European Journal of

Nuclear Medicine

Original article

Effects of tumour mass and circulating antigen on the biodistribution of 111In-labelled F(ab') 2 fragments of human prostatic acid phosphatase monoclonal antibody in nude mice bearing PC-82 human prostatic tumour xenografts M a r i t t a P e r & l & - H e a p e 1, Pirkko Vihko% A i r e L a i n e 2, J u h a n i H e i k k i l & 1, and R e i j o Vihko" I Biocenter and Department of Clinical Chemistry, University of Oulu, SF-90220 Oulu, Finland 2 Research Center of Farmos Group Ltd, Box 425, SF-20101 Turku, Finland Received 14 September 1990 and in revised form 14 January 1991

Abstract. We have evaluated the effects of tumour mass and circulating antigen (prostatic acid phosphatase, PAP) on the biodistribution and the incorporation of 111In-labelled F(ab')2 monoclonal antibody (MoAb) fragments directed against human PAP into human prostatic tumours (PC-82; 0.1-8.9 g) growing in nude mice. The radioactivities in the blood, liver, spleen, kidney and turnout were compared at 1, 3, 4 and 6 days after the intravenous administration of the antibody fragments. There was a significant correlation between the tumour size and the serum PAP concentration in the model employed. Even tissue of a small tumour (< 0.1 g) had a high concentration of PAP, but it was not secreted into the circulation in detectable amounts when measured by radioimmunoassay (the lowest standard was 0.5 gg/1). The percentage uptake by turnouts of the injected dose per gram of tissue (%ID/g) was inversely proportional to the tumour size at 24 h after the administration of l tlIn-labelled F(ab')2 fragments. This relationship had levelled off by 72 h and most likely reflected a better vascularisation of the smaller tumours. Our results show that the increase in tumour size and in the concentration of circulating antigen in the blood led to decreased turnout-to-blood ratios, since there was a tendency for higher blood activities in mice with larger tumours and higher serum PAP concentrations. There was no correlation between turnout size and label uptake by the liver during the follow-up over 144 h, although serum PAP concentrations ranged from 3.1 gg/1 to 352 lag/1. On the other hand, when compared with our previous data obtained with non-tumour-bearing mice, there was a significant increase in the uptake by the liver and spleen. These results indicate that even a small concentration of circulating antigen was able to trigger an abnormal change in the biodistribution of MoAbs. Offprint requests to . P. Vihko

Key words: Radioimaging cancer

Radiotherapy - Prostatic

Eur J Nucl Med (1991) 18:339-345

Introduction

In designing clinically useful methods for the radioimaging of malignant tumours, a central issue is to obtain a high target-to-background (turnout-to-blood) ratio at a time point suitable for imaging. When monoclonal antibodies (MoAbs) are used, the variables to be taken into account when optimizing a method include whether the parent antibody molecule or its fi'agment is to be employed and which conjugation method and radioactive nuclide are selected (Khaw et al. 1986; Andrew et al. 1988; Koizumi et al. 1989). We are interested in the radioimaging of prostatic cancer. Our previous report dealt with differences between a parent MoAb raised against human prostatic acid phosphatase (PAP) and its F(ab')z fragments, as well as differences between various chelating agents, when indium-Ill was used the label (Per/ilfi et al. 1990). In that study, the experiments were carried out in normal mice in order to reduce the number of variables in the model, and we selected F(ab')2 fragments conjugated with the cyclic anhydride of diethylenediaminepentaacetic acid (CA-DTPA) for further study. However, the antigen (PAP) acting as the target for the imaging agent frequently circulates in the patient's blood. In prostatic cancer (Babaian et al. 1987), as well as in gastrointestinal (Goldenberg et al. 1989) and ovarian cancers (Hnatowich et al. 1987) and malignant melanomas (Divgi and Larson 1989), the circulating antigen has been observed to influence the kinetics

© Springer-Verlag 1991

340 and biodistribution of MoAbs. The PC-82 human prostatic c a n c e r g r o w i n g in n u d e m i c e secretes P A P i n t o the b l o o d o f the h o s t ( v a n S t e e n b r u g g e et al. 1985), a n d this m o d e l gives a n o p p o r t u n i t y for d e t a i l e d s t u d i e s o f the effects o f t u m o u r size a n d c i r c u l a t i n g a n t i g e n o n the distribution a n d kinetics of our r a d i o i m a g i n g agent.

Materials and methods Preparation and purification of PAP-specific F(ab')2 fragments. F(ab')2 fragments of a MoAb raised against human PAP were generated, purified and characterized as previously described (Kurkela et al. 1988; Hakalahti and Vihko 1989). Preparation and purification of non-specific mouse F(ab')2 fragments from mouse immunoglobulin G. Mouse immunoglobulin G (IgG; Sigma 1-5381) 3 mg/ml)was dialysed overnight against 0.2 M sodium acetate buffer, pH 4.0, at 4 ° C. Pepsin (Sigma) was dissolved in the same buffer at a concentration of 1 mg/ml, and these solutions were then mixed to give an enzyme:mouse IgG weight ratio of 1:33 (Lamoyi and Nisonoff 1983). The reaction mixture was incubated at 37° C for 13 h. Digestion was stopped by adjusting the pH of the reaction mixture to 7.5-8.0 with 0.5 M NaOH. Pepsin-digested fragments were dialysed overnight against 0.02 M phosphate-buffered saline (PBS) buffer, pH 7.4, at 4 ° C, and purified on a Sephacryl S-200 (Pharmacia) gel filtration column (85 x 2.5 cm), using 0.02 M PBS buffer, pH 7.4, for elution. Fractions of 3.0 ml were collected (Kurkela et al. 1988).

plantation (Steenbrugge van et al. 1985), and the mean was 11 _+ 3.2days ( ± S D , n=10, range 7 17days). In these experiments, mice with turnouts of 0.1-8.9 g were used. A histological examination of the tumour tissue showed a moderately differentiated adenocarcinoma as originally described by Hoehn et al. (1980).

Biodistribution and imaging analyses. Biodistribution studies were performed in nude mice with PC-82 human prostatic cancer xenografts. The mice received an injection of 11tin-labelled F(ab')2 fragments (25 gg) and were sacrificed at time intervals (24, 72, 96 and 144 h) after administration. The tissues were dissected out and weighed, and their radioactivity was counted. Large turnouts were cut into smaller pieces, and the radioactivity of each piece was measured. Label uptake by the organs was expressed as a percentage of the injected dose per gram of organ (%ID/g). Nude mice were imaged using a gamma-camera (Elscint Apex 409 ECT, pinhole collimator) at 72 and 144 h after the administration of 6080 gCi of the 11lin.labelled F(ab')z fragments. Concentrations of serum and tissue PAP. Serum specimens from the xenografted nude mice were obtained by retro-orbital puncture 5-7 days before the injection of the radiolabelled conjugate. Serum PAP concentrations were determined by radioimmunoassay (PAPRIA) (Vihko et al. 1982). PAP concentrations were also measured in the supernatants of tumour homogenates. For this purpose, tumours were first homogenized in 0.1% Tween-80 solution with an Ultra-Turrax homogenizer for 1 rain and centrifuged at 40 000 x g for 30 min (Vihko et al. 1978).

Results Conjugation and radiolabefling of F(ab')~ fragments. DTPA was attached to F(ab')2 fragments using its cyclic anhydride (Hartikka et al. 1989). The conjugates of the F(ab')2 fragments were iabelled with indium-Ill as previously described (Hartikka et al. 1989). The specific activity of was 2-3 mCi/mg protein, and the labelling efficiency was more than 90% (Hartikka et al. 1989). Mice. Male nude mice (nu/nu-BALB/cABom), aged 5 8 weeks at the beginning of the experiments, were obtained from Bomholtgaard Breeding and Research Centre Ltd., Denmark. The animals were maintained in a barrier unit. All equipment and food entering the barrier were autoclaved. Establishment of in vivo tumours. Fragments of about 1-3 mm 3 of testosterone-dependent, PAP-producing, human prostatic adenocarcinoma tumour PC-82 (Steenbrugge van et al. 1985) were transplanted subcutaneously into the right shoulder of uncastrated mice. A silastic implant (length 1 cm, outer diamter 0.2 cm, internal diameter 0.15 cm; Silastic Medical-Grade Tubing, Dow Coming), filled with crystalline testosterone (Sigma, T 1500), was installed through the same incision (Steenbrugge van et al. 1984). The grafting was carried out under light ether anesthesia. The purpose of the implant s was to maintain sufficiently high plasma testosterone concentrations in the mice to support the growth of the androgendependent tumour. This has been shown to be advantageous (Steenbrugge van et al. 1985). Plasma testosterone was measured by the method described previously (J/inne et al. 1974). Tumours were allowed to grow for 3-5 months, by which time they had started to secrete PAP into the blood circulation. The slow growth rate of PC-82 turnout tissue (Steenbrugge van et al. 1985) was confirmed. Growing tumours were usually detected 6-8 weeks after transplantation. Turnout-doubling times were estimated from a semi-logarithmic plot of the tumour volume vs. time after trans-

E f f e c t o f tumour mass on P A P concentrations in tumour tissue and serum specimens T h e r e w a s a s i g n i f i c a n t c o r r e l a t i o n b e t w e e n t u r n o u t size a n d s e r u m P A P c o n c e n t r a t i o n s i n n u d e mice. T h e c o r r e l a t i o n coefficient, c a l c u l a t e d u s i n g P e a r s o n ' s c o r r e l a t i o n coefficient a n a l y s i s f r o m the l o g a r i t h m i c p l o t in Fig. 1, was 0.87 (n = 25 ; t u m o u r w e i g h t 0 . 0 1 4 - 7 . 4 g). P A P c o n centrations in tumour preparations (n=10; tumour w e i g h t 0 . 0 1 4 4 . 2 g) a n d sera were d e t e r m i n e d in m i c e t h a t h a d n o t received i n j e c t i o n s o f r a d i o l a b e l l e d F ( a b ' ) 2 f r a g m e n t s . T h e results a r e s h o w n in T a b l e i. O n l y t h o s e

100,0

S £1_ < G_

°~ " ~ ' ~ j

10,0

2~ c15 [aA q?

1.0

0.1

0

i

100

i

1000

10000

TUMOR WEIGHT (mg)

Fig. 1. Logarithmic plot showing the correlation between tumour size and serum prostatic acid phosphatase (PAP) concentrations in nude mice with human PC-82 tumour xenografts

341 Table 1. Effect of tumour size on PAP concentration in tumour homogenates and in sera

~124

h

~ 7 2

h

~ 9 6

h

~144

h

A

]

% I D / g BLOOD

Tumour (mg wet weight)

20 7

PAP concentration in tumour (p,g/g)

14 16 28 33 54 154 333 366 523 4246

19.1 74.8 21.7 38.9 34.5 74.2 84.8 78.2 61.9 33.5

in serum (l~g/1) < < < < <

0.5 0.5 0.5 0.5 0.5 3.8 20.3 2.5 11.2 178.2

,sJ10

5

0

.1

.2

.8 3.5 7,4

.1

.2

.8 1,5 5.1

,1

.2

.5 2.8

.4

1.1 6.5

TUMOR WEIGHT (g)

% ID/g TUMOR 14

B

12

tumours with a mass o f 0.1 g or m o r e secreted enough PAP to be detected in the sera, but P A P was detectable in both small and large tumours.

10

Influence of tumour size and circulating antigen on antibody uptake by blood and tumours For small tumours (0.1-0.5 g), the mean blood activity at 24 h was 11.3 % I D / g ( n = 4 ) , and for larger ( > 0 . 5 g) tumours, it was 11.9 % I D / g (n=5). At 72 h, the mean blood activities were 1.4 % I D / g ( n = 4 ) and 3.6 % I D / g (n=6), respectively (Fig. 2; P = 0 . 0 1 6 ) . At later time points (96 h and 144 h), there was also a tendency for higher blood activities in mice with larger tumours and greater serum PAP concentrations. When c o m p a r e d with our data obtained with non-tumour-bearing mice (Per/il/i et al. 1990), there was a significant difference ( P < 0 . 0 5 ) in blood activities. When circulating antigen is not present, as in the case of normal mice, the blood activity decreases faster than when circulating antigen is present, even at low concentrations, as in the case of mice with small tumours (Fig. 3). Label uptake by tumours, expressed as % I D / g , indicated a higher uptake ( P < 0 . 0 0 1 ) by smaller tumours (0.1-0.5 g) at 24 h than by tumours heavier than 0.5 g. For small tumours (0.1-0.5 g, mean PAP concentration in s e r u m = 8 . 8 gg/1, range 6.8-9.9 gg/1; n = 4 ) the m e a n label uptake was 7.9 _+ 1.6 % I D / g (n = 4), while for larger tumours (0.5 7.4 g; mean PAP concentration in ser u m = 155.1 gg/1, range 7.5-352 gg/1; n = 5 ) , the uptake was 2.5___0.7 % I D / g ( n = 5 ) at 2 4 h . At 9 6 h , the mean label uptakes were 4.1-t-2.9 % I D / g ( n = 5) and 4 . 0 + 1.8 % I D / g ( n = 3 ) , respectively (Fig. 2). Tumour-to-blood % I D / g ratios were higher for small tumours at all the time points. Mean t u m o u r - t o - b l o o d ratios were 0.8 (n = 4), 4.8 ( n = 4 ) , 12.5 ( n = 5 ) and 7.6 ( n = 2 ) for turnouts below 0.5 g and 0.2 ( n = 5), 1.5 ( n = 6 ) , 2.2 ( n = 3 ) and 6.3 ( n = 4 ) for tumours larger than 0.5 g at 24, 72, 96 and 144 h, respectively (Fig. 2).

.1

,2

.8 3.4 7.4

.1

.2

.8

1,5 5.1

,2

.3

.5

2.8

.4

1.1 6.5

TUMOR WEIGHT (g)

TUMOR-TO-BLOOD RATIO

C

2° I 15 10

0

.1

.2

,8 3.4 7.4

,1

.2

.8 1.5 5.1 .1 ,2 TUMOR WEIGHT (g)

,5 2.8

.4

1.1 6.5

Fig. 2A-C. Effect of tumour size on the uptake of radioactivity by blood (A) and tumour (B) expressed as a percentage of the injected dose per gram (%ID/g) and as tumour-to-blood ratios (C), following the administration of 111In-labelled PAP-specific F(ab')z fragments in nude mice with human PC-82 tumour xenografts

Influence of tumour size and circulating antigen on antibody uptake by liver, spleen and kidney There were no systematic correlations between t u m o u r size and label uptake by the liver, nor between serum PAP concentrations and label uptake by the liver, al-

342

[ m

anti-PAP-F(ab')2 (n)

~

anti-PAP-F(ab')2 (t)

~

mouse-F(ab')2 (t) ]

% IDlg BLOOD

A

14) 12 10 8 6 4

1

3

3 DAYS

5

% ID/g LIVER 50

B

40

30

20

10

T 1

3

5

1

3

5

1

3

Fig. 3A, B. Comparison of label uptake by blood (A) and liver (B) expressed as %ID/g using a t tin_labelle d PAP-specific F(ab')2 fragments in normal (from Per/il/i et al. 1990) and tumour-bearing mice and t t l in_labelled non-specific mouse F(ab')2 fragments in tumour-bearing mice. n, normal mice; t, tumour-bearing mice

5

DAYS

though the serum PAP concentration ranged from 3.1 to 352 lxg/1. At 24 h, the mean kidney activity was 54.6 %ID/g (n=4) for small tumours (0.1-0.5 g) and 24.7 %ID/g (n= 5) for large tumours (0.5-7.4 g). After that time point, there was no tumour size dependency of label uptake by the kidney. An inverse correlation between tumour mass and uptake was also seen in the spleen at 24h: for small tumours, the mean was 21.5 %ID/g (n=4), and for larger tumours, it was 9.2 %ID/g (n= 5). At later times, uptake by the spleen was not clearly dependent on tumour size. However, there was a significant increase in the uptake by the liver (P < 0.05; Fig. 3 B) and spleen (P < 0.05) when compared with non-tumour-bearing mice. As a control, we used non-specific mouse F(ab')2 fragments, which were conjugated and labelled identically to the PAP-specific fragments; they did not have any specificity towards the antigen in the tumours. Tumour-to-blood ratios showed a significant difference (P<0.05 at 96 h) between specific and non-specific F(ab')2 fragment uptakes. At 96 h, the tnmour-to-blood ratio for 111In-labelled PAP-specific F(ab')2 fragments was 13 compared with a value of 2 for 111In-labelled mouse F(ab')2 fragments (Fig. 4). Furthermore, in the liver, the uptake of

TUMOR-TO-BLOOD RATIOS 14

12

10

8

6

4

2

0

24 h

72 h

96 h

144 h

Fig. 4. Tumour-to-blood ratios for it tin-labelled PAP-specific F(ab')2 (e) fragments and for non-specific, 11l in_labelled, mouse F(ab')2 ( + ) fragments in nude mice with human PC-82 tumour xenografts

343

Fig. 5a, b. Gamma-camerascintigrams of PC-82-bearing nude mice obtained 3 (a) and 6 days (b) after intravenous administration of 111In-labelled, PAP-specific F(ab')z fragments. Tumour sizes were 8.9 g (left) and 2.5 g (right). Tumours are indicated by arrows

mouse F(ab')2 fragments was lower than the uptake of specific F(ab')2 fragments (Fig. 3).

Imaging analysis Gamma-camera imaging of nude mice injected with ill In-labelled F(ab')z fragments confirmed the results of the biodistribution studies. The large tumours were detected easily because of their size, but small tumours (< 0.5 g) were difficult to detect in spite of high tumourto-blood ratios (12 at 96 h), due to the detection limit of the gamma-camera. High uptakes by liver and kidney were observed in nude mice with small and large tumours. Figure 5 demonstrates the difference observed between images obtained for tumours of 8.9 g and 2.5 g, 3 and 6 days after injection.

Discussion The aim of this study was to investigate how tumour size and presence of circulating antigen affect the biodistribution of 11~in_labelled F(ab')z fragments after intravenous administration. In our tumour mouse model (human prostatic cancer PC-82), the target tumour antigen (PAP) is secreted by the tumour cells into the blood circulation. Detectable amounts of PAP were secreted by tumour cells into sera when the tumour reaches the size of 0.1 g. The smaller tumours (below 0.5 g) had serum PAP concentrations inferior to 30 lag/l, while for the larger tumours they increased to over 300 lag/1. Since there is a positive correlation between the tumour mass and the circulating PAP concentrations, the contribution of these two variables to the results must always be clearly distinguished. In previous studies, many investigators have reported the effect of tumour mass on the uptake of i.v. admin-

istered, labelled MoAb by tumours, and the relationship is known to be influenced by the radionuclide (Andrew et al. 1988) and the nature of the antigen (Watanabe et al. 1989). Hagan et al. (1986), Philben et al. (1986) and Watanabe et al. (1989) have shown the label uptake by tumours to be inversely proportional to the tumour size at 48 h. Baldwin and Pimm (1983) reported a linear uptake of ~25I-labelted MoAb in mice bearing human tumours. Hagan et al. (1985) investigated the effect of tumour size at multiple time periods, and they showed a greater per weight uptake of radiopharmaceutical in small than in large tumours at all time points. In our tumour model, the tumour-weight-related label uptake was higher for small tumours (weight 0.1-0.5 g) at 24 h than for larger ones, but by 72 h, large tumours manifested antibody uptakes quite similar to those of small tumours. This is interpreted to reflect a better vascularisation of the small tumours. The ability of the large tumours to reach the same weight-related uptake as smaller ones in the course of follow-up indicated that the large tumours were not necrotic. This was also evaluated by cutting them into smaller pieces and measuring the radioactivity in the individual parts. All the pieces of the same tumour had the same radioactivity per gram, demonstrating the homogeneous nature of the tumour. In several clinical and experimental studies of mice using carcinoembryonic antigen (CEA)-specific antibodies, it has been shown that circulating immune complexes are found following the antibody injection (Mach et al. 1981 ; Pimm et al. 1989; Beatty et al. 1990; Colcher et al. 1990). The same process may have taken place in the present study, because the accumulation of radioactivity in the liver was higher in mice bearing tumours than in normal mice (P<0.05; Fig. 3). Furthermore, we have used labelled, non-specific mouse F(ab')2 fragments as a control, and in tumour-bearing mice, their uptake by the liver was only about half of that observed with specific F(ab')2 fragments, again indicating the possible for-

344 mation of antigen-antibody complexes when particular P A P - s p e c i f i c F(ab')2 f r a g m e n t s are used. Liver activities in mice b e a r i n g small a n d large t u m o u r s were a b o u t the same, It is likely t h a t even a small c o n c e n t r a t i o n o f c i r c u l a t i n g a n t i g e n was able to trigger the c h a n g e o f liver activity c o m p a r e d w i t h the liver a c t i v i t y o f n o r m a l mice. Different, c i r c u l a t i n g a n t i g e n levels o b v i o u s l y influenced the c l e a r a n c e o f labelled a n t i b o d y f r a g m e n t s f r o m the b l o o d since w h e n c i r c u l a t i n g a n t i g e n was present o n l y in low c o n c e n t r a t i o n s , as in the case o f mice w i t h small t u m o u r s , b l o o d a c t i v i t y d e c r e a s e d faster t h a n in mice w i t h h i g h e r c i r c u l a t i n g a n t i g e n c o n c e n t r a t i o n s . This led to l o w e r t u m o u r - t o - b l o o d r a t i o s in the case o f large t u m o u r s c o m p a r e d with t h o s e o b s e r v e d for small t u m o u r s a t all the time p o i n t s studied. This is n o t a d e s i r a b l e p r o p e r t y o f a r a d i o p h a r m a c e u t i c a l int e n d e d for use as a r a d i o i m a g i n g agent. H o w e v e r , in i m a g i n g analysis, the large t u m o u r s c o u l d , b e c a u s e o f their size, be d e t e c t e d w i t h a g a m m a - c a m e r a , in spite o f s u b - o p t i m a l t u m o u r - t o - b l o o d ratios. We a r e p r e s e n t l y s t u d y i n g the effect o f u n l a b e l l e d M o A b s o n the b i o d i s t r i b u t i o n o f the labelled F ( a b ' ) 2 f r a g m e n t s in n u d e mice w i t h c i r c u l a t i n g a n t i g e n in o r d e r to increase i m a g i n g efficiency.

Acknowledgements. This study was supported by the Technology Development Center (Tekes, Finland) and the Research Council for Medicine of the Academy of Finland. We are grateful to Ms. Saini Stenfors and Miina Moilanen for their skilful assistance. The Department of Clinical Chemistry is a WHO Collaborating Centre supported by the Ministries of Education, Health and Social Affairs and Foreign Affairs, Finland.

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