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J. of Nuclear Sci. and Tech.
5319-101389No. 53, pp. 10-19, 2010
F(ab)2PR81m
Tc99
*1232452
2
421- -45156-58145:
-2 -14395-836:
3- 14115-331:
-4 14115-331:
-5 19969-11151:-
.:
2F(ab')PR81mTc
99
PR81. )(120('2F(ab. A.
2F(ab')mTc99.1/52/65%HYNIC
2/51/70% . BALB/c. 4
('2F(ab.PR81.
:PR812F(ab
)
-m
99
Production, Quality Control and Biodistribution Study of F(ab')2 Fragment ofAntibody PR81 Labeled with
99mTc for Diagnosis of Breast Carcinoma Bearing Mice
M. Salouti*1, M.H. Babaei
2, H. Rajabi
3, H. Forootan
2, M.J. Rasaee
4, A. Bitarafan Rajabi
5, F. Johari Daha
2,
M. Mazidi2, J. Mohammad Nejad4, M. Shafiee2
1- Department of Biology, School of Sciences, Islamic Azad University, Zanjan Branch, Postcode: 45156-58145, Zanjan - Iran2- Radioisotopes Department, Nuclear Science Research School, Nuclear Science and Technology Research Institute, AEOI, P.O. Box: 14395-836, Tehran - Iran
3- Department of Medical Physics, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box: 14115-331, Tehran - Iran4- Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box: 14115-331, Tehran - Iran
5- Department of Nuclear Medicine, Rajaei Cardiovascular, Medical and Research Center, Postcode: 19969-11151, Tehran - Iran
Abstract:Immunoglobulin fragments offer a number of advantages for intratumor penetration over theintact antibodies. The goal of this study was to prepare F(ab')2 fragmants from antibody PR81 and
99mTclabeling in order to produce a biological radiopharmaceutical for imaging of breast cancer in nuclearmedicine. The antibody PR81 was digested with 5% (w/w) pepsin at different times to find the best timefor a complete digestion. The prepared F(ab')2 fragments were purified by protein A columnchromatography. The immunoreactivity of the complex assessed by radioimmunoassay was determinedto be 65.2% 5.1. 99mTc radiolabeling of F(ab')2 fragment by using HYNIC as a chelator and tricine as aco-ligand resulted in a radiochemical purity of 70.1%5.2 after 1 hr with a high stability. The complexwas purified by the gel chromatography method to increase the radiochemical purity. Biodistribution andimaging studies in BALB/c mice with breast tumor xenograft demonstrated as a specific localization ofthe compound at the site of tumors 4 h post injection with high sensitivity and minimum accumulation innon target organs. The results showed that F(ab')2 fragment of PR81 is a promising candidate forradioimmunoscintigraphy of the human breast carcinoma.
Keywords:Breast Carcinoma, Radioimmunoscintigraphy, Antibody PR81, F(ab)2, Fragment,99m
Tc Labeling
:[email protected]*email14/6/89:1/9/88:
10
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531389
11
1-
22.
70%
]1[.PR81
MUC1
]2[.mTc
99
]36[.PR81
)1()HAMA(]7[.
-.
]8
12[.2F(ab')
PR81mTc
99
.
2-
2-1
T
hIgGSigma
:
FCS
RPMI 1640
)SDS(G-25
)DMSO(A
2/0
m
Tc99
I125
HYNIC
MCF-7
BALB/c
.
2-2
Omnifuge 2 RS/ Heraeus sepatech
EG & G/ ORTEC/ Model 4001 M
Gallenkamp
Nikon/Diaphot
Sartorius Ag Gottingen/PB 110s/ Max 110g,d=0.
UV/VIS
752 UV/VIS Grating
CO2/Water-Jacketed IncubatorNeuAir
FPLC (Fast Protein Liquid Chromatography)
Pharmacia LKB Biotechnology
Ependorf
Mediso, NuclineTM SPIRIT
pHM225CORNING
.
2-3
BALB/c
.
BALB/c.
]13[.
2030 810
.
222
.
.
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. . .
12
2-4PR81
PR81
23.
37. 9)FCS10(%
) )
)(RPMI
FCS20%.
.
PR81
.
.
.
20-
.
2-5PR812F(ab')
PR811/0
C4.pH
2
.2/4
mg/ml2120)(
C37
.121620242832
SDS-PAGE
.
Tris28=pH025/0
.
.
2F(ab')PR81
A
SDS-PAGE1417[[.
2-62F(ab')
MCF7)cells/ml107(
106
5106
5/2106
25/1106
625/0106
31/0
PBS)1%(BSA
1)3.(2F(ab')
I125
.]18[
5/0ng/ml40
)cpm10000(.
4 .
rpm20005.
.Lineweaver-Burk)
(
]19[.
2-72F(ab')
PR81mTc99
HYNIC
mTc
99]20[.)
(1/05/8=pH
HYNIC30.
50
4 24
)(.
2/5=pH.
.
mTc
99 .
-mTc99Tricine -HYNIC2F(ab')
20
.
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2-8
2F(ab')mTc
99
]212223[.
2-8-1
ITLC-SG.
cm14cm1
cm5/1)l5(.
cm10..
cm1
.
.5/0124
6824 .
.
2-8-2 ('F(ab2
2437
.FPLC)Superose 12 HR 10/30(
01/07=pH
.1002F(ab')
g/ml1
ml/min1
1.
.
2-8-32F(ab')
BALB/c
53BALB/c
.
)3(12F(ab')
mTc
99HYNIC
)
1015 20030048.)100200
24
...
.)ID/g%(
.
9-2
2-8-3
4824
)20(.
3-
3-1
SDS-PAGE
PR81120)(28
).1(IgG 2F(ab').
.
2F(ab')SDS-PAGE95%
)1.(
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. . .
14
-1
PR81 .:
)MW(:2F(ab')hIgG
:PR81
: 12162024
2832 .
3-22F(ab')
2F(ab')PR81
1/52/65%.
253/1
)2.(
3-3
ITLC-SG
5/01246824
.
%1/702/5
24
3.
2F(ab')
.
ITLC-SG
.1/593%
2-
MCF7.
3-2F(ab)PR81mTc
99
HYNIC24.
3-4
555%
.
)4.(
50
60
70
80
90
100
0 5 10 15 20 25
LabelingEfficiency(%)
Time Post Labeling (h)
y = 0.3075x + 1.5333
R2 = 0.9986
0
1
2
3
4
5
6
7
8
0 5 10 15 20
Totalactivity
/BondActivity
1 / Cell * 10E6
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-4 2F(ab)-HYNIC-mTc99 FPLC24 37
.
3-52mTc-HYNIC-F(ab')
99
BALB/c
52mTc-HYNIC-F(ab')99
BALB/c4824
.
4 .
6-3
BALB/c4
.
678BALB/c4824 .
-4
PR81IgG1 (k)
]2[.
PR81mTc
99
]36
[.
5-2F(ab)-HYNIC-mTc
99
ID/g)BALB/c%)4824 .
6- )()(BALB/c
4 2F(ab)-HYNIC-mTc99.
7- )()(BALB/c
8
2F(ab)-HYNIC-
mTc99
.
0
24
6
8
10
12
14
16
18
20
Blood
Liver
Kidney
Lung
Spleen
Heart
Stom
ach
Intestine
Colon
Sternum
Thyroid
Brain
Muscle
Bone
Pancreas
Skin
Bladder
Tum
or
Mean 4
Mean 8
Mean 24
%I
D/g
0
4000
8000
2000
6000
0 5 10 15 20 25 30
CountPerMinute
Fraction Number (ml)
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. . .
16
8- )()(BALB/c
24 2F(ab)-HYNIC-mTc99.
PR81
)HAMA(
]7[.PR81
2F(ab')
1415[23[. -
-mTc99
-]24[.
mTc99.]25[
2F(ab')]26[.
mTc
99
HYNIC
HYNIC. EDDA
mTc
99.
.]27[2F(ab')PR81
mTc
99
2/51/70%.
mTc
99
.
.
.
.
2F(ab')
.
]28[.
BALB/c
]13[. ('2F(abPR81
.
4824
2F(ab')-HYNIC-mTc
99
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4 .
24 .4
-mTc
9945
)
(15
.
4824
2F(ab')-HYNIC-mTc
99.
)(
.
5/12]15[.
445/066/2.1
..
]29[.
1-
4824 2F(ab)-HYNIC-mTc99
/4
/8
/24
05/226/288/1
12/
371/
053/
0
87/260/029/0
59/209/222/1
66/242/198/0
45/088/071/0
]56[
BALB/c
.
4 24
mTc99-
.SPECT
Planar
planar.MetzWienerButterworth
. .
.
2F(ab')PR81
HAMAPR81
.
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. . .
18
:
1- Human Anti-Murine Antibody Response
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