Download Chemical Engineering of the Monoclonal

[CANCER RESEARCH SI. 4310-4315. August 15. 1991]
Chemical Engineering of the Monoclonal Antibody A7 by Polyethylene Glycol for
Targeting Cancer Chemotherapy1
Kazuya Kitamura,2 Toshio I akahashi, Toshiharu Yamaguchi, Akinori Noguchi, Akira Noguchi,
Ken-ichiroh Takashina, Hiroshi Tsurumi, Masao Inagake, Tatsushi Toyokuni, and Sen-ichiroh Hakomori
First Department of Surgery, Kyoto Prefectura! University of Medicine, 465 Kawaramachi-hirokoji, Kamikyo-ku, Kyoto, 602 Japan, and The Biomembrane Institute,
Seattle, Washington 98104
and immunogenicity
ABSTRACT
The murine monoclonal antibody A7 (Mah A7) against human colon
cancer was chemically modified with methoxypolyethylene
glycol (PEG)
(M, 5000). A high substitution of PEG molecules on Mali A7 produced
a progressive reduction in antibody-binding activity. The pharmacokinetic
and immunological properties of PEG-modified monoclonal antibody A7
(Mab A7) and the PEG-modified F(ab')2 fragment, which retained their
antibody-binding activity, were assessed and compared with the parent
Mab A7 and the parent F(ab')2 fragment. Blood clearance of PEGmodifÃ-edantibodies appeared to be diminished by PEG modification and
was fitted by a two-compartment model. Low PEG-substituted Mab A7
showed less organ uptake in the liver and spleen and similar uptake in
the lung and kidney, compared with the parent Mab A7. PEG-F(ab')2
showed less uptake in the liver and kidney. Both preparations exhibited
less tissuerblood ratios in all resected organs as compared with parent
antibodies. Tumor localization was enhanced by PEG modification for
the F(ab')2 fragment, but not by PEG modification for the whole Mab
A7. Multiple i.v. administration
of PEG-modified
antibody to rabbit did
not appear to elicit a measurable immune response to the antibody portion
of the conjugate. In conclusion, PEG-modified antibodies are promising
reagents as drug carriers to the target tumor.
INTRODUCTION
The use of antibodies as a carrier of pharmacological agents
has become more practical since the advent of monoclonal
antibody technology, and several studies have demonstrated the
potential use of ¡mmunoconjugates in clinical application (15). We have reported immunotargeting chemotherapy using a
murine monoclonal antibody A7-drug conjugate for colorectal
carcinoma patients (6). One problem that has been encountered
clinically with the use of murine monoclonal antibodies is the
development of an immune response to the administered anti
body. This immune response has led to changes in antibody
pharmacokinetics and may lead to more serious problems such
as anaphylaxis (7). Thus, our study focused on chemical modi
fication of murine monoclonal antibody to reduce the murine
immune response.
Modification of proteins with a straight chain amphiphilic
polymer, methoxypolyethylene glycol, has been shown to elim
inate the immunogenicity of the proteins and increase their
circulating half-lives in vivo, resulting in augmentation of their
biological activity (8-15). We esterized methoxypolyethylene
glycol with yV-hydroxysuccinimide by activation of the hydroxy
group. Then we attached methoxypolyethylene glycol to Mab
A7 and its F(ab')2 fragment in order to eliminate the antimurine
of PEG'-modified
monoclonal antibody
A7.
MATERIALS AND METHODS
Reagents and Animals. Methoxypolyethylene glycol (M, 5000), dicyclohexylcarbodiimide, ¿V-hydroxysuccinimide,and glutaric anhydride
were purchased from Aldrich (Milwaukee, WI). Toluene, dicycloethane,
dry pyridine, diethyl ether, chloroform, dimethylformamide, benzene,
and petroleum ether were obtained from Nakarai Chemical, Ltd.
(Kyoto, Japan). All the reagents used were analytical grade. Female
BALB/c mice, BALB/c-nw/nu athymic mice and rabbits were obtained
from Shimizu, Inc. (Kyoto, Japan).
Preparation of Methoxypolyethylene Glycol /V-Hydroxysuccinimidylglutarate. Methoxypolyethylene glycol jV-hydroxysuccinimidylglutarate
was prepared in two steps. The first step followed the procedure of
Joppich and Luisi (16). Fifty g of PEG were dissolved in 80 ml of
toluene and distilled. After cooling, the solution was reacted with
glutaric anhydride (11.5 g) in dicycloethane (200 ml) and dry pyridine
(5 ml). The mixture was refluxed under nitrogen gas for 3 days. After
filtration and evaporation of the solvent, the residue was dissolved in
100 ml of water and washed twice with 50 ml of diethyl ether. PEGglutarate was then extracted from the water phase with two 50-ml
chloroform washes. Evaporation of the chloroform yielded about 38 g
of PEG-glutarate.
The second step followed a modified version of the procedure de
scribed by Anderson et al. (17). The PEG-glutarate was dissolved in
200 ml of dimethylformamide at 37°Cand was reacted with 0.94 g of
A'-hydroxysuccinimide. The solution was mixed with 1.7 g of dicyclohexylcarbodiimide in dry dimethylformamide and stirred vigorously in
an ice bath. The mixture was then stirred at room temperature for 24
h. Precooled benzene (100 ml) was added, and methoxypolyethylene
glycol iV-hydroxysuccinimidyl glutarate was precipitated by the dropwise addition of 200 ml of petroleum ether at 0°C.The precipitate was
collected on a sintered glass filter. Dissolution in benzene and precipi
tation with petroleum ether was repeated three times. NHS-PEG was
stored in a desiccator at -20°C.
Production of Monoclonal Antibody A7 and Its F(ab')2 Fragment. The
murine monoclonal antibody A7 which recognizes the M, 45,000 glycoprotein in human colon cancer (18) was developed as described by
Kotanagi et al. (19). The F(ab')2 fragment was produced by the pepsin
digestion of Mab A7 as described in a previous report (20). In brief, the
Mab A7-secreting hybridoma was injected i.p. into BALB/c mice and
then ascitic fluid was collected. Mab A7 was isolated from the fluid by
protein A ligand chromatography (Bio-Rad, Richmond, CA). The
F(ab')2 fragment of Mab A7 was produced by the pepsin digestion.
Pepsin was purchased from Sigma Chemical Co. (St. Louis, MO).
Incubation was performed at 37°Cfor 8 h in 100 mM acetate buffer
reaction and to increase tumor localization. The present report
describes the chemical properties, in vivo pharmacokinetics,
(pH 3.6) with an pepsin:antibody ratio of 1:50. These conditions were
considered optimal. The fragment preparation was dialyzed against
PBS and purified by gel filtration using a Sephacryl S-200 column
(Pharmacia, Uppsala, Sweden). Mab A7, the F(ab')2 fragment, LPEG-
Received 2/19/91: accepted 6/5/91.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' This work was supported in part by Grants-in-Aid for Cancer Research from
the Ministry of Education, Science, and Culture and from the Ministry of Health
and Welfare, Japan.
3To whom requests for reprints should be addressed.
3The abbreviations used are: PEG, methoxypolyethylene glycol; Mab A7,
monoclonal antibody A7; PBS. phosphate buffered saline; NHS-PEG, methoxy
polyethylene glycol jV-hydroxysuccinimidylglutarate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; LPEG-A7, low PEG substituted
Mab A7; MPEG-A7. medium PEG substituted Mab A7; HPEG-A7. high PEG
substituted Mah A7: %ID, percentage of injected dose/g of tissue; ELISA,
enzyme-linked ¡mmunosorbent assay; TNBS, trinitrobenzene sulfonate.
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POLYETHYLENE
GLYCOL MODIFICATION
A7, and PEG-F(ab')2 were radioiodinated with '"I (Amersham Inter
national, Amersham, United Kingdom) by the chloramine-T method
(21). The specific activity of Mab A7 and PEG-A7 was 6 x IO6 cpm/
jig, and that of the F(ab')2 fragment and PEG-F(ab')2 was 9 x IO6cpm/
Mg.
Attachment of PEG to the Antibody. NHS-PEG was reacted to a free
amino group of Mab A7 with NHS-PEG:antibody ratios of 1:15, 1:30,
and 1:50 in 0.1 M PBS (pH 7.5). The solution was stirred for 30 min
at room temperature, and any remaining activated ester was removed
by reaction with excess f-aminocaproic acid for 5 min. Unbound NHSPEG was removed from the reaction mixture by molecular-exclusion
chromatography on P-100 (Bio-Rad) in 0.1 M PBS (pH 7.5). The
protein concentration of PEG-modified antibody was determined from
its absorbance at 280 nm. PEG does not absorb at 280 nm. The
attachment of PEG to Mab A7 was confirmed by SDS-PAGE (26).
The gel was stained with Coomassie Blue R-250 (Nakarai Chemical,
Ltd., Kyoto, Japan). The degree of amino group modification in the
antibody molecule was determined by measuring the number of free
amino groups with TNBS (23).
In a separate experiment, NHS-PEG was added at a stoichiometric
molar ratio of 10 to a solution of F(ab')2 in 0.1 M PBS (pH 7.5). The
OF Mah A7
four rabbits. The resulting rabbit antimurine response was measured by
using a ELISA as described by Shawler et al. (4). Microtiter plate wells
were coated by exposure to 1 ¿ig
of the parent Mab A7 in 0.01 M PBS
(pH 7.5) for 2 h at 37°Cand washed thoroughly. Nonspecific binding
sites were blocked using a 5% solution of bovine serum albumin in 0.01
M PBS, (pH 7.5). Wells were incubated with serial dilutions of rabbit
sera for l h at 37°C,washed thoroughly, and then exposed to peroxidase-conjugated IgG fraction of goat anti-rabbit IgG (Cappel, West
Chester, PA). The color was developed with the addition of 1 mg/ml
o-phenylenediamine (Dakopatts, Glostrup, Denmark) in 0.1% (v/v)
hydrogen peroxide. Color development was quantified by measuring
the absorbance at 492 nm with a commercially available ELISA reader.
Titers were defined as the reciprocal of the highest dilution of rabbit
sera resulting in 50% of the maximum response.
RESULTS
Chemical Characterization of PEG-Antibody Conjugates. The
attachment of PEG to the antibody was confirmed by SDSPAGE. Under the conditions described in "Materials and Meth
ods," NHS-PEG:antibody molar ratios of 1:15, 1:30, and 1:50
next procedure was done as described above.
Antibody Binding Activity of PEG-Antibody Conjugates. The antibody
binding activities were determined by a competitive radioimmunoassay
using a human colon cancer cell line, SW1116. Aliquots of SW1116
cells (2 x IO5)were incubated with '"I-labeled Mab A7 (1 x IO5 cpm)
produced low, moderate, and high migration bands on SDSPAGE, respectively. We have designated these samples LPEGA7, MPEG-A7, and HPEG-A7, respectively (Fig. 1). Mab A7
and its F(ab')2 fragment were estimated to have 55 and 30 free
in the presence of serially diluted antibodies or PEG conjugates (power
of 1/5) in PBS at room temperature for 60 min. The concentration of
antibody ranged between 1 and (1/5)5 mg/ml for Mab A7, LPEG-A7,
MPEG-A7, and HPEG-A7 and 1/5 and (1/5)7 for F(ab')2 and PEGF(ab')2. The each reaction was performed in triplicate. After centrifu-
amino groups by TNBS assay, respectively. TNBS assay also
revealed that for LPEG-A7, MPEG-A7, and HPEG-A7, 5, 10,
and 15 PEG molecules were substituted on each Mab A7,
respectively (Table 1). The F(ab')2 fragment was reacted with a
NHS-PEG:antibody molar ratio of 1:15 and the resultant con
jugate had a substitution rate of 5 PEG molecules/antibody.
gation, the cell pellets were subjected to y counting and the percentage
inhibition was calculated as compared to the control.
Blood Circulation of PEG Conjugates. '"[-Labeled LPEG-A7 or '"!labeled parent Mab A7 (5 x IO6cpm) was injected i.v. into four BALB/
B
c mice of each group. Blood samples were drawn from the tail vein into
heparinized capillary tubes at various times after injection. The radio
activity of blood samples was measured with a gamma counter. Results
were expressed as a percentage of blood concentration at various times,
compared with that at 15 min after i.v. injection. The level of the
antibodies in the blood were analyzed using a two-compartment open
pharmacokinetic model. The analysis was performed using the nonlin
ear least-squares program SIMPLEX 4 (24).
In a separate experiment, '"I-labeled PEG-F(ab')2 or '"I-labeled
parent F(ab')2 (5 x IO6 cpm) was injected into four BALB/c mice of
D
each group and the next procedure was performed as the same manner
as described above.
Tissue Distribution of PEG-Antibody Conjugates. BALB/c athymic
mice, which were inoculated s.c. with SW1116 cells (5 x IO6),developed
a palpable tumor 2 weeks after inoculation. '"I-labeled LPEG-A7 or
the '"I-labeled Mab A7 was injected i.v. into four mice of each group.
Blood, tumor, and visceral organs were resected and subjected to y
counting to measure radioactivity. Visceral organs and tumor distribu
tions were examined for each preparation with the values expressed as
a %ID and tissue:blood ratio. Normal organ distribution of the two
preparations was compared as a %ID and tissue:blood ratio 24 h after
administration. Tumor localizations of the two were compared as %ID
at 1, 3, 5, and 7 days after injection.
In a separate experiment, tissue localization of PEG-F(ab')2 was
compared with that of the parent F(ab')2 in the same manner as
described above. The '"I-labeled F(ab')2 fragment or '"I-labeled PEGF(ab')2 was injected i.v. into four nude mice of each group. The data
were expressed as a %ID and a tissue:blood ratio. Normal organ
distributions of the two preparations was compared as a %ID and
tissue:blood ratio 12 h after administration. Tumor localization of the
two was compared as %ID at 6, 12, 36, and 60 h after injection.
Evaluation of the Immunogenicity of the Parent Mab A7 and the
MPEG-A7. Immunogenicity was evaluated by the i.v. injection of 1 mg
of each preparation three times at weekly intervals in two groups of
150K
Fig. 1. SDS-PAGE analysis of the parent Mab A7 and its PEG conjugates.
Solutions of the parent Mab A7 and PEG-modified antibodies were subjected to
SDS-PAGE (7.5%) in the absence of reducing agent. The gel was stained with
Coomassie Blue R-250. A, LPEG-A7; B, MPEG-A7; C, HPEG-A7; D, the parent
MabAV.
Table 1 Attached PEG molecules per antibody
The number of PEG molecules substituted on Mab A7 was estimated using a
TNBS assay (24). Data were expressed as mean triplicate measurement.
Attached PEG/antibody
LPEG
MPEG
HPEG
PEG-F(ab')2
5.1
9.8
14.8
4.9
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POLYETHYLENE
GLYCOL MODIFICATION
OF Mah A7
33.6 and 38.1 h, respectively (Fig. 3A). Those of F(ab'>2 and
PEG-F(ab')2 were tv,a of 3.6 and 5.6 h and tvßof 26.7 and 33.6
h, respectively (Fig. 3Ä).
Tissue Distribution. The tissue distributions of LPEG-A7 and
PEG-F(ab')2 were compared to those of the parent antibodies
in nude mice bearing transplantable xenogenic SW1116 tumors
to which the parent antibodies are known to localize (20). The
tumor localization profile of LPEG-A7 was identical to that of
the parent Mab A7 with respect to %ID in the tumor (Fig. 4).
PEG-F(ab')2 had a higher %ID in the tumor when compared
with the parent F(ab')2 fragment at various times after injection
(Fig. 5).
To clarify the specificity, visceral organ distributions of
LPEG-A7 and the parent Mab A7 were examined. The results
in Fig. 6/4, show %ID 24 h after injection. A higher %ID in the
liver and spleen was found for the parent Mab A7 compared to
LPEG-A7. There was no difference in kidney and lung distri
bution between the parent Mab A7 and LPEG-A7. Tissue:blood
ratios were higher for the parent Mab A7 than for the LPEGA7 in all resected organs (Fig. 6B). Additionally, the tissue
distribution of PEG-F(ab')2 was compared with that of the
parent F(ab')2 fragment with respect to %ID (Fig. 1A) and a
Antibody Concentration (mg/ml)
100
90.
80
TO
60
tissue:blood ratio 12 h after injection (Fig. IB). A higher %ID
in the liver and kidney was found for the parent F(ab')2 frag-
SO
!
I
40
30
20
10
fiV
IsJ
fit
UJ
fi)4
liY
(iV
fi)1
Uj
UJ
UJ
UJ
100908070-
Antibody Concentration img/mll
Fig. 2. Antibody-binding activity of PEG-modified antibodies. SW1 116 cells
were incubated with 1 X 10' cpm of '"1-labeled Mah A7 containing various
concentrations of antibodies and their PEG conjugates (diluted with power of I/
5). After washing and centrifugaron, the cell pellets »eresubjected to 7-counting
for the measurement of radioactivity. A, the parent Mab A7 (O). LPEG-A7 (•),
MPEG-A7 (A), HPEG-A7 (D). B. the parent F(ab')2 fragment (•).PEG-F(ab')2
60-
1
5040302010-
P).
0
12
24 hr
Antibody Binding Activities of PEG-Antibody Conjugates. The
antibody binding activities of LPEG-A7, MPEG-A7, and
HPEG-A7 were each compared to the parent Mab A7 by a
competitive radioimmunoassay. The results showed that while
LPEG-A7 retained the antibody-binding activity of parent Mab
A7, the binding activity of HPEG-A7 markedly reduced and
that of MPEG-A7 was reduced somewhat (Fig. 2A). In a
separate experiment, the binding activity of PEG-F(ab')2 was
examined in the same manners as described above. Fig. 2B
shows that PEG-F(ab')2 retained a binding activity nearly iden
tical to that of the parent F(ab')2 fragment.
Blood Clearance. Of the four PEG-antibody preparations
characterized thus far, LPEG-A7 and PEG-F(ab')2 were tested
for pharmacokinetics. These conjugates were selected because
they had the ability to preserve their antibody-binding activity.
Blood clearances of LPEG-A7 and PEG-F(ab')2 were each
compared to that of their respective parent, Mab A7 or the
F(ab')2 fragment. Fig. 3 shows the pharmacokinetic profiles of
LPEG-A7, the parent Mab A7, PEG-F(ab')2 and the parent
F(ab')2 fragment. In each case, an initial rapid decline was
followed by a much slower phase of decline. The observed
values closely fit the computer-generated curve for a two-com
partment system for 24 h of observation. Blood half-lives of
Mab A7 and LPEG-A7 were tv,a of 3.6 and 5.4 h and tv,ß
of
24 hr
Fig. 3. Blood circulation of PEG conjugates. '"I-labeled Mab A7, the '"Ilabeled F(ab')2 fragment, and their radiolabeled PEG conjugates (5 x 10' cpm,
each) were injected i.v. into four BALB/c mice of each groups. Blood samples
taken from the tail vein were subjected to i-counting for radioactivity. Data were
expressed as a percentage of blood concentration 15 min after injection. Points,
mean; bars, SE. A, Mab A7 (•),LPEG-A7 (D). The t*a values of Mab A7 and
LPEG-A7 equal 3.6 and 5.4 h, respectively. The rtt/3 values of Mab A7 and
LPEG-A7 equal 33.6 and 38.1 h, respectively. B, F(ab')2 fragment (•),PEGF(ab')2 (O). The frt«values of Mab A7 and LPEG-A7 equal 3.6 and 5.6 h,
respectively. The tv,ß
values of Mab A7 and LPEG equal 26.7 and 33.6 h,
respectively.
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POLYETHYLENE
GLYCOL MODIFICATION
100 •
1
3
5
7 day
Fig. 4. Tumor localization of the parent Mab A7 and LPEG-A7. '"I-labeled
Mab A7 and '"I-labeled LPEG-A7 (5 x 10' cpm, each) were injected i.v. into
four nude mice of each group. Mice were sacrificed at 1,3, 5, and 7 days after
injection and tumors were resected and weighed. The tumors were then subjected
to 7-counting for radioactivity. Data were expressed as a %ID. Points, mean;
bars, SE. Mab A7 (O), LPEG-A7 (A).
OF Mah A7
molar ratio of activated PEG to antibody, yielding conjugates
with an average of 5 to 20 molecules of PEG per antibody. An
initial stoichiometric molar ratio greater than 100:1 generates
the formation of high molecular weight polymeric aggregation.
We produced antibodies with low, moderate, and high degrees
of PEG substitution on Mab A7 (LPEG-A7, MPEG-A7,
HPEG-A7) and examined their antibody-binding activities. The
result showed that the antibody binding activity to antigenpositive cells was retained by LPEG-A7, lost by HPEG-A7, and
partially lost by MPEG-A7. The loss of antibody-binding activ
ity after extensive chemical modification suggests that lysine
residues may be involved at the antigen-binding site of the
antibody.
Since increasing the extent of PEG modification excessively
caused a progressive reduction in the antibody-binding activity,
LPEG-A7 and PEG-F(ab')2 were tested for the pharmacokinetics and tissue distributions. As was the case with some
enzymes, PEG attachment to Mab A7 or its F(ab')2 was shown
to markedly increase the circulatory half-life of the antibody.
I
I
SPLEEN
20
60 hr
40
Fig. 5. Tumor localization of the F(ab')2 fragment and PEG-F(ab')2. The 12!1labeled F(ab')2 fragment and '"I-labeled PEG-F(ab')2 (5 x 10' cpm, each) were
LIVER
injected i.v. into four nude mice of each group. Mice were killed at 6, 12, 36. and
60 h after injection and resected tumors were weighed, the tumors were then
subjected to -y-counting for radioactivity. Data were expressed as a %ID. Points,
mean; ears, SE. F(ab')2 fragment (O), PEG-F(ab')2 (•).
TUMOR
ment. The ratios were found to be higher for the parent F(ab')2
fragment in all resected organs.
Immunogenicity of PEG-modified Mab A7. The immunogenicity of MPEG-A7 was evaluated by measuring the immune
response. Rabbits were repeatedly immunized with either the
parent Mab A7 or MPEG-A7 as indicated in "Materials and
Methods" and the antimurine immune response was deter
BLOOD
mined by ELISA (Fig. 8). After three i.v. immunizations with
the parent Mab A7 (shown by arrow), the rabbits developed a
marked immune response against the parent Mab A7. In con
trast, when MPEG-A7 was administered i.v., a significant im
mune response was not observed although a slight response was
observed after the second injection. These findings suggest that
PEG modification of murine monoclonal antibodies reduces
the host immune response against the murine antibody.
LUNG
KIDNEY
SPLEEN
LIVER
DISCUSSION
TUMOR
Methoxypolyethylene glycol was esterized with 7V-hydroxysuccinimide by activation of the hydroxy group in two steps
(13). The active ester content was 77% as measured by Nhydroxysuccinimide release (24) (data not shown). This ester
reacts predominantly with free amino groups mainly derived
from the lysine residue of the antibody. The degree of PEG
substitution can be varied by adjusting the initial stoichiometric
0.2
0.4
0.8
1.0
TISSUE TO BLOOD RATIOS
Fig. 6. Tissue distribution of the parent Mab A7 and LPEG-A7. '"I-labeled
Mab A7 and I-labeled PEG-A7 were injected i.v. into four nude mice of each
group which were sacrificed 24 h after injection. Tissues were resected, weighed,
and subjected to 7-counting. Data were expressed as a %ID (A) and a tissue:blood
ratio (B). Bars, SE. Mab A7 (•),LPEG-A7 (m).
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POLYETHYLENE
GLYCOL MODIFICATION
OF Mah A7
Tissue:blood ratios were simultaneously calculated at the same
time points. Because our preliminary experiment have revealed
that tissue accumulation of F(ab')2 fragment and whole Mab
BLOOD
A7 reached maximum 12 and 24 h after injection, respectively.
LPEG-A7 was absorbed less by the liver and spleen than the
parent Mab A7. PEG-F(ab')2 was absorbed less by the liver and
kidney than the parent F(ab')2 fragment. The difference in tissue
distribution between PEG modified antibodies and the parent
antibodies was especially pronounced for the liver. The thera
peutic effect of the immunoconjugate A7-NCS was tested clin
ically on patients who had metastatic colorectal carcinoma in
the liver (6). In addition, radioimmunodetection of liver metas
tasis is being undertaken using whole Mab A7 and its F(ab')2
fragment. LPEG-A7 and PEG-F(ab')2 may be appropriate for
LIVER
targeting cancer chemotherapy or radioimmunodetection
of
liver metastasis, since it is distributed to the normal liver tissue
to a lesser extent and remains in the tumor at a high concentra
tion. Although it is unclear why PEG-modified antibodies ac
cumulate less in normal organs, an inherent property of PEG
or a removal of nonspecific binding to the Fc portion by a
masking effect of PEG may be responsible for this.
In tumor localization studies, LPEG-A7 showed a profile
identical to that of the parent Mab A7, while tumor localization
of the F(ab')2 fragment was enhanced by PEG modification. In
TUMOR
BLOOD
spite of enhanced blood retention, PEG modification did not
increase tumor localization for whole Mab A7. On the other
hand, PEG-F(ab')2 exhibited augmented tumor localization in
comparison with the parent F(ab')2 fragment. It is suggested
LUNG
KIDNEY
that the reason for this discrepancy between whole Mab A7 and
F(ab')2 fragment is the difference in molecular size. In other
words, the target tumor is more accessible to PEG-F(ab')2 since
SPLEEN
it can easily exude through the transcapillary route due to a
smaller molecular size. F(ab')2 fragment is thought to have
LIVER
TUMOR
0.2
0.4
0.6
0.8
1.0
TISSUE TO BLOOD RATIOS
Fig. 7. Tissue distribution of the F(ab')2 fragment and PEG-F(ab')2. 125Ilabeled F(ab')2 fragment and '"I-labeled PEG-F(ab')2 (5 x 10' each) were injected
i.v. into four nude mice of each group. Mice were sacrificed at 12 h after injection,
and the liver, spleen, kidneys, and lungs were resected to be subjected to ycounting. Data were expressed as a %ID and a tissue:blood ratios. Bars, SE.
F(ab')2 (•):PEG-F(ab')2 (E).
Those clearance curves were best fitted to a two-compartment
model. In both antibody preparations, PEG modification en
hanced «and ßhalf-lives of parent antibodies. Lobuglio et al.
(25) have reported that the a half-life represented the equilib
rium of IgG between the intra- and extravascular spaces. Lisi
et al. (26) have shown that PEG-modified proteins are more
resistant to proteolysis than the corresponding unmodified
protein, suggesting that the ßhalf-life is thought to be reflect
the catabolism of immunoglobulin. Therefore, the prolongation
of circulatory half-life by PEG modification may be accounted
for as follows: (a) PEG shields the proteolytic site on the
antibody with resulted in inhibiting catabolism of the antibody;
and (b) PEG modification decreases normal organ uptake of
the antibody.
Normal organ distribution of the parent antibodies and those
PEG conjugates were compared by examining a %ID 12 h for
F(ab')2 fragment and 24 h after injection for whole Mab A7.
rapid blood clearance resulting in less tumor accumulation as
compared with whole antibody. Chemical modification such as
PEG attachment may be especially beneficial for the use of the
F(ab')2 fragment in targeting cancer therapy.
When MPEG-A7 or the parent Mab A7 was repeatedly
injected i.v. into rabbits, the Mab A7-treated group exhibited a
strong antimurine reaction against the protein portion of the
conjugate, while no significant anti-murine antibody formation
was observed in the MPEG-A7-treated group. In this study, the
MPEG-A7 preparation was used instead of the LPEG-A7
I I
P
1
3
3
12345
1
Wwk»aft«rinjection
Fig. 8. Antimurine immune response. One mg of Mab A7 (•)or MPEG-A7
(O) was injected i.v. into three rabbits of each group. Rabbit anti-murine IgG
response was measured by ELISA as described in "Materials and Methods"
Arrows, indicate injection with 1 mg of Mab A7 or MPEG-A7. Titers were
expressed as the reciprocal of the highest dilution of rabbit sera resulting in 50%
of the maximum response. Points, mean. Arrows, infusion of I mg of antibody
preparations.
4314
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POLYETHYLENE
GLYCOL MODIFICATION
preparation since it was impossible to separate the unmodified
Mab A7 from the LPEG-A7 preparation. If the PEG-modified
antibody preparation contains even a small amount of unmod
ified antibody, an antimurine immune response would be in
duced in the same manner as observed in the parent Mab A7treated group. In preliminary experiments, a solid phase sand
wich ELISA revealed that anti-murine rabbit or goat serum
reacted with Mab A7 but not with LPEG-A7. This suggests the
PEG attached to the antibody may hinder a immunogenic
portion of antibody which would cause an antimurine reaction.
These findings indicate that PEG modification can apparently
reduce the host immune response against murine monoclonal
antibodies.
In summary, this report described the chemical modification
of Mab A7 by covalent attachment of PEG to generate conju
gates with low nonspecific uptake, low immunogenicity, and
high tumor uptake. This modified form of the antibody may be
useful in clinical applications of murine monoclonal antibodies.
11.
12.
13.
14.
15.
16.
17.
18.
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Chemical Engineering of the Monoclonal Antibody A7 by
Polyethylene Glycol for Targeting Cancer Chemotherapy
Kazuya Kitamura, Toshio Takahashi, Toshiharu Yamaguchi, et al.
Cancer Res 1991;51:4310-4315.
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