase. The scFv derived from a humanized 4D5
antibody [147] was fused to the N-terminus of barnase
through a peptide spacer (EFPKPSTPPGSSGGAP). The
spacer was derived from murine IgG3 hinge region. The
entire construct, scFv-barnase, had molecular mass of
42 kDa. Another immunoRNase contained the same scFv
and the “dibarnase” component as an effector moiety. The
latter was a barnase molecule directly fused to a variant of
the same enzyme with five N-terminal residues removed
(des1-5-barnase). The entire construct was scFv-dibarbase
(54 kDa). Both fusion proteins had “FLAG” and pentahis-
tidine tags at the N- and C-termini, respectively. They were
expressed in E. coli SB536 cells [146]. Since barnase is
lethal to bacterial cells, the constructs were co-expressed
with barstar [145,148,149]. Notably, an elegant method was
developed to separate the enzymes from the inhibitor and
purify the fusion proteins from cell lysates. This was accom-
plished with immobilized ion chromatography followed by
protein A-affinity chromatography. The 4D5 scFv-barnase-
His5 fusion protein was also successfully expressed in
human embryonic kidney (HEK) 293T cells and purified
from cell lysates [150].
In the earlier paper, the same group described the con-
struction and characterization of a fusion protein that