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Experimental Therapeutics, Preclinical Pharmacology |
Departments of Biochemistry and Molecular Biology [A. G., S. K. B.], Pathology and Microbiology [S. A., B. J. M. B., G. P., S. K. B.], and Radiation Oncology [J. B-K.], College of Pharmacy [S. A.], Eppley Institute for Research in Cancer and Allied Diseases [S. K. B.], University of Nebraska Medical Center, Omaha, Nebraska 68198; Coulter Pharmaceutical Incorporated, San Francisco, California 94080 [D. C.]; and University of California San Francisco Cancer Center, San Francisco, California 94115 [M. T.]
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ABSTRACT |
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INTRODUCTION |
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Most of the radioimmunoconjugates studied to date involve the use of
whole immunoglobulins, particularly IgG. Intact MAbs
(Mr 
150,000) remain in circulation
for an extended time and can therefore increase the radiation dose
delivered to normal tissues (16)
. Moreover, the poor tumor
penetration of MAbs results in the localization of only
0.0001–0.01%ID/g of tumor (17)
. Many solid tumors are
relatively radioresistant; therefore, RIT of such tumors requires high
radiation doses, which can result in significant nonspecific uptake by
normal tissue leading to toxicity, especially to the hematopoietic
system (18)
. Because the size of the antibody-based
molecule relative to the renal threshold for first-pass clearance is a
major factor in determining the residence time of the
radioimmunoconjugate in circulation, a range of IgG formats, like
F(ab')2, Fab', or Fab, have been investigated for
therapeutic potential at preclinical (19, 20, 21)
and clinical
levels (5
, 22
, 23)
. Decreasing the size of the antibody
molecule increased both the degree of penetration and the clearance
rate from the blood pool; however, these molecules were difficult to
generate at a large scale with clinical-grade purity and did not
provide a significant increment in quantitative tumor retention as an
intact antibody (24)
.
The advent of molecular biology has enabled the designing and purification in ample abundance of small, high-affinity, multivalent antibody-based molecules as carriers for radionuclides (25) . ScFvs are recombinant proteins composed of a VL amino acid sequence of an immunoglobulin tethered to a VH sequence by a designed peptide (26 , 27) . Compared with an intact antibody, scFvs can bind to a tumor cell in a more homogeneous distribution (28 , 29) . Such fragments lead to a higher tumor:normal tissue ratio, but the percentage of injected dose delivered to the tumor is usually poor because of their monovalent nature and faster removal from the circulatory system. Moreover, the high renal accretion of these small molecules can lead to severe nephrotoxicity at therapeutic doses (24) . To increase the functional affinity of scFvs, the valency of scFvs has been increased by connecting them together by either noncovalent or covalent interactions (28, 29, 30) . The divalent scFvs have shown improved avidity and efficacy for tumor targeting at preclinical levels (31, 32, 33, 34, 35, 36) .
CC49 is a second-generation murine MAb showing high affinity for the tumor-associated glycoprotein 72 (37) . CC49 MAb is under clinical trials for radiation-mediated therapy of ovarian, colorectal, breast, and prostate carcinoma (4 , 38, 39, 40, 41) . In the present study, we have investigated for the first time the therapeutic potential of divalent [sc(Fv)2] and tetravalent {[sc(Fv)2]2} CC49 scFvs under single and dose-fractionation schedules in athymic mice bearing human colon carcinoma xenografts. Fractionated therapeutic treatment was found to be clearly superior to single administration for both sc(Fv)2 and [sc(Fv)2]2. We believe that the multivalent CC49 scFvs hold potential toward the generation of optimum tumor-targeting reagents in radionuclide-mediated therapy.
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MATERIALS AND METHODS |
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A (Invitrogen,
Carlsbad, CA) and transformed into competent Pichia
pastoris KM71 cells
(his4arg4aox1
::ARG4) as described
earlier (36)
. Upon expression, 20–30% of divalent scFvs
were found to associate as tetravalent and/or higher aggregated forms.
ScFvs were purified by immobilized metal affinity chromatography using
the chelating resin Ni2+-nitrilotriacetic acid
Superflow (Qiagen Inc., Valencia, CA; Ref. 36
). A Superdex
200 column (1.6 x 60 cm; Pharmacia Biotech., Piscataway, NJ) was
used to separate divalent and tetravalent scFvs. Protein concentrations
were determined by the method of Lowry et al.
(42)
. CC49 IgG used for control studies was purified on
Protein G Sepharose Fast Flow resin (Pharmacia Biotech.) and dialyzed
into HEPES saline buffer [10 mM HEPES, 150
mM NaCl, (pH 7.4)].
Characterization of Purified Multivalent scFvs.
The purity of scFvs was assessed by SDS-PAGE and HPLC size exclusion
chromatography. SDS-PAGE was performed according to the method of
Laemmli (43)
under reducing and nonreducing conditions.
The gels were stained with Coomassie Blue R-250. HPLC analyses were
done on TSK G2000SW and TSK G3000SW (Toso Haas, Tokyo, Japan)
size-exclusion columns connected in series using 67 mM
sodium phosphate buffer (pH 6.8), 100 mM KCl as the mobile
phase. The columns were calibrated using the Gel Filtration Calibration
Kit (Bio-Rad, Hercules, CA). The elution was monitored by an in-line UV
detector at 280 nm.
The immunoreactivity of scFvs was analyzed by a solid phase competition ELISA using BSM (Sigma Chemical Co., St. Louis, MO) as the antigen (36) . The binding affinities of sc(Fv)2, [sc(Fv)2]2, and CC49 IgG for BSM were determined by SPR measurements using an upgraded version of BIAcore 1000 (Pharmacia Biosensor, Uppsala, Sweden) as described previously (36 , 44) . The kinetic rate constants (kon and koff) were measured, and the equilibrium association constant (KA) and equilibrium dissociation constant (KD) were derived.
Radioiodination of scFvs.
The scFv forms were labeled with either Na125I or
Na131I using
1,3,4,6-tetrachloro-3,6-diphenylglycoluril (IodoGen; Pierce
Chemical Co., Rockford, IL) as the oxidant (45)
. For
therapeutic labeling, iodinations were carried out with 15–20 mCi of
Na131I (NEN, Boston, MA)/mg of scFvs in
0.1 M sodium phosphate buffer (pH 7.2) with IodoGen (250
µg/mg of protein) and at a protein concentration of 2–3 mg/ml.
Unincorporated radioiodine was separated from the labeled protein by
size exclusion chromatography using a Sephadex G25 column
(Pharmacia, Piscataway, NJ). The specific activity of
125I- and 131I-labeled scFv
molecules was about 3–9 mCi/mg. The radiochemical purity of all of the
radiolabeled scFvs was 
98% as confirmed by ITLC.
Characterization of Radiolabeled scFvs.
Radiolabeled proteins were analyzed on SDS-PAGE gels, and the
radioactivity associated with the protein was measured using the
ImageQuant software of the PhosphorImager (Molecular Dynamics,
Sunnyvale, CA). Analytical size-exclusion HPLC was performed as
described above. Fractions (1 ml) of the radiolabeled protein were
collected, and the radioactivity was determined in a Packard Minaxi
Auto-Gamma 5000 gamma counter (Meriden, CT). The immunoreactivity of
radiolabeled CC49 scFv forms was assessed by RIA where BSM (surrogate
for TAG-72 antigen) and BSA (negative control) were attached to a
solid-phase matrix (Reacti-Gel HW-65F; Pierce; Ref. 36
).
Animals and Tumor Model.
Female athymic mice (nu/nu; 4–6 weeks of age) were used for
the in vivo studies (Charles River, Wilmington, MA).
The human colon carcinoma LS-174T cell line (American Type Culture
Collection, Rockville, MD) was implanted s.c. (4 x
106), and the mice were used 6 days (tumor
volume, 250–300 mm3
) after the injection of
cells. Mice were kept in microisolator cages and fed ad
libitum pathogen-free mouse diet and water. The procedures used
were in accordance with the USPHS Guidelines for the Care and Use of
Laboratory Animals and were also approved by the University of Nebraska
Medical Center IACUC. Potassium iodide (0.001%) was given in
the drinking water for 3 days before and terminated 3 days after the
administration of the radioiodinated scFv. This blocked the thyroid
radioiodine uptake that enabled the excretion of free iodine out from
the animal body without contributing substantially to the whole animal
dose or therapeutic dose to the tumor.
Biodistribution and Pharmacokinetics Studies.
Dual-label biodistribution studies were performed in LS-174T
human xenograft-bearing mice after a simultaneous i.v. injection via
the tail vein of 125I-labeled
sc(Fv)2 (5 µCi) and
131I-labeled
[sc(Fv)2]2 (2.5 µCi) or
125I-labeled CC49 IgG (5 µCi) and
131I-labeled
[sc(Fv)2]2 (2.5 µCi) as
described earlier (35
, 36)
. For the whole-body retention
studies, mice bearing the LS-174T xenografts (three/group) received
injections via the tail vein with 1.5 µCi of radioiodinated scFvs.
Each scFv was evaluated separately. The whole-body radioactivity was
determined at various times after injection in a custom-built NaI
crystal. The blood clearance studies were performed as described
previously (35
, 36)
.
Therapy Studies with 131I-labeled scFvs.
For the therapy studies, mice bearing established LS-174T tumors were
used. The animals were randomized, the initial body weight was
recorded, and tumor volume was measured by caliper. Two therapy
regimens were used: (a) a single i.v. dose of 1000 µCi of
131I-labeled sc(Fv)2
(n = 10) or
[sc(Fv)2]2
(n = 10) administered on day 6; and (b) four
i.v. doses of 250 µCi of 131I-labeled
sc(Fv)2 (n = 10) or
[sc(Fv)2]2
(n = 10) on days 6, 7, 8, and 9. For the control groups
(n = 5), mice received injections i.v. with PBS (pH
7.4) either as a single administration on day 6 or as four injections
on days 6, 7, 8, and 9. The tumor growth was monitored twice a week by
measuring the tumor in two dimensions where volume = (length of
short axis in mm)2 x (length of long axis in
mm)/2, as described earlier (35)
. The body weight of mice
was recorded twice a week. Mice were euthanized when the short axis of
the tumor was 12 mm, tumor ulceration was detected, or the animals
lost 20% of their original body weight.
Radiation Dosimetry.
The radiation-absorbed dose delivered to the tumors and normal organs
such as kidneys and liver was calculated according to the Medical
Internal Radiation Dose committee of the American Society of Nuclear
Medicine (46
, 47)
. For kidneys (average weight, 0.15
g), electron/ß were only included in the mean absorbed dose. For all
of the other organs and tumors, the self-absorbed dose was calculated.
The mouse organ-cumulated activity (µCi x h) was calculated by
integrating time-activity curves derived from the biodistribution data
after the first injection of 131I-labeled scFv.
The data were expressed in µCi/g and was not corrected for decay. It
was assumed that radionuclide localizes immediately, i.e.,
no lag time, in the organ of interest. It was further assumed that the
effective half-lives do not change with each consecutive injection of
the protein in the fractionated administration scheme. For calculation
of the cumulative tumor radiation doses, the absorption phase times
were Tabs = 2.2 h and 2.6 h,
for dimer and tetramer, respectively. The tumor was assumed not to grow
during the treatment period.
Statistical Analysis.
For comparing time with tumor quadrupling between various groups of
mice, the data were fitted to estimate the slope of the growth curve.
The Wilcoxon signed rank test was used to generate the two-tailed
Ps. The survival fraction of each treatment group was
evaluated according to the method of Kaplan and Meier. The survival
curves were compared, and a Logrank test was used to generate the
Ps using the GraphPad Prism, Version 2.01 (GraphPad Software
Inc., San Diego, CA).
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RESULTS |
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95%
pure as indicated by SDS-PAGE and HPLC analyses. The binding parameters
derived from the SPR studies were: kon
= 9.1 x 104
M-1s-1,
koff = 8.9 x
10-4 s-1, and
KA = 1.0 x
108
M-1 for
[sc(Fv)2]2; and
kon = 2.2 x
104
M-1s-1,
koff = 8.0 x
10-4 s-1, and
KA = 2.8 x
107
M-1 for
sc(Fv)2.
After radioiodination, the integrity of proteins stored at 4°C was
ascertained daily by SDS-PAGE and HPLC for at least 4 days. On
SDS-PAGE, both sc(Fv)2 and
[sc(Fv)2]2 migrated as
Mr 58,000 under nonreducing and
reducing conditions and were found to be stable when stored at 4°C in
1% mouse serum. HPLC analysis indicated that 95% of the
radioactivity was associated with the protein peak for both
sc(Fv)2 and
[sc(Fv)2]2 (Fig. 1)
. However, the amount of free 131I increased to
12% (72 h after labeling) and was corrected for therapeutic
administrations. The immunoreactivity of radiolabeled scFvs by solid
phase RIA was 85–95% (0.8–1.5% nonspecific binding). At 72 h
after labeling, a decrease of 15 and 20% in the immunoreactivity
was observed for sc(Fv)2 and
[sc(Fv)2]2, respectively.
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95% of both
radioiodinated scFvs cleared from the body, whereas the clearance was
only 75% for CC49 IgG. The tumor localization of
[sc(Fv)2]2 was 2-fold
higher than sc(Fv)2 beginning at 4 h after
injection (Fig. 2)
. Uptake at earlier time points (i.e. <4 h) was similar for
all of the proteins. At 24 h after administration, the %ID/g in
tumor was 10.5 ± 1.1 for
[sc(Fv)2]2 and 5.1 ± 0.7 for sc(Fv)2. The maximum tumor uptake of
[sc(Fv)2]2 occurred
between 6 and 16 h. CC49 IgG showed 28.4 ± 1.7%ID/g in
tumor at 24 h after administration. However, in blood CC49 IgG
showed 12.5 ± 0.9%ID/g as compared with 0.3 ± 0.1 and
0.2 ± 0.1%ID/g for
[sc(Fv)2]2 and
sc(Fv)2, respectively. RIs (%ID/g of tumor
divided by %ID/g of normal tissue) of the major organs were determined
for CC49 IgG, sc(Fv)2, and
[sc(Fv)2]2. For
well-perfused organs such as the liver and the spleen, the tumor:liver
and tumor:spleen ratios were 2.3:1 and 2.6:1 for CC49 IgG, 8.1:1 and
8.2:1 for sc(Fv)2, and 3.9:1 and 10.4:1 for
[sc(Fv)2]2 at 24 h
after administration.
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). The group administered with
[sc(Fv)2]2 showed tumor
regression for 14–16 days as compared with the control group, which
showed approximately a 6–8-fold increase in tumor volume (Fig. 3)
. The
times for tumor-quadrupling volume were 9.0 ± 0.8 and 16.6 ± 1.9 days for sc(Fv)2 and
[sc(Fv)2]2, respectively
(Table 1)
. The time for tumor-quadrupling volume for the control group was
8.3 ± 0.7 days (Table 1)
. A comparison of survival curves with
the control group for single dosing showed the median survival time as
26 (P > 0.5) and 32 days (P < 0.001)
for the sc(Fv)2 and
[sc(Fv)2]2, respectively
(Fig. 4)
. No apparent signs of systemic radiotoxicity were detected. Animals
did not lose >20% of their body weight with 1000 µCi as a single
dose (Fig. 5)
. The cumulative radiation doses delivered to the tumor for bolus
sc(Fv)2 and
[sc(Fv)2]2 were 8.5 and
36.0 rads, respectively (Table 2)
. Compared with sc(Fv)2, the higher uptakes of
[sc(Fv)2]2 in the liver
and kidneys resulted in a 2–3-fold increase in the absorbed radiation
dose.
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0.05) and
[sc(Fv)2]2
(P = 0.03) from the untreated control group (Fig. 3)
.
For both therapeutic regimens, tumors regressed for 24–28 days. During
the same time, the control group showed approximately a >18-fold
increase in tumor volume or was removed from the study (Fig. 3)
. The
times for tumor-quadrupling volume were calculated as 21.1 ± 2.9
days for sc(Fv)2, 32.9 ± 2.7 days for
[sc(Fv)2]2, and 8.4 ± 0.6 days for the control group (Table 1)
. Survival analysis showed a
significant tumor growth inhibition for sc(Fv)2
and [sc(Fv)2]2 with the
median survival time as 38 (P < 0.0001) and 53 days
(P < 0.0001), respectively, as compared with the
control group (Fig. 4)
. Mice treated with fractionated doses showed
a 
8% loss in body weight, in contrast to the mice that
received injections with a single dose of 1000 µCi, which showed a
10% body weight loss from 20 day onward (Fig. 5)
. In tumors, the
absorbed radiation doses for sc(Fv)2 and
[sc(Fv)2]2 were 38.2 and
168.9 rads, respectively, for each protein and were 4-fold higher than
doses after a single administration (Table 2)
. The cumulative activity
in the liver and kidneys also increased but was well below the
threshold levels. |
DISCUSSION |
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Alternatively, recombinant antibody-based molecules with high affinity, functional avidity, and optimal size are being engineered and evaluated for the improved RIT of solid tumors (35 , 56, 57, 58, 59) . However, only a few studies have investigated the tumor localization of trivalent antigen-binding antibody constructs (Fab')3 (60, 61, 62) and tetravalent IgG dimers (63 , 64) in xenografted mice.
We have previously compared the therapeutic efficacy of 131I-labeled noncovalent scFv dimers and intact IgG of MAb CC49 in athymic mice bearing human colon carcinoma xenografts. The maximum tolerated dose for IgG was 500 µCi and could be escalated to 1500 µCi with 131I-labeled sc(Fv)2. Even with the lowest dose of 131I-labeled sc(Fv)2, a statistically significant prolonged survival time was observed as compared with the control (P = 0.036; Ref. 35 ). For CC49 IgG, single administration of 250 or 500 µCi resulted in complete tumor regression in 30 and 60% of mice, respectively (35) . Reduced tumor growth in 80–100% of mice has been reported earlier with 131I-labeled CC49 IgG (50) . Although IgG has shown promising preclinical results, the clinical performance of intact antibody for cure of solid tumors has been limited (38 , 40 , 41) . This is primarily due to slower clearance of IgG from the blood pool that limits the maximum tolerated dose and dose escalation essential for the treatment of solid tumors.
A covalent dimeric CC49 scFv [sc(Fv)2] was subsequently constructed and characterized for in vitro antigen-binding affinity and in vivo tumor targeting (36) . Upon expression, 20–30% of sc(Fv)2 was found to associate as tetravalent and/or higher molecular weight aggregated forms. The tetravalent scFv {[sc(Fv)2]2} revealed a 3–4-fold higher KA than sc(Fv)2 in SPR studies. Also, [sc(Fv)2]2 showed a 2–3-fold slower koff as compared with CC49 IgG, indicating a stronger association with the antigen because of a higher functional affinity of the molecule. The gain in avidity due to tetravalency, along with a lower molecular weight than intact IgG, suggests this recombinant scFv as a good candidate for tumor targeting.
In biodistribution studies, [sc(Fv)2]2 exhibited an approximately 2-fold increase in tumor localization over sc(Fv)2 following 4 h after administration. Although tumor uptake of CC49 IgG was significantly higher at later time points (16 h onwards), the nonspecific retention of radioactivity was found to be elevated in the major organs studied because of the long circulation half-life of radiolabeled IgG (65) . The tumor:blood ratio for sc(Fv)2 and [sc(Fv)2]2 was detected as 1.5- and 15-fold higher, respectively, than CC49 IgG at 24 h after administration. Blood clearance studies showed the elimination half-life of sc(Fv)2 and [sc(Fv)2]2 as approximately 4- and 2-fold faster than intact CC49 IgG. The tumor:liver and tumor:spleen ratios for 131I-labeled sc(Fv)2 were found to be lower than the values observed for 131I-labeled sc(Fv)2 earlier (35) . The increased RI of noncovalent sc(Fv)2 resulted primarily because of its faster clearance from the blood than covalent sc(Fv)2 (36) . For [sc(Fv)2]2, the RI for tumor:liver ratio was found lower compared with that for sc(Fv)2 due to the faster clearance of the sc(Fv)2 from the blood. The liver might be the possible site for elimination as also evidenced by a lower kidney uptake of [sc(Fv)2]2 (Mr 120,000). High tumor localization with low nonspecific retention suggests that these multivalent scFvs may be preferred choices for RIT of solid tumors.
We have performed a direct comparison of the radioimmunotherapeutic
efficacy of dimeric and tetrameric CC49 scFvs under both single and
fractionated regimens. The outcome of RIT strongly depends upon the
initial size of the tumor because the uptake of radiolabeled antibody
decreases exponentially with the tumor size thereby reducing the tumor
dose (66)
. Impressive therapeutic results have been
documented in animals with small or micrometastatic tumors (51
, 67
, 68)
and in patients with small tumor burdens (13
, 14)
. In the present study, only larger tumors were used to
extrapolate the situation found in a clinical setting with higher tumor
burdens. We have reported previously that the therapy with
131I-labeled noncovalent dimers gave a tumor
regression lasting for 16 days at 1500-µCi radiation dose
(35)
. The treatment with 1000 µCi of covalent dimer
provided less effective therapy, probably because of the larger and
more aggressive tumors (250–300 mm3
at
approximately day 6). However, with tetravalent scFv, a single
therapeutic treatment of 1000 µCi showed a 2-fold slower tumor
volume-quadrupling time than the untreated group, with the median
survival time of the group increased by 1.3-fold. No significant
systemic toxicity was recorded for the single administration of 1000
µCi attributable mainly to the fast blood clearance properties of
multivalent scFvs (95% were cleared out in 48 h).
With radiolabeled IgGs, both a specific (targeting the tumor-associated antigen) and a nonspecific (emission properties of the radionuclide) components can be associated with tumor regression. However, the therapeutic advantage attained by the specific antigen binding is significant over the nonspecific cytotoxic properties of 131I (50) . Therefore, in the present study, PBS (pH 7.4) was used as the negative control rather than an irrelevant scFv. Also, the reference control of CC49 IgG was not included because the maximum tolerated dose for 131I-labeled CC49 IgG was 500 µCi for single administration (35) .
Fractionated therapy at medium dose levels has been suggested to
provide effective RIT (51
, 53
, 69)
. Therefore, a
dose-fractionation study was performed to directly compare the
advantages of RIT involving dose fractionation over single
administration. Fractionated doses were administered every 24 h as
whole-body clearance studies demonstrated that >90% of the
radioiodinated sc(Fv)2 and
[sc(Fv)2]2 was cleared
from the animal body. Also, at 24 h after administration the
specific accumulation of scFvs in the tumors was detected. For both
sc(Fv)2 and
[sc(Fv)2]2, dose
fractionation yielded slower tumor growth with the tumor-quadrupling
time about 2.5- and 4-fold slower than the control group, respectively.
In our study, the groups that received fractionated therapy of
radioiodinated sc(Fv)2 and
[sc(Fv)2]2 showed a
statistically significant lengthening of the median survival time,
which was 1.6- and 2.4-fold longer than in the untreated group.
As a reference, IgG was not included in the dose-fractionation
experiment because intact IgG remains in circulation for a much longer
period. At 24 h, the %ID/g in blood was >10 for IgG and could
have lead to potential bone-marrow toxicity upon multiple
administrations. In previous dose-fractionation studies with IgG,
usually the therapeutic regimen consisted of two or three
administrations of 300 µCi of 131I-labeled
B72.3/CC49 IgG at either a 7-day interval (51)
or a 3-day
interval (53)
.
Besides the molecular size and functional affinity of the antibody fragment, the therapeutic efficacy of a radioimmunoconjugate can depend on tumor vasculature, the total antibody protein dose, and the radiation dose administered (70, 71, 72) . Moreover, for fractionated RIT, morphological and physiological changes in the tumor blood vessels occur after the first administration of the radiolabeled antibody. This can significantly alter the localization of the subsequent dose of antibody (73) . Adams et al. (72) performed a study to compare the effect of dose escalation (50 µg to 1000 µg) and repeated i.v. administrations on the tumor localization of divalent scFvs. They demonstrate that a highly specific localization can be maintained with multiple i.v. bolus injections of divalent scFv administered 24 h apart. We believe that the increased therapeutic effect seen in the present study by fractionating 1000 µCi dosage to four doses of 250 µCi each, given 24 h apart, helps in overcoming the rapid clearance of the scFvs and in maintaining a better tumor localization. Nevertheless, the fractionated doses cannot be considered as additive because the irradiation-induced vascular changes can be significant for the tumor.
The tumor dosimetry showed: single administration of [sc(Fv)2]2 resulted in 4-fold higher radiation dose as compared with sc(Fv)2; the radiation dose with fractionated therapeutic scheduling of sc(Fv)2 was equivalent to single administration of [sc(Fv)2]2; and dose fractionation of [sc(Fv)2]2 further increased (5-fold) the tumor radiation absorbed dose. No radiation-related problems are anticipated on important target organs like the liver and kidneys based on the radiation dose calculations.
One of the major concerns of therapeutic strategies based on multiple dose scheduling is the development of human antimouse antibodies. ScFvs should have reduced immunogenicity because they do not contain CH2 and CH3 domains of intact immunoglobulins, or the CH1 or CL domains (responsible for antiallotype responses) found in Fab' or F(ab')2 fragments. We have recently developed a hu/muCC49 scFv containing the human subgroup IV germ-line VL and variable region of the murine CC49 heavy chain (74) . In vivo tumor-targeting studies showed similar biodistribution and pharmacokinetic properties of the shuffled and completely murine scFv (74) with possible implications in the reduction of human antimouse antibody responses in patients.
Although in the present study only a partial tumor regression occurred,
a definitive advantage of multivalency and dose fractionation was
noticed. There are increasing reports where either a partial or a
complete ablation of xenografted solid tumors have been shown using RIT
in conjunction with chemotherapy (75)
, radiotherapy
(76
, 77) , or blood flow-modifying agents
(78)
. Recently Behr et al. (79)
demonstrated that -emitters like 213Bi hold
advantage over conventional low linear energy transfer radionuclides
like 90Y in curing solid tumors with antitumor
Fab' fragments. Also, pretargeting RIT approaches that use either
radiolabeled bivalent haptens with bispecific antibody
(80)
or biotin-streptavidin as receptor-ligand pair
(81)
have shown improved cure rates. Studies are under way
to try further dose escalation and use of pretargeting approaches to
improve the therapeutic efficacy of the multivalent CC49 scFvs.
In summary, we investigated the potential of the genetically engineered, multivalent scFvs of MAb CC49 as candidates for RIT of colon carcinoma. Multiple administrations of the radiolabeled modality showed higher tumor:normal tissue ratios in a shorter time without normal tissue toxicity, leading to a statistically significant reduction in tumor progression and prolonged median survival times. Subsequent studies may combine the advantages of multivalency and pharmacokinetic properties of divalent and tetravalent scFvs with dose-fractionation administration to determine an optimum therapeutic treatment and improved diagnosis of cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the United States
Department of Energy (DE-FG02-95ER62024) and NIH (P5O CA72712 and RO1
CA78590). 
2 To whom requests for reprints should be
addressed, at Department of Biochemistry and Molecular Biology, Eppley
Institute for Research in Cancer and Allied Diseases, University of
Nebraska Medical Center, 984525 Nebraska Medical Center, Omaha, NE
68198-4525. Phone: (402) 559-5455; Fax: (402) 559-6650; E-mail: sbatra{at}unmc.edu
3 The abbreviations used are: RIT,
radioimmunotherapy; scFv, single chain Fv; sc(Fv)2,
covalent divalent scFv; [sc(Fv)2]2,
noncovalent tetravalent scFv; HPLC, high performance liquid
chromatography; MAbs, monoclonal antibodies; SPR, surface plasmon
resonance; BSM, bovine submaxillary gland mucin; %ID/g, % of injected
dose/g; RI, radiolocalization index; VL, variable light
chain; VH, variable heavy chain.
Received 7/26/00; revised 10/18/00; accepted 10/18/00.
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-interferon treatment. Cancer Res., 55(Suppl.): 5925s-5928s, 1995. versus low-LET ß emitters in radioimmunotherapy of solid tumors: therapeutic efficacy and dose-limiting toxicity of 213Bi- versus 90Y-labeled CO17–1A Fab' fragments in a human colonic cancer model. Cancer Res., 59: 2635-2643, 1999.This article has been cited by other articles:
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C. R. Divgi, J. A. O'Donoghue, S. Welt, J. O'Neel, R. Finn, R. J. Motzer, A. Jungbluth, E. Hoffman, G. Ritter, S. M. Larson, et al. Phase I Clinical Trial with Fractionated Radioimmunotherapy Using 131I-Labeled Chimeric G250 in Metastatic Renal Cancer J. Nucl. Med., August 1, 2004; 45(8): 1412 - 1421. [Abstract] [Full Text] [PDF]
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) and sc(Fv)2 (
) were eluted as single peaks of
Mr 
), sc(Fv)2 (•), and
[sc(Fv)2]2 (
) in groups of athymic mice
bearing LS-174T human colon carcinoma xenografts. The results are
%ID/g of tissue ± SD; n = 6.
). The control group (•)
received injections with PBS (pH 7.4). Arrow represents
the day of initiation of therapeutic regimen, i.e. day
6.