Localization of radiolabeled monoclonal antibodies …nopr.niscair.res.in/bitstream/123456789/23406/1/IJEB 42(4) 354-360.pdf · Localization of radiolabeled monoclonal antibodies

Indian Journal of Experimental Biology Vol. 42 April 2004, pp. 354-360

Localization of radiolabeled monoclonal antibodies in thyroid tumor xenografts

Archana A Damle, Archana A Narkar & Damayanti H Shah Laboratory Nuclear Medicine Section (BARC), clo Tata Memorial Centre Annexe, Parel, Mumbai 400 012 India

Received 26 JUlie 2003; revised 17 December 2003

Monoclonal antibodies to human thyroglobulin were produced using the hybridoma technique. Two monoclonal anti­bodies OSI and F91 were radiolabeled with 1251 and used for radioimmunolocalization studies in an immunosuppressed ani­mal model bearing xenografts of human thyroid tumor tissue. Biodistribution studies were carried out at various time inter­vals post-injection. Maximum tumor uptake was obtained at 72 hr after administration of the antibodies. The absolute tumor uptake (ATU) expressed as percentage of injected dose per gram of tissue (% ID/g) was 15.49 ± 2.47, 4.S1 ± 0.69 and 2.S0 ± 0.41 for OSI, F9I and control Igs respectively. The tumor to blood ratios (TIB) obtained were 3.01 ± 0.43 for OSI, 0.98±O.2 for F9I and 0.47 ± 0.12 for control Igs. ATU as well as TIB ratio obtained with OSI was significantly higher as compared to F91 and control Igs. The results indicated the potential application of radiolabeled monoclonal antibodies to human thyro­globulin for tumor targetting in patients of differentiated thyroid carcinoma, particularly those metastases which did not concentrate radioiodine.

Keywords: Monoclonal antibodies, Thyroglobulin, Thyroid tumor xenograft IPC Code: Int. Cl.7 A61 KS11l0

Conventional treatment of differentiated thyroid car­cinoma (DTC) is thyroidectomy followed by ablation of the residual thyroid tissue by 1311. Follow-up of these patients involves imaging with diagnostic 131I scan to localize metastases and the measurement of serum thyroglobulin (Tg) after withdrawal of thyroid hormones. Further, patients with metastatic disease are treated by administration of therapeautic doses of 1311. Significant number of these patients become re­fractory to 131I therapy as the metastatic tissue fails to concentrate 131I 1-5. However, the cells continue to produce Tg which is an established tumor marker for DTC 6. Since most of the tumors concentrate 131I, this is the primary mode of treatment, but problem arises mainly when the metastases do not concentrate 1311. Treatment modalities are then limited to either exter­nal radiation or chemotherapy which do not seem to be very effective in controlling the disease7

-1O

•

Monoclonal antibodies (MAbs) produced by hy­bridoma technology II are being labeled with various isotopes and used for radioimmunoscintigraphy (RIS) and radioimmunotherapy (RIT) in wide variety of human carcinomas I2

,13. Use of radiolabeled MAbs to carcino-embryonic antigen in the management of me­dullary thyroid carcinoma has b~en reported I4,15. Few reports are available for targeting OTC with radiola-

E-mail:archanadamle@hotmail .com

beled antibodies 16-20. A specific approach for the di­agnosis and therapy of non-iodine concentrating me­tastases could be the use of radiolabeled MAbs against specific thyroid antigens for tumor targeting. The aim of this study was to evaluate the usefulness of MAbs to human Tg (H-Tg) produced in our labo­ratory for in vivo localization of thyroid tumour xeno­grafts.

Materials and Methods Production of monoclonal antibodies-Human Tg

was purified from a single specimen of normal human thyroid by the method of Mouriz and Stanbury21. Monoclonal antibodies to H-Tg were produced by hy­bridoma technique as described earlier22. Briefly, Sp2/0 mouse myeloma cells were fused with spieen cells of Balb/c mice immunized with H-Tg. The clones ob­tained were screened for anti-H-Tg antibodies by an enzyme linked immunosorbant assay (ELISA)23. Two monoclonal antibodies 05I and F91 having high ELISA positivity were selected and single, stable clones were established by limiting dilution method and propagated in tissue culture flasks or grown as as­cites in pristane primed mice. Immunoglobulins (Igs) from culture supernatants and ascitic fluid from mice were precipitated with 50% (NH4hS04, pH 7.2 and dialyzed against 0.02M of phosphate buffered saline (PBS; pH 7.2). Affinity purification of MAbs was car-

DAMLE et al. : LOCALIZATION OF RADIOLABELED MONOCLONAL ANTIBODIES 355

ried out using MAPS II kits from Bio-Rad, USA as per the manufacturers protocol.

Radiolabeling of MAbs with125 I-Affinity purified MAbs were labeled with 1251 using iodogen method24

•

A glass vial was coated with 200 Jlg of iodogen (Pierce chemicals, UK) dissolved in 200 III of chloroform and air dried to form a thin film. Monoclonal antibody (10 Jlg) and 1 mCi of Nal251 (Amersham, UK) was added to the vial and the volume was made to 200 JlI with 0.2M phosphate buffer (PH 7.2). The reaction was car­ried out for 20 min at room temperature with intermit­tent shaking. The 1251 labeled MAb was separated from the free iodine by passing through a Sephadex G-75 column (Pharmacia, Uppsalla, Sweden). The immu­noreactivity of the1251 labeled antibodies was checked on H-Tg coated on solid phase. Normal mouse Igs pu­rified and labeled using the same procedure as that used for MAbs served as control Igs for biodistribution studies.

Evaluation of immunoreactivity of 125f labeled anti­bodies-Ninety-six wells polyvinyl plate was coated with 100 JlI of H-Tg (10 Ilg/ml) by incubating over­night at 37°C. The plate was then washed with 0.02 M PBS 5 times, and uncoated sites were blocked with PBS containing 1 % bovine serum albumin (BSA) for 1 hr at 37°C. After draining the BSA 1251 labeled MAbs 05I and F91 diluted in 1% BSA to give Ix105 cpm were added to individual wells and the plate was incu- . bated at 37°C for 2 hr. The unreacted MAb was then aspirated and plate was washed with PBS and air dried. The individual wells were then cut and the bound ra­dioactivity was counted in the multi gamma counter (Nuclear Enterprise, UK). Results were expressed as percentage activity bound as that of total counts added.

Establishing an experimental animal model-Four to eight weeks old female Swiss-white mice were an­aesthetized and the thymus was surgically removed. Two weeks after thymectomy, the mice were subjected to first dose of whole body irradiation of 2 Gy using a 6OCO source. A total cumulative dose of 10 Gy com­prising 5 doses of 2 Gy each were given at 2 weeks intervals25

• Total WBC as well as the lymphocyte counts were estimated, which were 60% lower than those of normal mouse. This confirmed the immuno­suppressed state of the mice. These mice were termed as T200x5R mice. Two weeks after the final dose the mice were used for xenografting.

Thyroid tumor tissue was collected from patients undergoing thyroidectomy for treatment of OTC. It

was cleansed of the attached connective tissue, blood clots and then cut into small pieces of 100 mg each. The mice were anaesthetized and the tissue was grafted sub-cutaneously (sc) in the right flank of the mice. Two weeks after xenografting the mice were used for localization studies by administration of 1251 labeled antibodies.

Biodistribution studies using 1251 labeled antibod­ies -The T200x5R mice were given Lugoi's iodine (0.1 % solution) in drinking water two days prior to the injection of 1251 labeled antibodies and throughout the period of experiment to saturate the thyroid tissue and block the uptake of free 1251.

Radiolabeled monoclonal antibodies 051 and F91 specific for H-Tg were used for biodistribution stud­ies. Normal mouse immunoglobulins were used as non-specific control antibody. Each of the above anti­body preparation (0.1 ml) was injected (iv) using·a 26 gauge needle in the tail vein of T200x5R mice bear­ing thyroid tumor xenograft (TTX). The amount of radioactivity injected per mouse was 10-20 JlCi corre­sponding to 0.3-0.5 Jlg of each antibody. The biodis­tribution studies were performed at 1, 3, 5 and 7 days respectively after administration of the antibodies.

The animals were subjected to ether anesthesia and blood was collected by cardiac puncture, weighed and measured radioactivity content in a gamma counter (ECIL, Bangalore, India) cali­brated for 1251 energy against a reference sample for accurate measurement of the activity in each tissue. The mice were sacrificed, dissected open, the individual organs as well as the grafted tumor was removed, weighed and radioactivity in the organ/tumor was counted. The radioactive uptake in each organ was express~d as percentage of the injected dose per gram (% ID/g) of tissue. In case of the tumor graft it was termed as an absolute tumor uptake (ATV) and expressed as % ID/g of tumor tissue. The mean value and standard de­viations were calculated for each group of mice.

Statistical analysis-ATV, the tumor to blood ratio (T/B) and the tumor to muscle ratio (TIM) of the three antibodies was compared using the Student's t test.

Results All the three antibody preparations could be suc­

cessfully labeled with 1251 using the iodogen method. The labeling efficiency of N-Igs, MAb 051 and MAb

356 INDIAN J EXP BIOL, ARRIL 2004

F9I was 73.81 ± 8.92, 66.38 ± 19.87 and 62.60 ± 19.62% respectively. The specific activity expressed as mCi/mg was 10.54 ± 2.18,34.94 ± 12.70 and 27.66 ± 13.86 for N-Igs, MAb D5I and MAb F9I respec­tively. Both the monoclonal antibodies retained their immunoreactivity to H-Tg, post-labeling. The per cent binding was 33.24 ± 5.14 for MAb D5I and 21.45 ± 4.31 for MAb F9I respectively.

The blood clearance of the three radio labeled anti­body preparations has been shown in Fig. 1. N-Igs re­mained in circulation for a longer time as compared to MAb D51 and MAb F9I. At 7 days post-injection the blood pool activity of N-Igs was 3.78 ± 0.18 % ID/g,

10 9 8 7

~ 6 g 5

4

~N-Igs --.. ---.-.. ---- ------ ---D51

----- -6-F91

3 -t-.-----2 1

----- .-- .. --- ------ --.... c----- --

_._--------- --.....",,-=---- -

0....1...--------------1 3 5 7

Days post-injection

Fig. I-Blood clearance of 1251 labeled antibodies in TIOO x 5R mice bearing thyroid tumor xenografts. Radiolabeled antibodies were injected (iv) in the tail vein of mice and their clearance was studied at various time intervals post-injection. [Values are expressed as % of injected dose per g of blood).

which was higher than MAb D51 (2.82 ± 1.48 % ID/g) and MAb F9I (0.49 ± 0.18 % ID/g). Amongst the two MAbs used, MAb F9I showed a faster clear­ance as compared to MAb D5I. TI/2 values for N-Igs was 8 days, for MAb D5I was 4.8 days and that for MAb F9I was 1.3 days. The rate of clearance of the three 1251 labeled antibody preparations was in the following order MAb F9I > MAb D5I > N-Igs.

Biodistribution of N-Igs in the animal model has been depicted in Table 1. The uptake in the tumor xenografts was 2.50 ± 0.41 % ID/g and 1.19 ± 0.12 % ID/g at 3 and 7 days post-injection. The tumor to blood ratios were < 0.5 at all the time intervals studied.

Biodistribution of MAb 051 has been shown in Table 2. Maximum ATU of MAb D5I in TIX (15.49 ± 2.47 % ID/g) was observed 3 days post-injection.

Table 1-Biodistribution of 1251 labeled N-Igs in T200 x 5R mice bearing thyroid tumor xenografts

[Values are mean ± SO of 4 animals]

Days post-injection

3 5 7

Blood 5.28 ± 0.99 4.48 ± 0.19 3.78 ± 0.18 Muscle 1.52 ± 0.06 1.34 ± 0.02 0.28 ± 0.09 Thyroid 3.32 ± 0.31 2.80±0.14 2.50 ± 0.39 Liver 2.63 ± 0.15 1.74 ± 0.08 0.55 ±0.05 Spleen 1.98 ± 0.32 1.53 ± 0.41 0.72 ±0.28 Small intestine 0.50 ± O.ll 0.42 ± 0.13 0.25 ±0.02 Large intestine 0.59 ± 0.28 0.35 ± O.ll 0.20± 0.03 Kidneys 5.57 ± 0.81 3.63 ± 0.63 1.42 ± 0.41 Tumor (ATU) 2.50 ± 0.41 2.10 ± 0.21 U9 ± 0.12 TumorlBlood ratio 0.47 ± 0.12 0.45 ± 0.05 0.32 ± 0.13 TumorlMuscle ratio 1.64 ± 0.13 1.49 ± 0.81 4.25 ± 1.40

Values are expressed as % of the injected dose per g of the tissue. ATU: Absolute tumor uptake.

Table 2-Biodistribution of 1251 labeled MAb 051 in T200 x 5R mice bearing thyroid tumor xenografts [Values are mean ± SO of6 animals]

Days post-injection

3 5 7

Blood 6.91 ± 0.96 5.15 ± 1.34 4.17 ± 1.37 2.82 ± 1.48 Muscle 1.41 ± 0.05 1.11 ± 0.42 0.63 ± 0.19 0.50 ± 0.22 Thyroid 7.03 ± 0.12 4.12±0.14 3.02 ± 0.08 1.00 ± 0.03 Liver 5.36 ± 0.57 3.39 ± 0.56 1.86 ± 0.40 1.48 ± 0.39 Spleen 1.80 ± 0.50 1.44 ± 0.31 0.90 ±0.25 0.09 ± 0.Q3 Small Intestine 1.45 ± 0.10 0.93 ± O.ll 0.53 ± 0.13 0.51 ±0.20 Large Intestine 1.95 ± 0.30 0.82 ± 0.09 0.83 ±0.08 0.31±0.10 Kidneys 5.20 ± 0.11 3.48 ± 0.09 2.30±0.30 1.63 ± 0.13 Tumor (ATU) 3.10 ± 0.52 15.49 ± 2.47 4.94 ± 1.87 2.82 ± Ll9 TumorlBlood ratio 0.14 ± 0.04 3.01 ± 0.43 U8 ± 0.06 1.01 ± 0.02 TumorlMuscle ratio 2.20 ± 0.81 13.95 ± 2.13 7.84 ± 2.45 0.18 ± 0_08

Values are expressed as % of the injected dose per g of the tissue.

DAMLE et al. : LOCALIZATION OF RADIOLABELED MONOCLONAL ANTmODIES 357

On the 7th day the ATU was 2.82 ± 1.19 % ID/g, whereas the uptake in other organs had reduced to < 2 %ID/g and the circulating activity in the blood was similar (2.82 ± 1.48 % ID/g) to that of the xenograft. The thyroid uptake was 4.12 ± 0.14 %ID/g at 3 days post-injection which reduced as a function of time. Maximum tumor to blood ratio (T/B) 3.01 ± 0.43 was obtained on 3rd day. after injection of MAb D5I. As the muscle uptake of MAb D51 was less 0 .11 ± OA2 %ID/g), the tumor to muscle ratio (TIM) was 13.95 ± 2.13 on 3rd day post-administration ofMAb D5I.

Biodistribution of MAb F91 has been summarized in Table 3. Maximum uptake in the tumor was ob­tained on 3rd day which reduced significantly on 7th day. ATU was 4.19 ± 0.54 % ID/g on lSI day and re­mained nearly same 4.51 ± 0.69 % ID/g on 3rd day after administration of MAb F9I. The thyroid gland of the mice also showed high uptake 6.0 ± 1.48 % ID/g at 3 and 7 days post-injection, the uptake was 3.5 ± 0.95 % ID/g. This was higher as compared to the other organs. The circulating activity in the blood was 8.36 ± 1.13 % ID/g at 24 hr, which reduced to 4.60 ± 0.23 % ID/g on 3rd day and further reduced to 0.49 ± 0.18 % ID/g on 7th day after administration of MAb F9I.

TIB ratio was highest at day 3 post-injection (0.98 ± 0.20) but was much less than that observed with MAb D51 (3.01 ± 0.43). The TIM ratio obtained was 6.94 ± 1.81 at day 3, indicating that there was no sig­nificant uptake in the muscles.

Comparison of various biodistribution parameters of three 1251 labeled antibodies has been shown in Fig.2a, b. Maximum A TU obtained was at day 3 after

administration of MAbs, hence the values at this time interval were used for comparison.

A TU was maximum with MAb D51 as compared to MAb F91 (p< 0.001) and N-Igs (p< 0.001). Both the MAbs i.e. MAb D51 and MAb F91 showed a signifi­cantly high uptake in the tumor xenograft as com­pared to N-Igs (p< 0.001 for both the MAbs).

TIB and TIM ratios obtained with MAb D5I and MAb F9I were significantly higher (p< 0.001 for MAb D51 as well as for MAb F91) as compared to N­Igs. TIB ratio of MAb D51 at 3 days post-injection was greater than that obtained with MAb F91 and was highly significant (p< 0.001).

Discussion Purpose of this study was to assess the potential

use of anti H-Tg monoclonal antibodies for in vivo localization of thyroid tumor tissue in an animal model. Therefore, an animal model of human thyroid carcinoma was established in our laboratory to carry out immunolocalization studies with 1251 labeled anti­H-Tg MAbs. Animal models bearing transplants of differentiated thyroid carcinoma as well as anaplastic thyroid carcinoma have been used for targeting with radiolabeled antibodies 17, 26-28. Biodistribution of ra­diolabeled antitumor antibodies in tumor xenografts growing in immuno-deprived mice provide prelimi­nary information prior to clinical trials on the local­ization of the antibodies in primary and metastatic tumors.

Earlier studies have used either polyclonal 16.17.29 or monoclonal antibodies directed against H-Tg 18. 19,27 or membrane antigen 26,28 to target thyroid tumor tissue in vivo. We have used MAbs against H-Tg as this is a

Table 3-Biodistribution of 1251 labeled MAb F9I in T200 x 5R mice bearing thyroid tumor xenografts

[Values are mean ± SD of 6 animals]

Days post-injection 1 3 5 7

Blood 8.36 ± 1.13 4.60± 0.23 1.21 ± 0.54 0.49 ± 0.18 Muscle 1.92 ± 0.60 0.65 ± 0.01 0.18 ± 0.06 0.12 ±0.04 Thyroid 13.5 ± 2.19 6.0 ± 1.48 4.00 ± 1.23 3.50 ± 0.95 Liver 3.57 ± 0.35 1.95 ± 0.32 0.51 ± 0.15 0.27 ±o.oi Spleen 2.16±0.41 1.44 ±0.44 0.45 ±0.09 0.23 ±0.06 Small intestine 1.63 ± 0.46 0.60 ± 0.31 0.15 ± 0.07 0.07 ± 0.03 Large intestine 1.88 ± 0.05 0.54 ±0.07 0.13 ± 0.03 0.07 ± 0.01 Kidneys 5.00 ± 1.08 3.37 ± 0.95 0.70 ± 0.05 0.30 ± 0.05 Tumor (ATU) 4.19 ± 0.54 4.51 ± 0.69 0.65 ± 0.30 0.32± 0.09 TumorlBlood ratio 0.50 ± 0.13 0.98 ± 0.20 0.54 ± 0.15 0.65 ± 0.18 TumorlMuscie ratio 2.18 ± 0.92 6.94 ± 1.81 3.61 ± 0.07 2.67 ± 0.63

Values are expressed as % of the injected dose taken up per g of the tissues.

358 INDIAN J EXP BIOL, ARRIL 2004

proven tumor marker for OTC patients 6. Preservation of immunoreactivity after labeling is also a key to the successful use of radiolabeled antibodies for tumor localization. SinceMAbs 05I and F9I used in this study retained their immunoreactivity to H-Tg as proved by binding to H-Tg on solid phase, it was pos­sible to use them for in vivo localization in tumor xenografts. Loss of immunoreactivity post-labeling has been reported30 even at low concentrations of the

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6' 12 -. 0

10 '::R. 0 -0 8 .~

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a .j OT/B 1_

--------------r--------mT~

N-Igs MAb 051 MAb F91

b " ~. " . .

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N-Igs MAb 051 MAb F91

Fig. 2-(a)-Tumor uptake of 12.51 labeled antibodies in T200 x 5R mice bearing thyroid tumor xenografts. The absolute tumor uptake CATU) of the three antibody preparations at 3 days post-injection is shown, [p< 0.001 for N-Igs vs MAb D51 and MAb F9I and p < 0.001 for MAb D51 vs MAb F9I] .

(b)-Target to non-target ratios of 1251 labeled antibodies in T200 x 5R mice bearing thyroid tumor xenografts. The values are of three days post injection of the antibodies. [TIB: tumor to blood ratio, p< 0.001 for N-Igs vs MAb D51 and MAb F9I, p < 0.001 for MAb D5I vs MAb F9I. TIM: tumor to muscle ratio, p< 0.001 for N-Igs vs MAb D5I and MAb F9I, p < 0.00 1 for MAb D5I vs MAb F9I].

isotope used31 if the modified labeled residues com­prise the part of the antibody combining site.

MAbs used in this study belonged to IgG I subclass of immunoglobulins. Oue to their ideal size and suit­able kinetics in vivo, most of the radioimmunolocali­zation (RIL) studies are carried out with MAbs be­longing to the IgG class, either whole IgG molecules or their fragments 32

,33.

The two important parameters for deciding the fi­nal suitability of MAb for RIL are the rate of clear­ance of the antibody from circulation as well as the specific uptake of the antibody in tumor tissue. For any antibody, a high A TU with minimum blood pool activity is best suited for RIL. Monoclonal antibody 05I cleared slowly from the blood as compared to MAb F9I. N-Igs used as controls remained in circula­tion for longer time as compared to MAbs 051 and F9I. In case of MAb F9I, though the circulating radio­activity was less, A TU was not very high as compared to MAb 051 and the tumor to blood (TIB) ratio was also low, indicating that it might not be an ideal anti­body for tumor localization. MAb F91 showed local­ization in the thyroid of the animal model used, since it also showed positivity on immuno-histochemistry to murine thyroid34

• This uptake could be due to cross-reactivity between human and murine Tg.

MonoClonal antibody 05I showed maximum TIB and TIM ratios as compared to MAb F91 and N-lgs. Though the rate of clearance of MAb 05I was slow as compared to MAb F9I, the TIB and TIM ratios were higher because of the high A TV of the antibody.

A TU of any antibody depends on various parame­ters like antibody affinity, antigen availability, and tumor antigen density. Tumor vascularity, blood flow and vascular permeability are other factors that affect the localization and distribution of radiolabeled anti­bodies in tumors 12,33. While the high ATU obtained with MAb 051 could be due to any of these factors, the higher affinity 2.4x109 MI of MAb 051 34 as com­pared to MAb F91 4.3 x 108 M J may have resulted in high A TU in the tumor tissue.

In conclusion, the studies indicated high tumor to non-tumor ratio and a significant ATV of MAb 051 in the thyroid tumor tissue xenografted in T200 x 5R mice indicating its future use for targeting tumors in patients of OTC especially those who produce H-Tg but do not concentrate radioiodine.

Acknowledgement We are grateful to Dr Vanaja Shetty, Senior Scien­

tist, The Foundation for Medical Research, Worli,

DAMLE et al. : LOCALIZATION OF RADIOLABELED MONOCLONAL ANTIBODIES 359

Mumbai, for her help in developing the immunosup­pressed animal model.

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