IMMUNORNASE FUSIONS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 330

TABLE 22.1 Anti-Cancer ImmunoRNase Fusion Proteins

Design Parent Antibody Target Antigen IC 50 (nM) Potential Indication References

CH2-Ang E6 CD71 0.05 Carcinomas [56,74,75]

Edn-scFv E6 CD71 0.2-1 Carcinomas [77]

scFv-bsR H17 hPLAP 0.3-4 Carcinomas [94,95]

Ang-sFv E6 CD71 4- 10 Carcinomas [80]

hpR-scFv E6 CD71 5-10 Carcinomas [79]

Edn-scFv E6 CD71 1- 8 Carcinomas [79]

scFv-hpR MgR6 ErbB2 50, 120 Carcinomas [60]

Ang-scFv Ki4 CD30 0.5-0.6 Lymphomas [84]

scFv-Ang Ki4 CD30 0.7-0.9 Lymphomas [84]

scFv-Bar 4D5 ErbB2 NA Carcinomas [146]

scFv-hpR PhL ErbB2 12.5-60 Carcinomas [103]

rpLR-scFv HMFG1 MUC1 80 Carcinomas [127]

V L -Bar F11 hFER NA Carcinomas [151]

rpLR-scFv RFB4 CD22 132-185 Lymphomas [129]

(rpLR-scFv) 2 RFB4 CD22 3-20 Lymphomas [129]

sFv-Ang MLT-7 CD22 360-380 Lymphomas [86]

(sFv-Ang) 2 MLT-7 CD22 74-118 Lymphomas [86]

Ang-scFv RFB4 CD22 57 Lymphomas [87,88]

(Onc) 2 -IgG LL1 CD74 0.6-1.3 Lymphomas [133]

scFv-hpR BerH2 CD30 100 Lymphomas [64,115]

(scFv-hpR) 2 PhL ErbB2 3.0 Carcinomas [107]

scFv-Fc-hpR 4E3, 2A1 CD30 3.3 Lymphomas [116]

scFv-Ang h22 CD64 0.2 Leukemias [89]

scFv-AD-Ang h22 CD64 0.03 Leukemias [89]

scFv-diBar 4D5 ErbB2 1.8-4.1 Carcinomas [152-154]

(scFv) 2 -Fc-hpR PhL ErbB2 NA Carcinomas [111]

(Onc) 2 -IgG RS7 Trop-2 1.3-8.5 Lymphomas [135]

Ang, angiogenin; Edn, eosinophil-derived neurotoxin; bsR, bovine seminal plasma RNase, hpR, human pancreatic RNase; Bar, barnase; rpLR, Rana pipiens
liver RNase; NA, not available; PhL, phage Library (fully human scFv isolated by phage display); Onc, onconase; diBar, dibarnase; AD, “adapter” (membrane
transfer peptide followed by a cytosolic cleavable peptide).

fusion protein with the latter linker was more active when
tested against renal carcinoma, colon carcinoma, and human
breast cancer cell lines. The respective IC 50 values are given
in Table 22.1. Elimination of the FB spacer between the
enzyme and antibody moieties resulted in distinctly lower
activity.
CD71, transferrin receptor used as a target surface anti-
gen in the construction of early immunoRNase fusions
discussed above is overexpressed in many cancers and
rapidly internalizes after antibody binding. However, it
was found to be able to cross the blood brain barrier together
with the toxic moieties associated with those constructs
[63,66]. Therefore, other target antigens were searched
for. A cell transmembrane type 1 glycoprotein CD30
(Ki-1), a 120 kDa member of the tumor necrosis factor
receptor family, is expressed in activated lymphocytes
and related tumors as well as in some carcinomas and
sarcomas [81-83]. St

kidney HEK293T) cells and secreted to the culture super-
natant. They were highly cytotoxic to Hodgkin-derived
CD30 positive L540 cells (Table 22.1).
CD22, a sugar binding membrane glycophosphoprotein,
is highly overexpressed in non-Hodgkin's lymphoma [85]
and most other B-cell lymphomas. It is therefore considered
to be a potential target in the treatment of hematologic
malignancies like non-Hodgkin's lymphoma, Burkitt's
lymphoma, multiple myeloma, chronic lymphocytic leuke-
mia, hairy cell leukemia, and prolymphocytic leukemia. An
anti-CD22 antibody was previously used for targeting
Onconase 1 [58,59] and, as discussed later in this chapter,
scFv of a similar antibody was fused to a close variant of this
RNase. Angiogenin was at first fused to a C-terminus of
MLT-7 scFv via (G 4 S 3 ) 2 spacer [86]. The product expressed
in E. coli periplasmic space was poorly active. Elimination
of the V L -V H linker from this fusion protein resulted in its
noncovalent dimerization through the scFv moieties and
substantially improved activity (Table 22.1). Kraus et al.
[87] generated several highly stable humanized single-chain
fragments of murine anti-CD22 RFB4 antibody. One of the
fragments was fused to the carboxyl terminus of angiogenin

oker et al. [84] were first to fuse the
anti-CD30 single-chain variable fragment of Ki4 monoclo-
nal antibody either to the C- or N-terminus of recombinant
angiogenin. For the first time, these fusion proteins were for
the first time expressed in eukaryotic (human embryonic

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