WO1987004164A1 - Technetium-antibody conjugate - Google Patents

Abstract

A conjugate of technetium with a radical having an antigen binding site wherein the technetium thereof is radioactive.

Description

TECHNETIUM-AHTIBODY CONJUGATE This invention relates to a technetium-antibody conjugate. The present invention provides a conjugate of technetium with a radical having an antigen binding site uiherein the technetium thereof is radioactive. The preferred technetium isotope is ^99mTc. In a particular aspect the present invention provides a technetium-antibody or antibody fragment conjugate which is preferential ly absorbed by a tumour cell as compared to a non-tumour cell. Preferably the conjugation is via a sulphide linkage. The present invention also provides a compound of formula Ab-Y-S-NTc(Hal)₃ where Hal is chlorine, bromine or iodine and including mixed halides, and Y is a conjugating chain and Ab is an antibody radical or a radical having an antigen binding site. In a preferred instance Y is of the formula

X Z " ' -[NH-C-(CH)_n]_z wherein Z is H, alkyl, aryl, carboxy,halide hydroxy or amino, n is 1-10, X is NH, O or S and z is 0 or 1. Alkyl groups preferably 1 - 6 carbon atoms, aryl groups preferably 5 - 16 carbon atoms. The present invention also provides compounds of formula Ab-S-NTc(Hal)₃ Ab-NH-Y-S-NTc(Hal)₃ wherein Ab, Ab-NH or Ab-S represents an antibody radical or a radical having an antigen binding site and Y and Hal have the meaning given above. The present invention also provides the intermediate compounds Ab-SH Ab-NH-Y-SH wherein Ab, Ab-NH or Ab-S and Y have the meaning given above. Compounds in accordance with this invention may be produced by taking one of said intermediate compounds and reacting with TcN(Hal)₄ wherein Hal has the meaning given above. The intermediate compounds may be formed by a) reducing an antibody to form free sulphydryl groups. Such reduction may be effected in a number of ways but it is presently preferred to use dithiothreitol (DTT), b) reacting an antibody succinimidyl pyridyldithiopropionate (SPDP) or an analogue thereto appropriate to the compound desired to obtain an antibody conjugate containing a -S-S-group, reducing the conjugate to form a -SH group, c) using S- acetylmercaptosuccinic anhydride (SAMSA) or SH introducing compounds to produce a side chain on an antibody containing a -S-linkage and reducing to form a -SH group, It is preferred that said radical is an antibody. The antibody may be a monoclonal antibody. Antibodies useful in the present invention included those showing specificity for breast, brain, melanoma, lung, pancreas and colon tumours. The antibody may be an intact immunogobulin or a fragment of an immunogobulin maintaining a sufficiency of an antigen binding site such that it is preferentially absorbed by a tumour cell as compared to a non tumour cell. Thus, in addition to whole antibodies, it is also possible to utilize F(ab')₂ and F(ab') fragments. Still further antibody polymers such as antibody pentamers IgM and derivatives of these such as immunogobulin monomers may be used. Also useable are IgG_2a, I_gG_2b, IgG₁ and IgG₃. The compounds of this invention may be combined with pharmaceutically acceptable carriers. The mode of administration of the compounds of this invention will be as selected. In particular, the compounds of this invention may be administered intravenously, intraperitonealy, intrapluraly, intrapericardialy, intracerebospinal fluid and subcutaneously. The technetium-antibody conjugates of the present invention may be formed into pharmalogical compositions with appropriate pharmaceutically acceptable diluents.

The technetium-antibody conjugates of the present invention are useful for in vivo detection of tumours such as by immunoscintigraphy.

Part A

Radioactive isotopes of technetium coupled to MoAb have been used by us in the search for specific methods of diagnosing sma l l tumours. In this l ight we have successfully located tumours in both mouse and man, and antibodies have been administered either intravenously or subcutaneously, or by other routes. It is clear that radiolabelled MoAb can indeed localise in tumours in vivo and with the use of computer assisted tomography, with subtraction for non-specific effects, this method can then be utilized for the specific detection of tumours - both primary and secondary. However, there are problems of specific activity, specificity and high blood background which need attention before this technique can be accepted as a usefu l diagnostic tool. Major advances in the diagnostic radiolocalization of tumours should result from the production of better MoAb , better methods of radiolabelling and finally, design of methods to reduce the background provided by circulating radiolabelled antibodies. It is expected that more specific antibodies wil l become available with time. We have shown that the use of second antibody (anti-immunoglobulin), is able to clear the circulating pool of antibody, and thereby significantly lower the background.

Prior use by us of ^{1 31} I, or a combination of ^{1 31} I, or a combination of ¹³¹I and ¹²⁵I in experimental models has shown that ¹²⁵I cannot be used in man because of high tissue attenuation but there are however serious drawbacks with the use of ¹³¹I. This nuclide provides a poor quality image, it produces significant radiation exposure due to its beta emissions and has a short biological half life - presumably due to the de-iodination of the MoAb. The use of ^99mTc for label ling MoAb as in this invention of f ers several advantages: it has a reasonably short half life; it is cheap, easy to produce, and is readily available. The isotope has an optimal gamma energy (140keV) for detection with currently available scintigraphic instrumentation and produces very little radiation exposure to patients undergoing scanning procedures. Materials and Methods Mice: Mice used were: RF/J, C57BL/6 and ( C57BL/6 x BALB/c)F₁ (=BCF₁) bred in our colony. Athymic, BALB/c mice (nu/nu) were obtained from the Royal Dental Hospital (Melbourne, Australia). Tumour Cell Lines: Two tumour cell lines were used: one, the E3 clonal variant of the thymoma ITT(1) 75 NS(1) which was obtained by three successive rounds of fluorescent activated cell sorting of ITT(1)75NS cells stained with monoclonal Ly-2 antibodies and selected for the most fluorescent 1 % of cells. The murine cell line E3 was maintained in vitro in DME supplemented with 10 % heat inactivated newborn calf serum (Flow Laboratories, Sydney, Australia), 2mM glutamine (Commonwealth Serum Laboratories, Melbourne, Australia), 100 IU panicillin/ml and 100 mg streptomycin/ml (Glaxo Laboratories, Melbourne, Australia). E3 cells were washed twice in DME (without additives) and twice in DME containing 0.5 % BSA and used in the in vitro binding assays. The E3 cell line was maintained in vivo by the passaging of cells from ascites fluid produced in BCF₁ mice. Ascites were washed in DME and PBS, solid tumour grew after the s.c. injection of 10⁶-10⁷ cells. The second cell line used was a human colonic carcinoma, COLO 205 (2), maintained in culture with RPMI containing the same additives; adherent cells were harvested with 0.125% trypsin (Commonwealth Serum Laboratories, Australia) washed with RPMI and injected s.c. into nude mice, where tumours appeared after the injection of 2 x 10⁶ - 1 x 10⁷ cells. MoAb: Two MoAb were used: (i) anti-Ly-2.1 (IgG2a), an antibody raised against the murine alloantigen Ly-2.1 (3); and (ii) 250-30.6 (IgG2b), an antibody to human colonic secretory epithelium (4). The MoAb were isolated from ascitic fluid by precipitation with 40% ammonium sulphate, followed by dissolution in 0.01 M Tris buffer pH 8.0 and extensive dialysis against the same buffer and further purif ied by af f inity chromatography using protein -A Sepharose (Pharmacia Inc., Piscataway, NJ, U.S.A.)and purity was confirmed by gel electrophoresis and antibody activity assayed by a rosetting test (5).

^{9 9m}Tc Label l ing of MoAb) : Sodium Pertechnetate Injection B.P. produced from fission product chromatography generators was used for all preparations. Generators were obtained either from the Australian Atomic Energy Commission (Lucas Heights, Sydney, Austral ia), or from Mal linckrodt

Inc. (St. Louis, MO. USA). MoAb were label led with ^99mTc using two methods - the new method described herein, and a method using stannous chloride.

(a) Labelling using ^99mTcNCl₄ - : ^99mTcNCl ₄ - was prepared as a dry salt residue as described in detail in (9). For labelling, the MoAb was first reduced with the addition of DTT (20 microl, 115 mg/ml in PBS) to 200microg of MoAb (1 mg/ml in PBS) and allowing the mixture to stand at room temperature for 30 minutes when the reduced MoAb was separated from DTT by gel chromatography using 0.1 M sodium acetate pH 4.0 as eluant on an 8cm x 1cm column of Biogel P-6 (Biorad Laboratories, Richmond, U.S.A.). The fractions (1 ml) containing the protein peak were added to the dried ^99mTCcl₄- salt residue and the mixture brought to pH 3.0 with 0.2M hydrochloric acid; after 2 minutes at room temperature, 0.1 M sodium phosphate was added and the pH adjusted to 7 by the careful addition of sodium hydroxide. Purification of the labelled MoAb was then achieved by gel chromatography with a Sephadex G-25 co lumn ( PD-10, Pharmacia).

(b) Stannous Chloride Reduction Method: Stannous chloride (20-200 microg) was added to 200 microg MoAb (1mg/ml), 4-6 mCi of pertechnetate was added, and the mixture allowed to stand at room temperature for 30 minutes. The labelled MoAb was purified by gel filtration on a PD-10 sephadex column. Radioiodination of MoAb: MoAb (100 microg, 1 mg/ml) were labelled using the chloramine-T method (6): 2.5mCi of carrier-free Na ¹²⁵I (Amersham International Ltd., Amersham, England) and 3 microl of chloramine-T (1 mg/ml) were mixed with protein for 2 minutes at room temperature and the reaction then terminated by the addition of 3 microl of sodium metabisulf its (2.4 mg/ml). Iodinated MoAb was separated from free iodine by gel filtration using a PD-10 column. Serological Analysis: A binding assay was developed to determine the stability and specificity of the ^99mTcN-MoAb complexes. MoAb complexes were tested in one of two ways - either a) using one MoAb and two cell lines; or b) using two different MoAb and one cell line - both MoAb being labelled identically, one being reactive with the cell line, the other non-reactive. Polyvinyl chloride 96 well plastic plates (Pynatech Laboratories, Inc., Alexandria, Va) were washed with 1% bovine serum albumin (BSA) in PBS. In this assay either the number of cells or the quantity of MoAB could be kept constant while the other was varied. Either: (1) after serial dilution of 25 microl of the labelled MoAb, 1 x10⁷-2x10⁷ target cells (either tumour cell lines or thymocytes) suspended in PBS were added to each well; or (2) different cell numbers (10⁸- 2 x 10⁴) contained in the same volume of the target cells were added and an equal amount of radiolabelled MoAb was then added to each well. In both assays the contents were mixed and the plate incubated on ice for 30 minutes; after 3 washes, the plates were dried and the cell pellets were counted for one minute in a gamma counter. All assays were performed in duplicate. Biodistribution: Nude mice bearing COLO 205 xenografts or BCF₁ mice bearing the E3 thymoma were used. The first study compared the distribution of two ^99mTcN-MoAb in BCF₁ mice; groups of 4 mice were sacrificed at 20 hrs, 30 hrs and 35 hrs after the injection of labelled MoAb. The second study compared the binding of a ^99mTcN-MoAb complex to two different tumours - the E3 thymoma and COLO 205 xenografts. In these studies, the biodistribution of ^99mTc label was determined by gamma counting of blood, heart, lungs, spleen, stomach, intestine, kidneys and tumour from these mice. The distribution of isotope is reported as a localization ratio (tissue (cpm/g)/blood (cpm/g)). All mice received approximately 115 microCi of ^99mTc activity (approximately 10 microg of protein) by tail vein injection. Imaging : BCF₁ mice bearing the E3 thymoma (0.3 1.0cm) were given i.v injections of either approximately 115 micro Ci of ^99mTcN-labelled anti-Ly-2.1 (specific MoAb) and 200 micro Ci (4microCi/micro g) of ¹²⁵I labelled anti colon (non-specific MoAb) or 115 micro Ci of ^99mTc-labelled anti colon MoAb. Twenty-seven hours after injection mice were anaesthetised by intraperitoneal injection of 4% chloral hydrate (0.01 ml per g body weight). Vertical views of the mice were taken using a Toshiba GC 402A gamma camera and a low energy parallel hole collimator. A setting of 50 keV with an 80% window and 140 keV with a 20% window was used to image the ¹²⁵I and ^99mTc photons respectively. Data were stored in digital form by an MDS Modumed computer.

RESULTS

The study was conducted in three phases: the establishment of the conditions for the coupling of ^99mTcNCl₄-to MoAb; the testing of the stability of the TcN-MoAb complexes in vitro; and the measurement of the distribution of the complexes in vivo to determine whether tumours could be detected specifically. 9^9mTc-MoAb (SnCl₂) : ^99mTc labelled anti-Ly-2.1 MoAb was tested in a binding assay using thymocytes from two strains of mice - RF/J (Ly-2.1 ⁺) and C57BL/6 (Ly-2.1-). In this assay the amount of labelled MoAb added to each well was kept constant, the cell number varied (10⁵-10⁸ cells per well). There was clearly no noticeable dif ference in the uptake of radioactivity by the two cell types i.e. the ^99mTc-MoAb complexes bound to non reactive and reactive cells equally. Varying the amount of SnCl₂ used in the reduction procedure had no effect on the result. This is shown in Fig.1 which shows the binding of ^99mTc labelled anti-Ly-2.1 prepared by SnCl₂ reduction of RF/J and C57BL/6 thymocytes. The amount of radioactivity incorporated as a function of cell number is shown. 9^9m TcN-MoAb: An alternative method of labelling was designed, utilising the unique property of ^99mTcNCl₄- to form a stable covalent linkage to sulfur atoms. MoAb were partially reduced with DTT to generate free sulfhydryl sites and mixed with the ^99mTc Cl₄ -, leading to the formation of ^99mTcN-MoAb complexes. The ^99mTcN-MoAb complexes were then tested in different serological assays to determine whether the label ling procedure damaged or altered the binding or specificity of the MoAb, and whether the complexes formed were stable.

The binding curve obtained when ^99mTcN-labelled Ly-2.1 MoAb was incubated with thymocytes from mouse strains RF/J (Ly-2.1 ⁺) and C57BL/6 (Ly-2.1 -) is shown in Fig.2 which shows the specific binding of anti-Ly-2.1 labelled with ^99mTcNCl₄- on RF/J and C57BL/6 thymocytes; Amount of radioactivity bound as a function of antibody in the reaction mixture. There was clearly a major difference in the binding of the labelled MoAb to the reactive cells (RF/J) when compared to the non-reactive cel ls (C57BL/6) with specific ratio (=cpm RF/J / cpm C57BL/6) of greater than 20. Thus with the use of the one antibody and two difference sources of cells the procedure used to couple ^99mTcNCl₄- to MoAb produced a stable complex which bound only reactive target cells as shown in Fig.2.

The results are clearly superior to those obtained with the complex formed with the use of SnCl₂. In this respect, reference is made to Fig.1 which shows the binding of ^99mTc labelled anti-Ly-2.1 with SnCl₂ reduction on RF/J and C57BL/6 thymocytes; amount of radioactivity incorporated as a function of ce l l number. In the second assay, two different MoAb, one directed against colonic secretory epithelium (250-30.6) and the second, the anti-Ly-2.1 MoAb, were label led with TcNCl₄- under identical coupling conditions and the two complexes were tested for their ability to bind to the murine T cel l thymoma E3;, which is Ly-2.1⁺ but does not react with the anti-colon MoAb (250- 30.6). Again the specific MoAb complex ^99mTc Ly-2.1 bound more efficiently than the TcN-anti-colon complex as shown in Fig.3 with a specific ratio of 10. Fig.3 shows the specific binding of ^99mTcNCl₄- laballed anti-Ly-2.1 and anti-colon Mo-Ab on ITT(1) 75NS E3 target cells. Thus in these assays, it appeared that a stable bonding of ^99mTc to MoAb had been produced, so that only the binding of antibody to the appropriate target cell was detected.

Stability of ^99mTcN-MoAb: Aliquots of ^99mTc labelled Ly-2.1 MoAb were stored at 4°C for 20 hrs and then tested in binding assays with RF/J and C57BL/6 thymocytes. No loss of binding reactivity was observed when the binding curve (as shown in Fig 4) was compared with that obtained at 6 hrs (as in Fig.2). Fig.4 shows the binding of anti-Ly-2.1 labelled 24 hours previously with ^99mTcNCl₄, on RF/J and C57BL/6 thymocytes. Effect of ^99mTc Concentration on the Activity of ^99mTcN-MoAb complexes: At the time when ^99mTc is obtained from a ^99mTc generator, there is approximately 0.7 micro g

^99mTc/ml of ⁹⁹Tc eluted (8). As the number of labelled binding sites on the antibody molecule is determined by the chemical quantity of technetium present, the effect of labelling with increased quantities of Tc may be studied by the addition of ^99mTcCl₄- carrier to the ^99mTcO₄ - used for labelling. The addition of 2 micro g ⁹⁹Tc to the reaction mixture was thus equivalent to increasing the ^99mTc activity used by a factor of 200. This approach was adopted to avoid the radiation hazards associated with the handling of high levels of activity and to overcome "dead-time" problems which would arise in the gamma counting of very high activities.

The binding curves obtained using ^99mTcN-anti-Ly-2.1 containing added ⁹⁹Tc carrier are shown in Fig.5. Fig.5 shows the binding of two ati-Ly-2.1 conjugates - one containing added Tc carrier, the other carrier free on

RF/J and C57BL/6 thymocytes. The binding observed to RF/J

(Ly-2.1⁺) and C5781/6 (Ly-2.1 -) cells was the same as that observed for the preparation containing no added ⁹⁹Tc as in Fig.2 and again, a specificity ratio of greater than 20 was observed. Hence these results show that increasing the specific activity of the preparation by a factor of 200 would not effect the binding specificity of the MoAb.

Biodistribution: The in vivo localization and biodistribution of ^99mTc-MoAb complexes was examined by injecting mice with ^99m-Tc-MoAb and determining the relative amounts of radiolabel accumulated in the tumour or the tissue. These results were used to calculate the localization ratio derived as follows: tissue (cpm/g) / blood (cpm/g).

In the first study, two groups of 16 BCF₁ mice bearing the E3 (Ly-2.1⁺) tumour (0.23 - 1.11g) were injected i.v. with either ^99mTc-Ly-2.1 or ^99mTc-250-30.6 MoAb - each mouse received 115 micro Ci ^99m Tc and 10 micro g MoAb. Four mice from each group were sacrificed at different time intervals after injection (20, 30.5, 35 hrs) and the distribution of the two MoAb determined. After 20 hours, the tumour localization was observed to be 3 times greater for the specific MoAb than that observed for the non specific antibody Ly-2.1 ) with the localization ratios in liver, spleen and kidney being less than or similar to that of b l ood. The non-specific antibody (250-30.6) the localization ratio of the liver, spleen and kidney were observed to be higher than that of the blood and at 30.5 hours the liver localization ratio was 5 times greater than that of blood - the reason for this high ratio is unknown, but may be due to the different reactivity of the MoAb.

In the second study, the localization of ^99mTc-Ly-2.1 was compared in two different tumours -nude mice bearing Colo 205 xenografts (Ly-2.1 -) or the E3 thymoma (Ly-2.1⁺) were used. After 20 hours the E3 thymoma (Ly-2.1⁺) was observed to take up to 4 times more radioactivity than the Colo 205 xenografts (Table 1) and there was at least 4 times more radioactivity in the tumour than in other tissues - except the liver. The ^99mTc-MoAb complexes coul d specifically localise to tumours in vivo. Radio-Imaging A. Nuclear image obtained after the injection of ^99m Tc-anti-Ly-2.1 into a mouse bearing the E3 thymoma on the right thigh (as visualized).

B. Nuclear image obtained after the injection of ^99mTcNanti-Ly-2.1 into a mouse hosting a thymoma on its left anterior side and ¹²⁵I-anti-colon.

C. ¹²⁵I nuclear image of the same mouse as in B.

In the initial imaging experiments, tumours (0.23-1.2g) could be visualized with the use of a small animal scanner as early as 2 hrs after injection of the specific ^99mTcN-MoAb (results not shown) the visualization became well defined with time. The mouse in B. had an E3 tumour (1.0cm in diameter) which was easily seen as a distinct single entity on the right hind leg. The tumour was dissected and found to have a localization ratio (tumour to blood approximately 2.0). Radioactivity in this image is also concentrated in the central region of the mouse, indicative of significant distribution of antibody to large vascularized organs such as liver, lung and heart; a phenomenon that tends to obscure visualization of small tumours. The second image B. was of a mouse with a much smaller thymoma (0.4cm in diameter) on the left anterior side and again the tumour is again clearly visualized. The tumour was dissected and found to have a localization ratio of approximately 1-2. At the time of injection with ^99mTc-MoAb this mouse was also injected, C., with a non-reactive MoAb (250-30.6) labelled with ¹²⁵I, however scanning failed to localize the tumour with this MoAb. It was noted that the tumours were readily visualized and that the contribution of the reactive MoAb to the overall blood pool radioactivity did not obscure the visualization of the tumour and consequently a computer assisted subtraction of the image provided by the non-reactive MoAb (^{1 25}I labelled) - the control blood pool was not required. Discussion

Immunoscintigraphy, with the use of radiolabelled MoAb is a new method for the in vivo detection of tumours and thus cancers of colon, breast and other tissues have been detected with some degree of success. However there is not a marked increase in the detection rate of tumours, except in a few cases only previously identified tumours could be detected and clearly the sensitivity of the procedure needs to be increased. As the specificity and sensitivity is determined by the ratio of the amount of radiolabelled MoAb bound v ersus the background blood pool , the ways of increasing this ratio are either to directly increase the primary signal (by altering MoAb and/or isotope) or to reduce the background. We are adopting both approaches through the use of multiple MoAb and MoAb fragments (Fab or F(ab')₂). In this manuscript we report on the advantages of using ^99mTcN-MoAb to detect tumours. In this study a new method of coupling ^99mTcNCl₄ to MoAb and the subsequent use of these complexes to detect tumours in vivo is described. As previously indicated, ^99mTc offers a number of advantages for radioisotopic localization studies in patients, as it has a short half life (6 hrs) and thus limits the radiation exposure to patients and has an optimal gamma energy (140 keV) ideal for currently used scintigraphic instrumentation. ^99mTcO₄- from a portable generator and must be reduced prior to coupling with MoAb. Many methods have been described for the reduction of pertechnetate; these procedures generally lead to the reduction of pertechnetate to the Tc (IV) or TC (V) oxidation state. At present, the most frequently used reducing agent for preparation of ^99mTc labelled compounds is SnCl₂. Problems have been experienced with this agent when used f or l abel ling MoAb, such as hydrolysis, instability towards oxidation and competition of Sn for binding sites. Indeed, in our hands pertechnetate reduced with SnCl₂ readily bound to MoAb but such complexes showed no specificity when tested in our in vitro binding assay or when tested in vivo. Because of the problems experienced with the ^99mTc labelling of MoAb with the use of SnCl₂- we have used ^{99 m}Tc NCl _4A - to produce ^{99 m}TcN-MoAb by a substitution reaction. Important features of our study have been to show that MoAb may be labelled without loss of activity to yield a highly specific complex which retains its stability far at least 24 hours and which yield superior results when tested in vivo by immunoscintigraphy. Tumours could be visualised as early as two hours after injection, and small tumours (approximately 0.4cm in diameter) located near large vascular organs could be visualised without the need for blood pool subtraction.

The complexes formed with the use of TcNCl₄- are clearly different to those made using SnCl₂ as the presence of the nitrido group attached to Tc modifies the chemical behaviour of the Tc atom and makes it more favourable for co-ordination with certain ligands. Ligands which bind through sulfur atoms form more stable complexes with the TcN core than do ligands binding through nitrogen. In our initial experiments, attempts were made to bind the TcN group directly to amino groups of the MoAb and while ^99mTcN-labelling of MoAb was achieved, there was considerable loss of specificity. To utilise the known preference of the TcN group for sulfur atoms, we developed a partial reduction procedure used for the coversion of disulfide linkages to sulfhydryl residues. Such an approach was also attractive because it was known that the sulfhydryl groups involved in the coupling were likely to be distant from the sites responsible for antibody binding. Thus ^99mTCNCl₄- was prepared in a stable dry form without the presence of any contaminating metal ions and was successfully complexed to partially reduced MoAb in a simple one step procedure that resulted in a stable covalent complex which retained MoAb activity. It should be noted that the reduction step critical to this procedure used DTT to produce sulfhydryl residues on the MoAb. The chemical stability and activity of TcNCl₄- complexes was determined in several serological assays which involved MoAb reactive and non-reactive cells; either one MoAb, two different target cells or conversely, two MoAb and one call target. In all studies specific binding of radiolabelled MoAb to target cells was demonstrated, the complexes were not non specifical ly "sticky" nor unstable with the release of ^99mTc to bind to other non reactive target cells. In vitro studies have shown that ^99mTcNCl₄- may be used to produce chemically stable MoAb complexes that retain their activity for at least 24 hours. Furthermore these complexes may be prepared at a clinically useful specific activity without any changes in the in vitro properties. For example, it was possible to increase the amount of ^99mTc bound to b 200 fold without affecting MoAb activity (text fig.5).

An important finding obtained in the study was to demonstrate that ^99mTcN-MoAb complexes localized to tumours in vivo.

With the use of nuclear imaging equipment large tumours

(0.8 - 1.1 cm in diameter) could be easily visualised (A.) but the ultimate sensitivity of this technique lay in the detection of small tumours (0.3 - 0.6cm in diameter) that were located near vascular organs, such tumours being detected without the requirement of a blood pool subtraction

(B.). Incidentally, the same mouse received a simultaneous injection of a ^{1 25}I label led non-reactive MoAb and subsequent scans could not visualize the same tumour (C.). Similar results were obtained for mice scanned with a non-reactive TcN-MoAb, where tumours (0.6 - 1.5cm in diameter) could not be visualized.

Our studies showed that the ^99mTcNC_l4- complex could be successfully coupled to MoAb, in a simple one step procedure that resulted in stable covalent complexes which retained MoAb activity. An alternative ^99mTc labelling procedure described in the literature involves the coupling of DTPA to MoAb prior to the coordination of reduced technetium. There are however several difficulties with this procedure. As metal ions are able to compete with ^99mTc far the DTPA coordination sites, reduction systems using heavy metals are thus undesirable. For this reason sodium dithionite has f ound f avour as a reducing agent f or this system. Hydrolysis of reduced Tc still remains a problem. Another difficulty is the limitations produced by the number of DTPA molecules that can be coupled to the MoAb before activity is affected. This leads to a restriction on the specific activity of the labelled MoAb. Our method eliminates the need for DTPA coupled MoAb and the ^99mTcNCl₄- used for labelling is free of any heavy metal contamination, thus enabling high specific activity to be obtained.

Thus TcNCl₄- monoclonal antibody can be simply produced and have high activity for specifically localizing tumours both in vitro and in vivo. At present we consider the coupling method to be superior to other methods of coupling ^99mTc to antibody and the immunoscintigraphic findings to be superior to that obtained with radiolabelled iodine. On this basis we are now conducting a chemical trial to determine the clinical usefulness of the new reagent.

TABLE 1 Biodistribution and Localization of a Tc radiolabeled anti-Ly-2.1 in BCF₁ mice bearing the E3 thymoma and nude mica bearing COLO 205 tumor xenografts.

Tissue Localizatie Ratio E3/COLO 205 Ratio

ITT(1)E3 COLO 205

Blood 1.0 1.0 1.0 Tumor 1.23 0.30 4.10

(.39 - 1.11 g) (0.5 - 1.5g)

Stomach 0.08 0.2 0.04

Spleen 0.59 0.60 0.98

Kidney 0.77 0.76 1.01

Heart 0.33 0.39 0.35

Liver 0.84 0.56 1.50

Lung 0.38 0.42 0.90

Intestine 0.09 0.15 0.50

Referencee

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8. BONNYMAN, J. Effect of milking efficiency on ⁹⁹Tc content of ^{9 9 m}Tc deriv ed from ^{9 9m}Tc generators. Int. J.Appl.radiat.Isot.1983;34:901-906.

9. DeLAND, F.H., KIM.E.E., CORGAN R.L., CASPER, S., PRIMUS, F.J. SPREMULLI, E., ESTES, N. and GOLDENBERG, C.M. Axillary Lymphoscintigraphy by Radioimmunodetection of a Carcinoembryonic Antigen in Breast Cancer. J. Nucl. Med.1979; 20 :1243-1250. Part B We now describe the use of the above method to label a panel of MoAb and these have been tested to show that the procedure established has general use, and can be used to label all subclasses of MoAb with ^99mTc with retention of

Immunoreactivity; the complexes formed can be used to localize tumors in vivo. MATERIALS AND METHODS Mice: RF/J, CBA, AKR, C57BL/10(B10), BALB/c, C57BL/6(B6), and (C57BL/6xBALB/c) F1 (B6CF1) mice were brdd in our colony. Tumor Cell Lines: Human tumor cell lines (CEM and Bordin - an EBV induced B cell line) were cultured in RPMI 1640 medium with L-glutamine. BW 5147 and several clonal variants (E3,D1) of the murine thymoma ITT(1)75NS (20) were cultured in DME with L-glutamine. The clonal variant ITT(1)75NS.E3 (E3) was maintained by serial passage in ascitic fluid in (B6CF1) mice. For imaging experiments 10⁶ -10⁷ cells injected subcutaneously into B6CF1 mice and reached a size of 0.5-1.0 cm in diameter prior to experimentation. Monoclonal Antibodies: The details of MoAb are shown (Table 1). IgM antibodies were isolated from ascitic fluid by dialysis against water at 4°C, after which the precipitate was collected and resuspended in phosphate buffered saline (PBS, pH 7.3): IgG antibodies were prepared by precipitation with 40% ammonium sulfate [NH₄(SO₄)₂], followed by dissolution of the preparation in 0.01 M Tris buffer (pH 8.0); after dialysis against the same buffer, the IgG fraction was further purified by either: (i) adsorption onto Protein-A-Sepharose, washing with PBS and eluting with either 0.2 M glycine-HCl (pH 2.8) or citrate buffers (pH 5.0, 4.0, 3.0) and neutralization with saturated Tris, after which antibodies were dialyzed against PBS; or (ii) ion-exchange chromatography on DEAE-Sephacel and with elution using a linear gradient of 0.5 M NaCl In 0.01 M Tris buffer (pH 8.0). The purity of the antibody (>90%) was confirmed either by high pressure liquid chromatography (HPLC) or gel electrophoresis, and antibody activity assayed by a rosetting test (29) or by the immunoperoxidase method on tissue sections (27). Preparation of ^99mTcNCl₄- and Labelling of MoAb ^99mTcNCl₄- was prepared as a dry salt residue (18). Sodium azide (15-20 mg) was added to 1 ml sodium ^99mTcO₄-, produced from a fission product chromatography generator, in 6-7 mls of concentrated hydrochloric acid (specific gravity, 1.18). The solution was refluxed for 5 minutes to destroy excess azide and then evaporated to dryness in a rotary evaporator. MoAb were labeled with ^99mTcNCl₄- by one of two procedures: (i) 100-200mug of MoAb (1 mg/ml) was directly added to the dried ^99mTcNCl₄- salt residue, or (ii) a modified version of the method previously described (19) where 20 mul of dithiothreitol (DTT, 115 mg/ml) was added to 200 mug MoAb (1 mg/ml) and the solution allowed to stand for 30 minutes at room temperature; it was then transferred to Biogel P6 to remove unreacted DTT and the column eluted with 0.1 M sodium acetate (pH 4.0). The protein fraction (1.5 mis) was added to the dry TcNCl₄- residue, reacted for 2 minutes at room temperature prior to adjusting the pH to 7 with sodium hydroxide. Prior to use, the ^99mTcN- labeled MoAb was purified by passage over Sephadex G-25 (PD-10) and sterilised using a 0.22 mum membrane filter. Serological Analysis: In vitro cel l binding studies were performed on cultured tumor cell lines or mouse thymocytes (19). ^99mTcN- MoAb complexes were tested in one of two ways: (i) using one MoAb and two different target cells; or (ii) using two different MoAbs and one target cell line. The ability of the MoAb to bind to target cells was assessed after each step. In this assay cultured tumor cells or thymocytes (3x10⁵) were incubated for 30 minutes on ice with one of the following: (i) untreated MoAb; (ii) DTT treated MoAb; or (iii) ^99mTcN- labeled MoAb. The cells were then washed 3 times with PBS (0.5% BSA), resuspended in PBS and then treated with iodinated sheep anti-mouse immunoglobulin (¹²⁵I-SAM) for 30 minutes on ice. The cells were then washed 3 times with PBS (0.5% BSA) to remove unbound ¹²⁵I-SAM and the amount of ¹²⁵I-SAM bound determined. Immunoscintigraphy: Mice bearing E3 tumors were used in two studies. The first compared two identically labeled different MoAb and one tumor; the second compared a specific MoAb in mice bearing several tumors. B6CF1 mice bearing the E3 thymoma (0.5 - 1.0cm in diameter) were given intravenous injections of approximately 115muCi (12muCi/mug) of ^99mTcN- labeled anti-Ly-2.1 (specific MoAb) or anti-Ly-1.1 (nonspecific MoAb). Each animal was given an intraperitoneal injection of 4% chloral hydrate (0.01 ml/g body weight) imaged 4-28 hours after injection. Vertical views of the mice were taken using a Toshiba GC 42A gamma camera and a low energy parallel hole collimator using a setting of 140 keV with a 20% window to image the ^99mTc photons. Data were stored in digital farm by a MDS modumed computer. RESULTS Radiolabeling of MoAb with ^99mTcNCl ₄-: After radiolabeling, unbound reaction products were removed by passage of the final reaction mixture through a gel permeation column of Sephadex G-25 (PD-10). The yield of ^99mTcN passing through the column was then a measure of the success of radiolabeling and typical yields were 80-90%. (A typical example of the elution profile is shown in Figure 6). It was noted that 98-99% of the radioactivity present in the protein fraction could be precipitated with trichloracetic acid (TCA). Analysis of ^99mTcNCl₄- complexed to amino groups: The methods of complexing ^99mTcNCl₄- to MoAb were evaluated in serological assays to determine whether the labeling procedure used damaged or altered the binding or specificity of the MoAb. ^99mTcNCl₄- complexed directly to amino groups of Ly-2.1 MoAb was tested in a binding assay using thymocytes from 2 strains of mice: RF/J (Ly-2.1+) and B10 (Ly-2.1-). ^99mTcN-anti-Ly-2.1 achieved almost identical binding to both cell types (Figure 7a), with a specific ratio (cpm bound RF/J / cpm bound B10) of approximately 1.2. In a second assay ^99mTcNCl₄- directly bound to 2 different MoAb: anti-Ly-2.1 reactive with the E3 cell line, the other nonreactive (anti-colon carcinoma) produced a specific ratio (cpm anti-Ly-2.1 bound / cpm anti-colon bound) of 2-3 (Figure 7b). The conclusion is that the ^99mTcN-MoAb complexes produced in this way were either unstable or "sticky" and on exposure to target cells the ^99mTcN bound nonspecifically.

^99m_TcNCl₄- complexed to sulfhydryl groups after partial reduction: As the former label ling method gave law specificity (previously due to nan-specific labelling) an alternative method of labeling was designed, utilizing the known ability of ^99mTcNCl₄- to form a stable covalent linkage to sulfur atoms. MoAb were partially reduced with D T T to generate free sulfhydryl sites and mixed with the ^99mTcNCl₄-, leading to the formation of ^99mTcN-MoAb. These complexes were shown by sodium dodecyl sulfate polyaerylamide gel l1ectrophoresis (SDS-PAGE) to consist of intact IgG. The binding assay demonstrated ^99mTcN-MoAb complexes produced in this way to be specific and to yield workable specificity ratios. The E3 (Ly-3+) cell line bound

8-10 times more ^99mTcN-anti-Ly-3 than did the BID5147 (Ly-3-) cell line (Figure 8a). Similarly two different MoAb, one directed against colonic secretary epithelium (250-30.6) and the other directed against Ly-2.1 , were labeled with TcNCl₄- under identical conditions and the complexes were tested for their ability to bind to the murine thymoma E3 (Ly-2.1⁺, Ca Colon Ab-) (Figure 8b), when the reactive MoAb complex bound 6-8 times more efficiently than did the

^99mTcN-anti-colon complex. In separata experiments the ^99mTcN-colon MoAb, unable to bind to the E3 thymoma could bind reactive human tumor cell lines, Colo 397 and Colo 205 incorporating 90-250 times more radioactivity than nonreactiva control cells (Figure 9).

The partial reduction method was then used to radiolabel a panel of eleven different MoAb, including some of the same specificity but of different isotypes. All MoAb were testsd in the binding assay, and bound specifically to reactive target cells (Table 2). Four different Ly-2.1 MoAb were tested; there was a 10-15 fold difference between the binding of ^99mTcN-labeled IgG_2a and IgM anti-Ly-2.1 (monomer) MoAb on reactive target cells (CBA, RF/J) compared to that found with nonreactive target cells (BALB/c, C57BL/6), whereas the IgG₁ and IgG₃ Ly-2.1 MoAb produced specificity ratios of 30-50 and 50-70 respectively. Other MoAb were also highly selective e.g. anti-Ly-1.1 (IgG_2a) and H129-19 (IgG₁) produced specificity ratios of 70-130 and 110-130 respectively (Table 2). Radiolabeling of IgM MoAb (Pentamers) with ^99mTcNCl₄ -: In contrast to the preceding results obtained by direct labeling (using amino groups) the direct complexing of TcNCl₄- via amino groups with IgM class of MoAb produced ^99mTcN-MoAb complexes that gave 10 times more specific binding than similar complexes made with IgG MoAb. ^99mTcNCl₄- directly complexed with anti-Thy-1.2 (IgM) MoAb via amino groups produced specificity ratios of 15-20 (cpm bound CBA / cpm bound AKR) when tested on thymocytes from CBA (Thy-1.2⁺) and AKR (Thy-1.1⁺) strains (Figure 10). However the partial reduction procedure, complexing TcNCl₄- through sulfur atoms, resulted in superior specificity ratios, and CBA thymocytes (Thy-1.2⁺) incorporated more than 90 times more TcN-anti-Thy-1.2 than nonreactiv e AKR thymocytes (Thy-1.1 ⁺). The immunoperoxidase method was also used to assess the MoAb activity of two IgM MoAb, before and after labeling with ^99mTcNCl₄-: (a) 3E1.2, which reacts strongly with membrane and cytoplasm of breast carcinoma and with the luminal membrane of normal breast and (b) 5C-1 , which reacts with colonic carcinoma; the labeling procedure used did not significantly alter the binding ability of the radiolabeled MoAbs (Table 3). Immunoreactivity of ^99mTcNCl₄- labeled MoAb: It was necessary to show that the partial reduction procedure used to label MoAb with ^99mTcNCl₄ - did not significantly compromise the binding ability of the MoAb to bind reactive target cells and this was demonstrated in three ways. First it was important to assess the immunoreactivity of the MoAb retained after labeling and so the percentage of binding of the radiolabeled ^99mTcN-MoAb was determined. ^99mTcN-anti-Ly-2.1 was added to an increasing number of E3 cells (Ly-2⁺), and the amount of MoAb binding to the cells was determined (Figure 11 ). The degree of nonspecific binding was determined by running in parallel a nonreactive ^99mTcN-anti-Ly-1.1 isotype control labeled under identical conditions. The reactive ^99mTcN-anti-Ly-2.1 achieved significantly higher binding to E3 cells (60% in the plateau region) than the control ^99mTcN-anti-Ly-1.1 (5%) (not shown). Secondly the binding of unmodified MoAb and ^99mTcN-labeled MoAb was determined using the binding assay, where the amount of MoAb bound to reactive cells was measured using a second antibody (anti-immunoglobulin) which was iodinated and reactive with the first. Increasing concentrations of unmodified anti-Ly- 2.1, DTT treated anti-Ly-2.1 , ^99mTcN-anti-Ly-2.1 labeled to a high (100 muCi/mug) or low specific activity (12 muCi/mug) had equal binding capacity on E3 cells (Figure 12a), which suggests that this technique had not impaired the binding activity of the MoAb. This was also confirmed by the rosetting assay, when the initial MoAb titer of 1 :16,384 w a s unaltered after radiolabeling (Figure 12b). Binding of ^99mTcN-anti-Ly-2.1 to tumor cell lines withdifferent concentrations of Ly-2.1 : To clarify that the binding of the TcN-MoAb complexes was primarily dependent on the concentration of reactive antigen binding sites on the target cel ls, the binding assay was used, with 3 different tumor cell lines E3, 01 and 8-15147, that differed in concentrations of Ly-2 present on the cel l surface. E3 and 01 are high and low Ly-2⁺ variants of the ITT(1 )75NS cell line, and BW 5147 being Ly-2- was used as a control. The amount of binding was in proportion to the antigen density and ^99mTcN-anti-Ly-2.1 bound the E3 cell line 8 times more antibody than the D1 cell line and incorporated up to 100 times more radiolabel than did the nonreactivs BW 5147 cell line (Figure 13). Imaging: The four different Ly-2.1 MoAb (IgG₁, IgG_2a, IgG₃ and IgM) were used in imaging experiments using B6CF1 mice bearing E3 tumor grafts to determine which subclass best localized the tumor in vivo. In the first experiment mice with E3 tumor (0.82 cm in diameter) located on one thigh were given intravenous injections of ^99mTcN-anti-Ly-2.1 (IgG_2a) or ^99mTcN-anti-Ly- 1.1 (IgG_2a), the control antibody. Scintigrams images obtained 28 hours after injection demonstrated the specific localization of the ^99mTcN-anti-Ly-2.1. Radioactivity was concentrated in the central region of the mouse, indicative of the significant antibody distribution to vascularized organs such as the liver, lung and heart but the tumor was easily defined. Iilhen 99mTcN-anti-Ly-1.1 was used as a nonreactive isotype control, the definition of the tumor was poor relative to the images obtained with specific MoAb and only blood pool activity in the tumor was observed, with no specific localization. In another experiment B6CF1 mice hosting three E3 tumors were scanned 28 hours after the intravenous administration of 99mTcN-anti-Ly-2.1 (IgG2a), and all three tumors could be visualized. However the high blood pool activity hindered visualization of the tumor close to the vascular organs such as the heart and liver. The IgM Ly-2.1 MoAb (monomer) was used to specifically localized E3 tumors in B6CF1 mice, and mice with two E3 tumors (0.62 cm and 0.55 cm in diameter) were scanned 4 and 28 hours after an intravenous injection. Both tumors could be visualized 4 hours after injection, and the tumors became progressively better defined with time. From the scans obtained it was apparent that the IgG, Ly-2.1 MoAb resulted in superior images compared to the images obtained with the IgM MoAb and also those obtained with the IgG1 and IgG3 MoAb (data not shown). DISCUSSION Th e u s e o f ^{9 9 m}Tc a s a r ad io l ab e l f or immunoiscintigraphy with MoAbs has been advocated as it is one of the most useful radionuclides because of its ideal nuclear properties (T1/2=6 hr, energy 140keV, with no beta emmission). However little use has been made of this radionuclide for radiolabeling MoAb because of the complex chemistry involved in satisfactorily attaching it to antibody. Several different methods have been used to complex ^99mTc to MoAb. The first relied on the conjugation of the metallic radionuclide via bifunctional chelates, of proven success for ¹¹¹In in both experimental (5-8) and clinical application (9-11) but of little value for ^99mTc. The second approach involved the direct complexing of ^99mTc to MoAb to produce complexes with either amino groups or sulfhydryl groups, the latter shown to form stable complexes in vitro (14,15). However, problems are associated with the methods used to reduce ^99mTc prior to coupling with MoAb, such as the production of colloid and the instability of the ^99mTc-labeled MoAb complexes (16,19). We now describe a simple one step method of radiolabeling MoAb with ^99mTcNCl₄- based on a substitution reaction and have complexed ^99mTcNCl₄- to MoAb via two different ligands, either the amino groups or sulfhydryl groups and during our studies we observed that the two complexes behaved differently. The complexes produced by reacting TcNCl₄- directly with amino groups of MoAb was characterized by low specificity ratios as the ^99mTcN bound equally well to reactive and nonreactive target cells (Figure 7). The complexes formed with the use of sulfyhydryl groups are clearly different from those formed with the use of amino groups and ligands that bind through sulfur atoms produce more stable complexes. To utilize the known preference of the TcN group for sulfur atoms we developed a partial reduction procedure used for the conversion of disulfide linkages to sulfhydryl residues. Such an approach was also attractive because it was known that the sulfhydryl groups involved in the coupling were likely to be distant from the sites responsible for antibody binding.

Important features of our study here show that the coupling procedure used to attach ^99mTcNCl₄- to MoAb is simple, efficient and reliable and can be applied to a number of MoAbs of either IgG or IgM classes. There is no need for long incubation times required with the pretinning method necessary for the reduction of ^99mTc (16). The labeling efficiency of ^99mTcN-labeled MoAb ranged between 80-90% (Figure 6) and results obtained from TCA protein precipitation determinations of radiolabeled MoAb indicated that up to 98-99% of the ^99mTcN is bound. The labeling procedure adopted did not damage the binding specificity of the antibody molecule and did not alter the antibody antigen binding capacity (Figure 12) as can occur with the conjugation of metallic radionuclides via bifunctional chelates (6). Up to 60% of the radiolabeled preparation was able to bind specifically to target cell (Figure 11). Furthermore, the degree of antibody binding wa3 dependent on the antigen density of the target cells, hence the Ly-2HIGH E3 cell line bound 8 times more ^99mTcN-anti-Ly-2.1 than the Ly-2LOW D1 cell line (one has approximately B times the antigen density of the other) and 10 times more than the nonreactive BW5147 (Ly-2.1-) cell line (Figure 13). Finally, high specific activities were achieved which allowed the specific localization of labeled MoAb in the appropriately reactive murine tumors.

Specific localization by immunoscintigraphy of murine tumors was demonstrated with ^99mTcN-labeled MoAb by imaging studies and was illustrated in two ways. First, imaging studies showed that the E3 (Ly-2.1⁺) tumors were visible when mice were injected with reactive ^99mTcN-anti-Ly-2.1 whereas identical tumors could not be localized with nonreactive ^99mTcN-anti-Ly-1.1 an isotype identical control.

Second ^99mTcN-MoAb could specifically detect more than one tumor and this study showed that several tumors in the one mouse could be specifically localized. The method is useful to detect murine tumors and results indicate value in patients with cancer.

TABLE 1

Characteristics of murine monoclonal antibodies used for radiolabeling with ^99mTcNCl₄-

Antibody Antigen Antibody Class Antibody purification (Ref) Specificity and Subclass

anti-Ly-2.1 (21) Ly-2.1 ^IgG2a Protein A IgG₁ Protein A

IgG₃ Protein A

IgM(monomer) DEAE

anti-Ly-3.1(22) Ly-3.1 IgG₁ DEAE anti-Ly-1.1(23) Ly-1.1 ^IgG2a Protein A anti-Ly-15.2(24) Ly-15.2 IgG_2a DEAE anti-Thy-1.2 Thy-1.2 IgM(pentamer) NH₄(SO₄)₂

H129-19(25) L3T4 IgG_2a DEAE HuLy-m9 human transferrin IgG₁ Protein A receptor

250-30.6(26) colon IgG_2b Protein A 3E1.2(27) breast IgM(pentamer) precipitation in water 5C-1 (28) colon IgM(pentamer) precipitation in water

TABLE 2

Specificity ratios of ^99mTcN-labeled MoAb, prepared by the partial reduction of MoAb with DTT and obtained from in vitro binding studies.

Origin of

Antibody Target cells Specific

Designation Reactive Nonreactive Ratio1

anti-Ly-2.1

IgM(monomer) CBA BALB/c 10-15

IgG₃ CBA BALB/c 50-70

IgG₁ CBA BALB/c 30-50

IgG_2a RF/J C57BL/6 10-15

anti-Ly-3.1 E3 thymoma BW5147 7-11 anti-Ly-1.1 CBA BALB/c 70-130 anti-Ly-15.2 RF/J BALB/c 50-60 anti-Thy-1.2 CBA AKR 60-90

HuLy-m9 CEM E3 120-200

250-30.6 COLO 205 BORDIN (EBV) 150-250

COLO 397 BORDIN (EBV) 90-180

H129-19 E3 thymoma BW5147 110-130

1 Specific ratio = cpm bound reactive cells / cpm bound nonreactive cell.s.

TABLE 3 Effect of conjugation with ^99mTcNCl₄- on the reactivity of IgM MoAb. A. Reaction of MoAb 3E1.2 with carcinoma of the breast.1

Antibody Dilution Treatment

none DTT ^99m TcN-labeled

-NH2 -SH

10-3 ++++ ++++ ++++ ++++ 10-4 +++ +++ +++ ++ 10-5 ++ ++/+ ++ + 10-6 + +/- +/- +/-

B. Reaction of MoAb 5C-1 with normal colon.1

Antibody Dilution Treatment

none DTT ^99mTcN-labeled

-NH2 -SH

10-2 ++++ ++++ ++++ ++++ 10-3 +++ +++ +++ ++ 10-4 + + + +

1 Tissues wsre tested by immunoperoxidass and specificity graded: 0 = negative, + = weak, ++ = moderate, +++ = strong, ++++ = very strong. REFERENCES 1. Hnatowich DJ, Davis TW, Griffin TW, et al: Radioactive labeling of antibody: a simple and effective method. Science 220:613-615, 1983. 2. Krejcarek GE, and Tucker KJ: Covalent attachment of chelating groups to macromolecules. Biochem. Biophys. Res. Comm. 77:581-585, 1977. 3. Paik CH, Ebbert MA, Murphy PR, et al: Factors influencing DTPA conjugation to antibodies via cyclic DTPA anydride. J Nucl Med 24:1158-1163, 1983. 4. Meares CF, McCall MJ, Reardan DT, et al: Conjugation of antibodies with bifunctional chelating agents: Isothiocyanate and bromoace tamide reagents, methods of analysis and subsequent addition of metal ions. Analytical Biochem 142:68-78, 1984. 5. Khaw BA, Strauss HW , Cahill SL, et al: Sequential imaging of Indium-111-labeled monoclonal antibody in human mammary tumors hosted in nude mice. J Nucl Med 25:592-603, 1984. 6. Fawwaz RA, lilang TST, Estabrook A, et al: Immunoreactivity and biodistribution of Indium-111-labeled monoclonal antibody to a human high molecular weight melanoma associated antigen. J Nucl Med 26:488-492, 1985. 7. Murray JL, Rosenblum MG, Sobol RE, et al: RadIoimmunoimaging in malignant melanoma with ¹¹¹In-labeled monoclonal antibody 96.5. Cancer Res 45:2376-2381, 1985. 8. Perkin AC, Pimm MV and Birch MK: The preparation and characterization of ^{1 11}In-labeled 791T/36 monoclonal antibody for tumor immunoscintigraphy. Eur J Nucl Med 10:296-301 , 1985. 9. Rairweather DS, Bradwell AR, Dykes PW, et al: Improved tumor localization using indium-111 labeled antibodies. Br Med J 287:167-170, 1983. 10. Rainsbury RM, Ott RJ, Uestwood JH, et al: Localization of metastatic breast carcinoma by a monoclonal antibody chalats labeled with Indium-111. Lancet :934-938, 1983. 11. Hnatowich OJ, Kosciuczyk GC, Rusckowski M, et al: Pharmacolcinetics of an indium-111-labeled monoclonal antibody in cancer patients. J Nucl Med 26:849-858, 1985. 12. Pettit WA, Deland FH, Bennett SJ, et al: Improved protein labelling by stannous reduction of Pertechetate. J Nucl Med 21:59-62, 1980. 13. Rhodes BA, Torvstad DA, Breslow K, et al: ^99mTc-labeling and acceptance testing of radiolabeled antibodies and antibody fragments. In Burchiel SW, Rhodes BA, Friedman B. (Eds): Tumor Imaging: The Radioimmunochemical detection of cancer., New York, Masson Publishing USA Inc., 1982, pp 111-123. 14. Paik CH, Phan LNB, Hong JJ, et al: The labeling of high affinity sites of antibodies with ^99mTc. Int J Nucl Med Biol 12:3-8, 1985. 15. Rhodes BA, Zamora PO, Newel l KD, et al: Technetium-99m labeling of murine monoclonal antibody fragments. J Nucl Med 27:685-693, 1985. 16. Pettit WA , Deland FH, Pepper GH, et al: Characterization of tin-technetium colloid in technetiumlabeled albumin preparations. J Nucl Med 19:387-392, 1978. 17. Pettit WA , Deland FH, Bennett SJ, et al: Improved protein labeling by stannous tartrate reduction of pert echnetate. J Nucl Med 21:59-62, 1980. 18. Baldas J, and Bonnyman J: Substitution reactions of ^99mTcNCl₄- - a route to a new class of ^{99 m}Tc-radiopharmaceuticals. Int J Appl Radiat Isot 36:133-139, 1985. 19. Kanellos J, Pietersz GA, McKenzie IFC, et al: Coupling of the ^99mTechnietium-nitrido group to monoclonal antibody and use of the complexes for the detection of tumors in mice. J Natl Cancer Inst 77:431-439, 1986. 20. Smyth MJ, Pietersz GA, Classon BJ, et al: Specific targeting of chlorambucil to tumors with the use of monoclonal antibodies. J Natl Cancer Inst 76:503-510, 1986. 21. Hogarth PM , Edwards J, McKenzie IFC, et al: Monoclonal antibodies to murine Ly-2.1 cell surface antigen. Immunology 46:135-144, 1 9 8 2. 22. Murray BJ, Mercer W, McKenzie IFC, et al: The polypeptide structure and assembly of the Ly-2/3 heterodinters. Immunogenetics 21:510-527, 1985. 23. Hogarth RM, Potter TA, Cornell FN, et al: Monoclonal antibodies to murine cell surface antigens. J Immunol 125:1618-1624, 1980. 24. Potter TA, Hogath PM, and McKenzie IFC: Ly-15: A new murine lymphocyte alloantigenic locus. Transplantation 31:339-342, 1981. 25. Pierres A, Naquet P, Agthoven AV, et al: A rat anti-mouse T4 monoclonal antibody (H129.19) inhibits the proliferation of la-reactive T cell clones and delineates two phenotypically distinct (T4+, Ly-2/3- and T4-, Ly-2/3+) subsets among anti-la cytotoxic T cell clones. J Immunol 132:2775-2782, 1984. 26. Thompson CH, Jones SL, Pihl E, et al: Monoclonal antibodies to human colon and colorectal carcinoma. Br J Cancer 47:595-605, 1983. 27. Stacker SA, Thompson CH, Riglar C, et al: A new breast carcinoma antigen defined by a monoclonal antibody. J Natl Cancer Inst 75:801-811, 1985. 28. Teh JG, Stacker SA, Thompson CH, et al: The diagnosis of human tumors with monoclonal antibodies. Cancer Surveys 4:149-184, 1985. 29. Parish CR and McKenzie IFC: A sensitive resetting method for detecting subpopulations of lymphocytes which react with alloantisera. J Immunol Methods 20:173-183, 1978. FIGURE LEGENDS Figure 6 Purification of ^99mTcNCl₄--labeled MoAb using Sephadex G25. The column was equilibrated with PBS and 0.5 ml fractions were collected. Figure 7 Binding of ^99mTcNCl₄--labeled anti-Ly-2.1 prepared by direct labeling of MoAb on RF/J (●) and BIO (o) thymocytes (Fig. 7a) or anti-Ly-2.1 (●) and anti Ca. colon (o) on E3 thymoma cells (Fig. 7b). The amount of radioactivity bound as a function of antibody concentration is shown. Figure 8 Specific binding to E3 target cells (8a) anti-Ly-3 labeled with ^99mTcNCl₄- to DTT treated E3 (●) andBW5147 (O) target cells; or (8b) anti-Ly-2.1 (●) and anti-colon antibody (O).

Figure 9 Specific binding of DTT treated anti-colon antibody labeled with ^99mTcNCl₄- to Colo 397 (●), Colo 205 (O), and a non-reactive control (EBV derived tumour) (▲).

Figure 10 Binding of 2 different anti-Thy-1.2 conjugates - one prepared with direct labeling of MoAb with ^99mTcNCl₄ ^" (—) and the other prepared with DTT treated MoAb (- - - -) or CBA (●) and AKR (O) thymocytes.

Figure 11 Percentage binding of ^99mTcNCl₄--labaled anti-Ly-2.1 (●) to E3 target cells; the control anti-Ly-1.1 antibody did not bind >5% at any dilution (not shown). The amount of radioactivity incorporated as a function of cell number is shown.

Figure 12 Binding of anti-Ly-2 to E3 target cells, using unmodified anti-Ly-2.1 (▲); DTT treated anti-Ly-2.1

(□), ^99mTcNCl₄- labeled anti-Ly-2.1 , high specific activity

(100muCi/mug) (●) and low specific activity (12muCi/mug) (■). (12a) detection using ¹²⁵I-sheep anti-mouse Ig; (12b) rosetting using sheep anti-mouse Ig.

Figure 13 Binding of DTT treated anti-Ly-2.1 labeled with ^99mTcNCl₄- to E3 (●), D1 (Δ) and BW5147 (□) target cells. The amount of radioactivity as a function of antibody concentration is shown..

Claims

The claims defining the invention are as follows: 1. A conjugate of technetium with a radical having an antigen binding site wherein the technetium thereof Is radioactive. 2. A conjugate as claimed in claim 1 , wherein the technetium is ^99mTc. 3. A conjugate as claimed in claim 1 or claim 2, wherein said radical is an antibody or antibody fragment which is preferentially absorbed by a tumour cell as compared to a non-tumour cell. 4. A conjugate as claimed in any preceding claim, wherein the conjugation is via a sulphide linkage. 5. A conjugate as claimed in any preceding claim, and of formula I Ab-Y-S-NTc(Hal)₃ Formula I wherein Hal is chlorine, bromine or iodine and including mixed halides, Ab is a radical having an antigen binding site and Y is a conjugating chain. 6. A conjugate as claimed in claim 5, wherein Y is of formula II

wherein Z is H, alkyl, aryl, carboxy, halide, hydroxy or amino, X is NH, O or S and z is 0 or 1. 7. A conjugate as claimed in claim 5 and of formula Ab-S-NTc(Hal)₃ wherein Ab and Hal have the meaning given in claim 5 or Ab-S represents an antibody radical or a radical having an antigen binding site. 8. A conjugate as claimed in claim 5 and of formula Ab-NH-Y-S-NTc(Hal)₃ wherein Ab and Ab-NH represent an antibody radical or a radical having an antigen binding site and Y and Hal have the meaning given in claim 5. 9. A conjugate as claimed in any preceding claim, wherein said radical is an antibody, an antibody polymer, an antibody monomer or an antibody fragment having an antigen binding site. 10. A conjugate as claimed in any preceding claim, wherein said radical is an antibody, an antibody polymer, an antibody monomer or an antibody fragment having an antigen binding site selected from the group showing specificity for one of breast, brain, melanoma, lung, pancreas and colontumours. 11. A conjugate as claimed in claim 9, wherein said radical is an antibody fragment having an antigen binding site and selected from F(ab')₂, F(ab'), IgG₁, IgG_2a, IgG_2b and IgG₃. 12. A pharmaceutical composition comprising a compound in accordance with any preceding claim together with a pharmaceutically acceptable diluent. 13. A compound of formula Ab-SH or Ab-NH-Y-SH wherein Ab, Ab-NH, Ab-S and Y have the meaning given in claims 7 and 8. 14. A method of making a conjugate in accordance with any one of claims 1 - 11 comprising taking a compound in accordance with claim 13 and reacting it with TcN(Hal)₄- wherein Hal is chlorine, bromine or iodine and including mixed halides. 15. A method as claimed in claim 14, including obtaining the compound of claim 13 by reducing an antibody or a compound having an antigen binding site to form free sulphydryl groups. 16. A method as claimed in claim 14, including obtaining the compound of claim 13 by reacting an antibody or compound hav ing an antigen binding site succinimidy l pyridyldithiopropionate (SPDP) or an analogue thereto appropriate to the compound of claim 13 desired to obtain an antibody or compound having an antigen binding site conjugate containing a -S-S- group and reducing the conjugate to form a -SH group. 17. A method as claimed in claim 14, including obtaining the compound of claim 13 by using S-acetylmercaptosuecinic anhydride (SAMSA) or SH introducing compounds to produce a side chain on an antibody or compound having an antigen binding site containing a -S- linkage and reducing to form a -SH group. 18. A conjugate or method of making same substantially as hereinbefore described with reference to any one of the preparations. 19. The articles, things, parts, elements, steps, features, methods, processes, compounds and compositions referred to or indicated in the specification and/or claims of the application individually or collectively, and any and all combinations of any two or more of such.