The Atomic Energy Organization of Iran (AEOI) a-10!1!46-25f8902

7/31/2019 The Atomic Energy Organization of Iran (AEOI) a-10!1!46-25f8902

1/10

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

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)DMSO(A

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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

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2-3

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Tris28=pH025/0

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106

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.FPLC)Superose 12 HR 10/30(

01/07=pH

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g/ml1

ml/min1

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5/10

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-1

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)MW(:2F(ab')hIgG

:PR81

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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

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.

678BALB/c4824 .

-4

PR81IgG1 (k)

]2[.

PR81mTc

99

]36

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5-2F(ab)-HYNIC-mTc

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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

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Brain

Muscle

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Pancreas

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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)


7/10

. . .

16

8- )()(BALB/c

24 2F(ab)-HYNIC-mTc99.

PR81

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]7[.PR81

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.


9/10

. . .

18

:

1- Human Anti-Murine Antibody Response

References:

1. .

)1382.(

2. M. Paknejad, M.J. Rasaee, F. Karami, S.Kashanian, M.A. Mohagheghi, K. Omidfar,

Production of monoclonal antibody, PR81,

recognizing the tandem repeat region of MUC1

mucin, Hybridoma and Hybridomics, 22(3):

153-158 (2003).

3. M. Salouti, H. Rajabi, H. Babaei, M.J. Rasaee,R. Najafi, M. Paknejad, Z. Mohammad Hasan,

T.M. Altarihi, 99

mTc direct radiolabeling of

PR81, a new anti-MUC1 monoclonal antibody

for radioimmunoscintigraphy, Iran J. N. M,

23: 7-16 (2005).

4. M. Salouti, H. Rajabi, M.H. Babaei, M.J. Rasaee,R. Najafi, M. Paknejad, Z. Mohammad Hasan,

T.M. Altarihi, J. Mohammad Nejad, A NewMonoclonal Antibody Radiopharmaceutical for

Radioimmunoscintigraphy of Breast Cancer:Direct Labeling of Antibody and Its Quality

Control,DARU; 14(1): 14-19 (2006).

5. M. Salouti, H. Rajabi, M.H. Babaei, M.J. Rasaee,R. Najafi, M. Shafiee, M. Mazidi, Z.M. Hasan, A.

Bitarafan Rajabi, N. Namvar, T.M. Altarihi,Mohammad Nejad, Radioimmunoscintigraphy of

Breast Tumor Xenografts in mouse model by99mTc Direct Radiolabeling of a Monoclonal

Antibody PR81, The Iranian Journal of MedicalPhysics, 2(8): 27-34, 60 (2005).

6. M. Salouti, H. Rajabi, M.H. Babaei, M.J.Rasaee, R. Najafi, M. Shafiee, M. Mazidi, Z.M.

Hasan, A. Bitarafan Rajabi, N. Namvar, T.M.

Altarihi, Mohammad Nejad,

7. S. Potamianos, A.D. Varvarigou, S.C.Archimandritis, Radioimmunoscintigraphy

and radioimmunotherapy in cancer: principles

and application, Anticancer Research, 20:

925 948 (2000).

8. M. Mariani, M. Camagna, L. Tarditi, E.Seccamani, A new enzymatic method to obtain

highyield F(ab')2 suitable for clinical use frommouse IgG1, Molecular Immunology, 28(1/2):

69-77 (1991).

9. D.W. Rea, M.E. Ultee, A novel method forcontrolling the pepsin digestion of antibodies,

JIM, 157: 165-173 (1993).

10.S. Demignot, M.C. Garnett, R.W. Baldwin,Mouse IgG2b monoclonal antibody

fragmentation. Preparation and purification ofFab, Fc and Fab/c fragments, JIM, 121: 209-217(1989).

11.D.E. Milenic, J.M. Esteban, D. Colcher,Comparison of methods for the generation of

immunoreactive fragments of a monoclonal

antibody (B72.3) reactive with human

carcinomas, JIM, 120: 71-83 (1989).

12.R. Kurkela, L. Vuolas, P. Vihko, Preparationof F(ab')2 fragments from monoclonal mouse

IgG1 suitable for use in radioimmunoimaging,JIM, 110: 229-236 (1988).

13.T. Ghazanfari, Z. Mohammad, R. Yaraie,Recognition and determination of

immunogenicity of a spontaneous tumor in a

BALB/c mouse, Daneshvar, 24: 65-72 (1998).

14.B.A. Rhodes, D.A. Torvestad, K. Breslow,99mTc labeling and acceptance testing of

radiolabeled antibodies and antibody fragments.

In: Burcheil SW, Rhodes BA, editors. Tumor

Imaging. New York: Mason, 111 (1982).

15.M.H. Babaei, R. Farshidfar, R. Najafi,Evaluation of tumor targeting with radiolabeledF(AB')2 fragment of a humanized monoclonal

antibody, DARU, 10(2): 49-53 (2002).

16.T.M. Behr, S. Memtsoudis, R.M. Sharkey, R.D.Blumenthal, R.M. Dunn, S. Gratz,

Experimental studies on the role of antibody

fragments in cancer radio-immunotherapy:

influence of radiation dose and dose rate on

toxicity and anti-tumor efficacy, Int. J. Cancer,77: 787-795 (1998).


10/10

531389

19

17.R. Kurkela, L. Vuolas, P. Vihko, Preparationof F(ab')2 fragments from monoclonal mouse

IgG1 suitable for use in radioimaging, JIM,

110: 229-236 (1988).

18.H.J. Haisma, J. Hilgers, V.R. Zurawski,Iodination of monoclonal antibodies fordiagnosis and radiotherapy using a convenient

one vial method, J. Nucl. Med. 27: 1890-1895

(1986).

19.T. Lindmo, E. Boven, F. Cuttitta, J. Fedorko,P.J. Bunn, Determination of the

immunoreactive fraction of radiolabeled

monoclonal antibodies by linear extrapolation

of binding at infinite antigen excess, J.

Immunol Methods, 65: 55-63 (1983).

20.M.J. Abrams, M. Juweid, C.I. TenKate,Technetium-99m human polyclonal IgGradiolabeled via the hydrazino nicotinamide

derivative for imaging focal sites of infection in

rats, J. Nucl. Med. 31: 2022-2028 (1990).

21.A. Johnstone, R. Thorpe, Basic techniques. In:Johnstone A, Thorpe R, editors.

Immunochemistry in practice. Blackwell

Science Ltd, 1-34 (1996).

22.H.J. Lee, W. Pardridge, Monoclonal antibodyradiopharmaceuticals: cationization, pegylation,radiometal chelation, pharmacokinetics, and

tumor imaging, Bioconjugate Chem, 14: 546-

553 (2003).

23.M.J. Verhaar, B.A. Zonnenberg, J.M.H. Klerk,G.H. Blijham, Radioimmunodiagnosis and

therapy, Cancer Treatment Reviews, 26: 3-10

(2000).

24.Y. Yamaguchi, H. Kim, K. Kato, K. Masuda, I.Shimada, Y. Arata, Proteolytic fragmentationwith high specificity of mouse

immunoglobulin, G. JIM, 181: 259-267

(1995).

25.L.M. Lamki, Radioimmuniscintigraphy ofcancer, problems, pitfalls and prospects. In:

Freeman, L.M. (ed) Nuclear Medicine Annual

1990, Raven Press, New York, 113-150 (1990).

26.A.M. Verbruggen, Radiopharmaceuticals:state of the art, Eur. J. Nucl. Med. 17: 346-364

(1990).

27.S.W. Schwarz, J.M. Connett, C.J. Anderson, etal, Evaluation of a direct method for

technetium labeling intact and F(ab')2 1A3, an

anticolorectal monoclonal antibody, Nucl.

Med. Biol. 21: 619-626 (1994).

28.S.K. Larsen, G. Caldwell, J.D. Higgins, M.S.Abram, H.F. Solomon, Technetium complex

of tricine: useful precursor for the 99mTc

labelling of hydrazino nicotinamide modified

proteins, J. Label Compd Radiopharm, 35: 1-2(1994).

29.B.W. Wessels, Current status of animalradioimmunotherapy, Cancer Res, 50, 970-973

(1990).

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The Atomic Energy Organization of Iran (AEOI) a-10!1!46-25f8902