�9 1994 by Humana Press Inc. All rights of any nature whatsoever reserved. 0163-4992/94/24-25/001-007/$5.40

Linking Radiosiiver to Monoclonal Antibodies Reduced by Ascorbic Acio

Comparison of Results with Stable Silver Using Gravimetric Technique and Silver llO-M

Using Radiotracer Technique

D. K. HAZRA,* V. L. LAHIRI, A. K. GUPTA, N. K. PAINULY, MANISH PATHAK, PANKAJ KtlANNA,

R. K. GUPTA, P. KHANNA-HAZRA, AND SHABD SARAN

Nuclear Medicine and Ria Unit, Postgraduate Department of Medicine, S.N. Medical College, Agra 282 002 India

ABSTRACT

Radiosilver-111 and Radiogold-199 were proposed by us (1) as suit- able isotopes for radioimmunotherapy in areas such as India by reason of their suitable half-lives and B-emissions (Ag-111 T,/2 = 7.45 d and Au-199 T1/2 = 3.15 d). Since silver is monovalent, it is difficult to link to conventional bifunctional chelates. We therefore explored the use of sulfur-based linkers (2). Encouraged by the Thakur and De Fulvio Technique (3) of linking technetium to disulfide groups in antibodies reduced by ascorbic acid that is eminently biocompatible, we have explored the linkage of silver to immunoglobulin reduced by ascorbic acid. The linkage of silver was assessed with stable Ag-108 using dial- ysis to quantify the free silver after the reaction of silver and reduced immunoglobulins in various molar ratios (1:1, 1:2, 1:5, 1:10). The silver quantity was estimated gravimetrically after precipitation as chloride. It was observed that using these molar ratios there was negligible silver effiux into the dialysate, suggesting stable linkage. We also assessed the linkage using Ag-110M as radiotracer. The com- parative results with the two techniques are described.

Index Entries: Radiosilver-111; radiogold-199; monoclonal anti- bodies; ascorbic acid.

*Author to whom all correspondence and reprint requests should be addressed.

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INTRODUCTION

Radioimmunotargeting is a rising area of research seeking to define new modalities for diagnosis and treatment of cancer. Interest is focusing on the selection of proper radioisotopes as progress is being made in gen- erating tumor-specific antibodies. The choice of radionuclide for radio- immunotherapy basically depends on:

1. Desired physical characteristics of the radioisotopes, i.e., half- life and energy of photon or particulate emission;

2. Chemical reactions involved for introducing the radionuclide into the antibody; and

3. Appropriate biocompatability of the selected radionuclide, and, where necessary, of the daughter/parents/contaminant isotopes, as well as of all the reagents contained in the material being injected in vivo.

This includes considerations such as lack of bone-seeking behavior, in vivo stability of the radioisotope-chelate linkage, lack of transchelation, and the resistance of the radionuclide antibody combination to biode- gradation, e.g., dehalogenase for radioiodinated compounds.

Since the accumulation of antibody in the tumor takes 3 d, the half-life of the radioisotopes is of crucial importance in relation to tumor residence time. Too short a radioisotope half-life would be undesirable.

The use of beta emitters in radioimmunotherapy has attracted atten- tion because of their high linear energy transfer. Apart from the other beta emitters under investigation, Ag-111 has been investigated because of its physical characteristics, such as suitable half-life (7.43 d) and beta emission (68.0%) with high energy of 1.04 Mev and associated gamma imaging possibility. Ag-111 has been considered by us to be an appropriate candidate for radioimmunotherapy (4), especially under conditions in India, where there are inherent time lags between production of the iso- tope at the reactor at Bhabha Atomic Research Centre (BARC) and supply to distant areas of the country over 1000 km away.

Another consideration pertinent to the choice of an isotope is its avail- ability in carrier-free form because the stable isotopes compete with the radioisotope for the antibodies/chelate compounds. We have previously shown that it is possible to obtain silver-111 in practically carrier free form using the neutron irradiation reaction and bombardment of a Palladium target at the APSARA reactor of the BARC Bombay (5).

Inspired by the aforementioned facts and considering the existing problems, we have selected radiosilver as a candidate isotope for our present work (see Table 1) (4).

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Linking Radiosilver to Antibodies

Table 1 Characteristics of Radiosilver as a Candidate Isotope

3

Emission Radionuclide chosen and half-life fl 3'

Internal conversion

UlAg 0.73 (6%) 0.743 (1%) T1/2 = 7.5 d 0.81 (1%) 0.340 (6%)

1.06 (93%) m

m

PROBLEMS INVOLVED IN LINKING SILVER: POSSIBLE APPROACHES

It is known that silver can exist in three oxidation states I, II, and III, out of which (Ag-I) is found to be the most stable state. However, silver as a monovalent cation creates problems in forming stable complexes with the conventional bifunctional chelates like DTPA, EDTA, and so on.

Other possible agents like certain cryptates (Crown ethers, macro- cyclic agents) (6) and heterobifunctional agents such as paraaminobenz- oic acid (PABA) have been suggested that can form lipophilic chelates with the radiometal cations. The cryptates are macrobicyclic cages having a cavity in which the radiometal sits.

Further, according to Schwarenbach classification on the basis of rela- tive complexing ability of metals, silver and sulfur should link in a soft acid-soft base interaction, and the bifunctional chelate N-succinimidyl-3- (2-pyridyl-dithio) propionate (SPDP) can also be exploited for radiolabel- ing Ag-111 to antibodies. It should be possible to link silver to the sulfur naturally occurring in the antibody. Encouraged by the Thakur and De Fulvio technique (3) of linking technetium to disulfide groups in antibody, we successfully tried the simple reduction of disulfide groups in immuno- globulins to allow linkage of radiosilver (110 m) to monoclonal antibodies.

ASCORBIC ACID USE IN LINKING TECHNETIUM TO ANTIBODY (3)

1. Incubate 25-120 #g of the protein with 3500 times molar excess of ascorbic acid.

2. Adjust pH of ascorbic acid solution to 6.5 with equimolar solu- tion of sodium ascorbate.

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Table 2 Ratios of Reduced [gG to Silver

Wt of IgG Wt of ascorbic acid Wt of silver

Set I I rng 3 mg 0.54 ag (X1) Set II Img 3 mg 1.08 #g (X2) Set III I rng 3 mg 2.7 #g (X5) Set IV I rng 3 mg 10.8 #g (X20)

3. After 60 min incubation, reduce freshly eluted Tc-99m solution with 0.6-0.8 ~g/Na2S204 and further allow to incubate at 22~ for 30 min.

METHOD

In the present study, cold silver (108) is linked with immunoglobulin with the same procedure and the reaction product was dialyzed using cuprophane membrane with the model developed earlier (4) by us.

The linkage of silver (108) with the antibody reduced by ascorbic acid was estimated by dialyzing the reduced IgG silver reaction-product against distilled water. The effiux of silver was measured by taking aliquots of the dialyzate every 5 min. The amount of silver in the dialyzate was quanti- tated by precipitation with dilute hydrochloric acid followed by gravi- metric estimation of the precipitate. For control purposes, silver and the silver + IgG mixture were also similarly dialyzed. The reaction was per- formed using the ratios of reduced IgG to silver as shown in Table 2.

It was observed that the efflux of silver in the dialyzate was negligible compared to the control, indicating that ascorbic acid reduced IgG was effectively and stably linked with the silver.

RADIOTRACER EXPERIMENT

In order to quantitate the linkage of silver with antibody it was con- sidered useful to set up a radiotracer experiment. Since the half-life of Ag-111 is only 7 d, we thought that longer lived isotopes of silver would be useful for these studies. Possible longer lived isotopes of silver are listed in Table 3.

Of these, 110-m silver was selected because of its long half-life, per- mitting not only chemical but also detailed pharmaceutical and biodistri- bution studies.

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Linking Radiosilver to Ant ibodies

Table 3 Possible Isotopes of Silver

5

Isotope and half-life Emitted radiations and their frequency

l~ Er (Kev) 64.0 280.4 344.5 443.4 T1/2 = 41.3 d lr (%) 10.5 29.5 40.9 11.4 l~ Er(Kev) 406.2 429.2 451.0 411.8 T,/2 -- 8.5 d lr (%) 13.5 13.2 28.4 88.2

Er(Kev) 748.4 804.3 824.7 1045.8 lr (%) 20.7 12.4 15.4 29.7

11~ Er(Kev) 657.7 677.6 706.7 763.9 T1/2 = 255 d lr (%) 94.7 10.7 16.7 22.4

Er(Kev) 884.7 937.5 1384.0 1505.0 lr (%) 72.9 34.3 24.3 13.1

644.5 11.4

616.2 21.7

1128.0 11.8

717.3 29.1

1199.4 1527.0 11.3 16.4

EXPERIMENT WITH AG-110M

One mill igram of immunog lobu l in was reduced with 3 m g of ascorbic acid, this co r responded to a molar excess of ascorbic acid 3/176:1/200,000, 1:2.93 x 10 -4 (dissolved in 500 #L of distilled water). This was reacted with Ag-110m (1 mCi) in a vo lume of 1 mL cor responding to a ratio of 1 a tom of radiosi lver/molecule of immunoglobu l in . The reaction mixture was dialyzed and this s h o w e d that 50% of the silver had been b o u n d to the reduced IgG. The product was also submi t ted to co lumn chromatog- raphy on a PD-10 Sephadex co lumn (Pharmacia, Uppsala, Sweden) as well as paper chromatography . The results are s h o w n in Fig. 1. Each frac- t ion is two drops, i.e., 50 #L. C o l u m n was pre-equil ibrated wi th 1% BSA. The reaction produc t was t ransferred to a PD-10 co lumn and eluted wi th distilled water containing 1% BSA.

The reaction produc t was also subjected to paper ch roma tography us ing W h a t m a n filter paper No. I and 90% methano l in distilled water as a solvent. Care was taken that all the media were free of chloride ions. Results are s h o w n in Fig. 2.

DISCUSSION

It appears that there is significant b inding of the radiosilver with the reduced immunoglobu l in , but the nature of b inding remains moot , as NMR studies to fur ther characterize the b inding are awaited. It is also p l anned to alter the ratio of silver, ascorbic acid, and immunog lobu l in from the present ratio (1:1:1) to see whe the r b inding can be enhanced .

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200 COUNTS PER MINUTE

160

100

50

0 I | I I I I I I t I I I I I I I I I I

2 3 4 5 6 7 8 g 10 11 12 13 14 15 16 t7 18 19 20

CHROMATOGRAM Fig. 1. PD-10 chromatography of Ag-110m with reduced IgG. Each fraction is

two drops, i.e., 50 #L. Column was pre-equilibrated with 1% BSA.

COUNTS PER MINUTE (Thousand,s) 200

150

100

60

I l I I I I • �9 L L l

2 8 4 6 6 7 8 g 10 11 12 13 14 16 16 17 18 lg 20

CAqROMATOGRAM

Fig. 2. Radiochromatography complex formed by Ag-110m + reduced IgG. Each fraction is two drops, i.e., 50 #L. Column was pre-equilibrated with 1% BSA.

Further, the stability of the Ag + IgG complex under physiological conditions when exposed to an environment where chloride is ubiquitous needs to be examined. Biodistribution studies are also planned.

ACKNOWLEDGMENTS

This work was supported by DST, Govt. of India, Research Project No. SP/SO/B.10/86.

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Linking Radiosilver to Antibodies

REFERENCES

1. Hazra, D. K., Dass, S., Lahiri, V. L., Kumari, M., Saran, S., and Singh, R. (1986) Br. J. Cancer 54, 550,551.

2. Hazra, D. K., Chaturvedi, G. K., Hazra, P., Sachdeva, Y., Saran, S., Wahal, P. K., and Lahiri, V. L. (1991) Int. Hammersmith Symposium, Greece, May 7-12.

3. Thakur, M. L. and De Fulvio, J. D. (1990) Biotechniques 8, 512-516. 4. Hazra, D. K., Doss, S., Saheb, D., Arvind, B., Kumari, M., Lahiri, V. L.,

and Chaturvedi, G. K. (1986) In Vitro Model for Assessing Bifunctional Chelates for Radioimmunoimaging, Society of Nuclear Medicine 18th Annual Con- ference (Souvenir) India, Abst. No. 67.

5. Hazra, D. K., Lahiri, V. L., Kumari, M., Khanna, P., Arvind, B., and Singh, R. (1988) Radiosilver (111 Ag) Separation for Labeling Monoclonal Antibodies for Radioimmunotherapy. Proceedings of the Hammersmith Symposium on the Advances in the Application of Monoclonal Antibodies in Clinical Oncology, Royal Postgraduate Medical School, London, May 25-27.

6. Burgerweister, W. and Oswatish, R. W. (1977) Quoted in 8th International Hammersmith Meeting on Advances in the Application of Monoclonal Antibodies in Clinical Oncology, May 8-13; Porto Carras, Greece: Role of Alcoholic Solvents in Silver Complexation, Hazra, D. K. and Chaturvedi, G. K., et al. (1991).

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