Biomedical Engineering Reference
In-Depth Information
contained the antiferritin antibody (F11) light chain variable
domain and barnase [151]. The enzyme was fused to the
C-terminus of the V L through a 13 amino acid spacer
(ALGTVDSASAGIL); the hexahistidine tag was placed at
the C-terminus of barnase. The construct was expressed in
E. coli, mostly as a soluble protein in a dimeric form
(
[156-161]. Shortening the flexible peptide linker connecting
V H and V L domains to less than 12 (usually five) residues
prevents the scFv to assemble in a normal monovalent
orientation and, as a result, two scFvs form a bivalent dimer
(diabody, 60 kDa) [131,162,163, and references therein].
Similar diabodies were also designed by engineering a
C-terminal cysteine residue to scFvs to form a disulfide
bond [158]. Reducing the linker to less than three residues
leads to the formation of trimers or tetramers with molecular
masses of 90 or 120 kDa, respectively [162]. Tumor reten-
tion improves with multimerization. This is a result of an
increase in size and/or valency [158,162,163]. On the basis
of these and similar observations, an optimal molecular size
range of 60-120 kDa for therapeutic molecules targeting
solid tumors has been proposed [161] and widely accepted
[65,66,163] as reasonably balancing tumor penetration,
retention, and clearance from the circulation. Monovalent
immunoRNases comprising scFvs and monomeric enzymes
(42-46 kDa) fall below the lower limit of this range but some
were found quite effective in vitro (Table 22.1) as well as
in vivo. Single Fv-dibarnase [152,153,155] with molecular
mass of 54 kDa is slightly closer to the optimal range but
dimeric/bivalent immunoRNases (82-92 kDa) fit in per-
fectly and, indeed, are profoundly more cytotoxic to cancer
cells that their monomeric/monovalent counterparts [63-
65,86,107,129]. ImmunnoRNases comprising scFvs and Fc
fragments of compact [111] or full size [116] antibodies
(140 and 150 kDa, respectively) are outside of the optimal
molecular mass range and may be more suitable for the
treatment hematological malignances (if properly targeted)
rather than solid tumors. The largest fusion protein of
Onconase 1 and the anti-Trop-2 antibody (180 kDa) targets
lymphomas [135].
53 kDa). It was able to specifically bind ferritin and
remained active as an RNase.
The only barnase fusion protein actually tested for anti-
cancer activity is the 4D5 scFv-dibarnase. Edelweiss et al.
[152] found that this construct as described above [146] was
2800-fold more active against SKOV-3 ovarian carcinoma
cell line overexpressing ErbB2 antigen (IC 50 1.8 nM) than
did barnase alone (Table 22.1). This fusion protein was
almost as active toward another highly ErbB2 positive breast
carcinoma BT-474 cell line. On the other hand, embryonal
kidney HEK293 cells, with normal level of this antigen,
were over 270-fold less sensitive. ErbB2 negative human
peripheral blood mononuclear cells were unaffected [152].
The sensitive cells displayed typical features of apoptosis,
including membrane blebbing, DNA fragmentation, and
caspase activation. Apoptotic activity was inhibited by either
4D5 scFv or barstar. This confirmed that both, binding and
effector moieties of this immunoRNase contributed to cyto-
toxicity. In the next papers [153,154], the authors reported
high activity against ErbB2-overexpressing human breast
cancer cell lines, SKBR-3 or BT-474 (IC 50 4.1 and 2.4 nM,
respectively) (Table 22.1) as well as the in vivo anticancer
activity of 4D5 scFv-dibarnase in mice bearing human breast
cancer xenografts. Treatment of BALB/c nude mice bearing
SKBR-3 breast cancer with a tolerated dose of the immu-
noRNase reduced the tumor growth by 77%.
22.3 ASPECTS OF IMMUNORNASE DESIGN
AND PRODUCTION
22.3.2 Interaction with Mammalian RNase Inhibitor
RNase inhibitor is an
50 kDa, leucine and cysteine rich
protein present in the cytosol of mammalian cells (reviewed
in References 164-166). It forms extremely stable com-
plexes with most mammalian RNases (K d values 10 16 -
10 14 M) and thus, abolishes their enzymatic activities.
RNases with innate cytotoxic activity toward cancer cells
are not inhibited by this protein [17]. As discussed earlier,
some of them (Onconase 1 , Rana pipiens liver RNase or
barnase) were used as effector moieties of ImmunoRNases
and these constructs are certainly able to evade the inhibitor
when translocated to cytosol. Mammalian RNases, angio-
genin, RNase 1, RNase 2, or a single-chain subunit of bovine
seminal plasma RNase used in the construction of immu-
noRNases strongly interact with the inhibitor. Therefore, the
effectiveness of these fusion proteins could be potentially
affected by this interaction. As discussed by Rutkoski and
Raines [32], fusion of various binding moieties to RNases
may or may not influence their affinity for the inhibitor. For
22.3.1 Molecular Size and Valency
Single-chain Fv fragments are the smallest constructs com-
prising the entire antigen binding sites of parent antibodies.
They are composed of variable heavy and light chain
domains linked with flexible peptide segments. Their molec-
ular masses range from 27 to 30 kDa. When compared with
larger antibody formats like entire IgG (150 kDa), F(ab') 2
(110 kDa), or Fab' (55 kDa) [155], tumor xenograft studies
demonstrated superior penetration of a radiolabeled scFv
fragment from the vascular system into a solid tumor. While
most of the entire IgG penetrated tumor regions directly
adjacent to the vasculature, the scFv was more evenly
(homogeneously) distributed in the tumor mass. The remain-
ing forms penetrated the tumor in a size-related manner. On
the other hand, tumor retention of scFvs is low due to their
rapid clearance from the circulation and monovalent binding
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