TECHNETIUM-ANTIBODY CONJUGATE

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 techπetium-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 " m Tc. In a particular aspect the present invention provides a t echn e t iu -an t ib ody or antibody fragment conjugate which is referential l absorbed by a tumour cell as compared tα a non-tumour cell. Preferably the con ugation is via a sulphide linkage. The present invention also provides a compound of formula Ab-Y-S-NTc(Ha 1 )-- 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 n t -[NH-C-(CH) n ] z wherein Z is H, alkyl , aryl , c a r b o x y , h a 1 i d e hydroxy or amino, n is 1 -10, X is NH, 0 or 3 and z is 0 or 1. Alkyl groups preferably 1 - S 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 where"! n 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 T c N ( H a 1 ) 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 dithiothrei o 1 (DTT), b) reacting an antibody succinimidyl pyridy ldithio- propionate (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-acety 1 mercaptosucciπic anhydride (SANSA) or SH introducing compounds tα 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 i u n o g o b u 1 i n or a fragment of an immunogobu 1 in maintaining a sufficiency of an antigen binding site such that it is preferentiall absorbed by a tumour cell as compared tα a non tumour cel l. Thus, in addition to whole antibodies, it is also possible tα utilize F(ab') 2 an F ( al _') fragments. Stil l further antibody polymers such as antibody pentamers I gfl and derivatives of these such as immuno obu 1 in monomers may be used. Also useable are IgG 2 a» I 9 G 2b» X 9 G 1 aπd I 9 G 3 ' The compounds of this invention may be combined with phar aceu ically acceptable carriers. The mode of adminis ration of the compounds of this invention will be as selected. In particular, the compounds of this invention may be administered intra enously, i n t r a p e r i t o n e a 1 y , i n t r a p 1 u r a 1 y , i n t r a p e r i c a r d i a 1 y , iπtracereb ospiπa 1 fluid aπd subcutaπeous 1 y. The t e c h π e t i u m - a n t i b o d conjugates of the present invention may be formed into phar a 1 ogica 1 compositions with appropriate pharmaceutically acceptable diluents. The t e c h π e t i urn - a n t i b α d y conjugates of the present invention are useful for in vivo detection of tumours such as by immunoscintigraphy . Pa_rt _\ Radioactive isotopes of technetium coupled to 1-lσAb have been used by us in the search f or specif ic methods of d ia gno s ing s ma l l t umo ur s. I n thi s l i gh t w e h a v e successful l y located tumours in both mouse and man, and antibodies have been administered either intravenousl or s u c u t a n e o u s 1 y , or by other routes. It is c l ear that radio 1 ab e 11 ed 1-loAb can indeed localise in tumours in _/____£ and with the use of computer assisted tomography, with subtraction for non-specific effects, this method can then be utilized for the ££££.if i£ detection of tumours - both primary aπd secondary. Howev er, there are prob l ems of specif ic activ ity , specif icity aπd high b l ood background which need attention before this technique can be accepted a s a u s e f u l d i agn os t ic t oo l . a jo r a d v anc es in the diagnostic r ad io 1 oca 1 iza t ion of tumours should result from th e pro duc tio n o f b e t ter 1-l o A b , b ett er meth ods of r adio 1 ab e 11 in g aπd final ly, design of methods to reduce the background provided by circulating r adi o 1 abe 11 ed antibodies. It is expected that more specif ic antibodies wil l become a vail able with time. Ill e have shown that the use of second antibody (aπti-immuπog l obu l iπ) , is ab l e to c l ear 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 ' 3 ' I and * I in experimenta l model s has 1 P 5 shown that > £ -- J ι cannot be used in man because of high tissue attenuation but there are however serious drawbacks with the use of 3 ' I. This nuclide provides a poor quality image, it produces signif icant radiation exposure due to its beta emissions and has a short biological half life - presumably due to the d e - i o d i n a t i o n of the i-l o A b . The use of 99m Tc for label ling M o A b 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 (l40keV) for detection with currently available sc in tig rap hi c instrumentation and produces very little radiation exposure to patients undergoing scanning procedures. _.£I££_.£_-_. SH_1 _!_L_-_i_-_-_L _!__£_Li --ice used were: RF/J, C57BL/6 aπd ( C57BL/6 x 8ALB/c)F 1 (sBCF.-) bred in our colony. Athy ic, BALB/c mice (ΠH HH) --- - obtained from the Royal Dental Hospital (Melbourne, Australia). I___!2_-E £lϋ kiπ£_L τ ω ° tumour cell lines were used: one, the E3 cloπal variant of the thymoma I T T ( 1 ) 75 S (1 ) which was obtained by three successive rounds of fluorescent activated cell sorting of ITT(1)75NS cells stained with monoclonal Ly-2 antibodies aπd selected for the most fluorescent 1 % of cells. The muriπe cell line E3 was maintained in ____£££ in D fll E supplemented with 10 % heat inactivated newborn calf serum (Flow Laboratories, Sydney, Australia), 2mM glutamine (Commonwealth Serum Laboratories, Melbourne, Australia), 100 IU paπicil lin/πl aπd 100 mg streptomycin/ml (Glaxo Laboratories, Melbourne, Australia). E3 cells were washed twice in DME (without additives) aπd twice in DME containing 0.5 % BSA and used in the j.ιι v , i.t££ binding assays. The E3 cell line was maintained in vivo by the passaging of cells from ascitεs fluid produced in BCF. mice. Ascites were washed in DME and PBS, solid tumour grew after the s.c. injection of 10 6 -10 7 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 (Co on ealth Serum Laboratories, Australia) washed with RPMI aπd injected s.c. into nude mice, where tumours appeared after the injection of 2 x 10 s - 1 x 10 7 cells. ϋ_£fi_Li " Two MoAb were used: (i) anti-Ly-2.1 (IgG2a), an antibody raised against the muriπe alloaπtigen Ly-2.1 (3); aπd (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.)aπd purity was confirmed by gel e 1 ec t ro h or esis and antibody activity assayed by a rosetting test (5). 9 il_lT£ L bel _l iπ ££ __£___.._.i Sodium P e r t e c h n e t a t e 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, M0. USA). MoAb were label led with 99m Tc using two methods - the new method described herein, aπd a method using staπnous chloride. (a) Labelling using 99m TcNC1 4 ~ : 99m TcNC1 4 ~ was prepared as a dry sa l t residue as described in detai l in (9 ). For labelling, the MoAb was first reduced with the addition of DTT (20microl , 115m g / m 1 in PBS) to 200microg of MαAb (1 mg/ml in PBS) and allowing the mixture to stand at room temperature for 30minutes when the reduced MoAb was separated from DTT by gel chromatography using 0.1 M sodium acetate pH 4.0 as eluaπt on an 8cm x 1 cm column of Biogel P- 6 (Biorad Laboratories, Richmond, U.S.A.). The fractions (1 ml ) containing the protein peak were added to the dried 9m TCcl 4 ~ salt residue and the mixture brought to pH 3.0 with 0.2M hydroch loric acid; after 2 minutes at room temperature, 0.1 M sodium phosphate was added and the pH a justed to 7 by the careful addition of sodium hydroxide. Purification of the labelled MoAb was then achieved by gel c h roma t o gr a p h y w i t h a S ep h a d e x G -25 c o l umn ( P D - 10, Pharmacia). (b) Stanπous Chloride Reduction Method: Stannous chloride (20-200 microg) was added to 200 microg MoAb (I mg/ml), 4-6 mCi of p e r t e c h π e t a t e was added, and the mixture al lowed to stand at room temperature for 30 minutes. The labelled MoAb was purified by gel filtration on a PD-10 sephadex column. - £.£___-£._.£ dill _._-_. £11 2.L J_£_.i_± MoAb (100 microg, 1 mg/ml) 2 were labelled using the chloramiπe-T method (6): 2.5mCi of 3 carrier-free Na I (Amersham International Ltd., Amersham, 4 England) and 3 microl of chloramine-T (1 mg/ml) were mixed 5 with the protein for 2 minutes at room temperature and the 6 reaction then terminated by the addition of 3 microl of 7 sodium etabisulf its (2.4 mg/ml). Iodiπated MoAb was B separated from free iodine by gel filtration using a PD-10 9 column . 0 _.£_.£__££_.£_.__ flU £_.__ ___.£_. A binding assay was developed to 1 determine the stability and specificity of the 99m TcN-MoAb 2 complexes. MoAb complexes were tested in one of two ways - 3 either a) using one MoAb and two cell lines; or b) using two 4 different MoAb and one cell line - both MoAb being labelled 5 identically, one being reactive with the cell line, the 6 other π o n - r e a c t i v e . Polyviπyl chloride 96 well plastic 7 plates (Pynatech Laboratories, Inc., Alexandria, Va) were 8 washed with \% bovine serum albumin (BSA) in PBS. In this 9 assay either the number of cells or the quantity αf MαAB 0 could be kept constant while the other was varied. Either: 1 (1) after serial dilution of 25 microl of the labelled MoAb, 2 1 x10 -2x10 target cells (either tumour cell lines or 3 thy ocytes) suspended in P8S were added to each well; or (2) 4 different cell numbers (10 - 2 x 10. ) contained in the same 5 volume of the target cells were added aπd an equal amount of 6 r a d i o 1 a b e 11 e d MoAb was then added to each well. In both 7 assays the contents were mixed aπd the plate incubated on 8 ice for 30 minutes; after 3 washes, the plates were dried 9 and the cell pellets were counted for one minute in a gamma 0 counter. All assays were performed in duplicate. 1 ____££ " iϋi „ ___i_____.i££._, Nude mice bearing COLO 205 xeπografts 2 or BCF.. mice bearing the E3 thymoma were used. The first 3 study compared the distribution of two 99m TcN-MoAb in BCF., 4 mice; groups of 4 mice were sacrificed at 20 hrs, 30 hrs aπd 5 35 hrs after the injection of label led MoAb. The second 6 study compared the binding of a 99m TcN-MoAb complex to two 7 different tumours - the E3 thymoma and COLO 205 xenografts. 8 In these studies, the b i α d i s t r i b u t i o π of 99 m T c label was determined by gamma counting of blood, heart, lungs, spleen, stomach, intestine, kidneys aπd tumour from these mice. The distribution of isotope is reported as a localization ratio (tiss e (cpm/g)/blood (cpm/g)). A l l mice received approxima ely 115 microCi of 99m Tc activity (approximately 10 microg of protein) by tail vein injection. _ £_.ϋϋ_ki23 : BC F., mice bearing the E3 thymoma (0.3 1.0cm) were given i.v injections of either approximatel 115 micro Ci of 99m TcN- 1abe 11ed anti-Ly-2.1 (specific MoAb) and 200 micro Ci (4microCi/micrα g) of 25 ι labelled aπti colon (non-specific MoAb) or 115 micro Ci of 99m Tc- 1 abe 11 e anti colon MαAb. Twenty-seven hours after in ection mice were anaesthetised by iπtraperitoπeal injection of ? 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 colli ator. A setting of 50 keV with an 80? window and 140 k e V with a 20? window was used to image the 125 ι an d Tc photons respectivel . Data were stored in digital form by an MDS Modu ed computer. RESULTS The study was conducted in three phases: the establishment of the conditions for the coupling of 99 TcNCl 4 " to MoAb; the testing of the stability of the TcN-MαAb complexes iri vij j £i and the measurement of the distribution of the complexes i ^ ri v.iv/£ to determine whether tumours could be detected s ecifically. ---!£_._! 2iL_- _L-_.H__._k 2-1-i. " mTc 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 C578L/6 (Ly-2.1 " ). In this assay the amount of labelled MoAb added tα each well was kept constant, the cell number varied (10 -10° cells per well). There was clearly no noticeable dif ference in the uptake of radioactivity by the two cell types i.e. the 99m Tc-MoAb complexes bound to πon reactive and reactive cells equally. Varying the amount of SπCl, used in the reduction procedure had no effect on the result. This is shown in Fig.1 which shows the binding of 99 m T c labelled anti-Ly-2.1 prepared by SnC1 2 reduction of RF/J aπd C57BL/6 thymocytes. The amount of radioactivit incorporated as a function of cel l number is shown. ---!.£_-_- M_£_L_-.L An alternative method of labelling was designed, utilising the unique property of 99m TcN C 1 4 ~ t o form a stab l e cov al ent l inkage to su lf ur atoms. MoAb were partial ly reduced with DTT to generate free sulfhydryl sites and mixed with the 99m Tc Cl 4 ~, l eading to the f ormation of 99m TcN-MoAb complexes. The 99m TcN-MoAb complexes were then tested in different serological assays to determine whether the labe l ling procedure damaged or al tered the binding or specificity of the MoAb, aπd whether the comp lexes formed were stable. The binding curve obtained when 99m TcN - 1 ab e 11 e d Ly-2.1 MαAb 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 specif ic binding of anti-Ly-2.1 l abe l l ed with 99 m TcN C 1 4 " on RF/J and C57BL/6 thymocytes; Amount of ra di o ac ti v i ty b oun d a s a f unctio n of a ntib ody in th e reaction mixture. There was cl early a major dif f erence in the binding of the l abe l l ed M oAb to the reactiv e cel ls (RF/J) when compared to the non-reacti v e ce l l s (C57BL/6) with specif ic ratio (=cpm RF/J / cpm C57BL/6) of greater than 20. Thus with the use of the one antibody and two dif f erence sources of cel ls the procedure used to coup le TcNCl 4 ~to MoAb produced a stable complex which bound only reactive target cel ls as shown in Fig.2. The results are clearly superior to those obtained with the complex formed with the use of SnCl 2 - ∑ π this respect, reference is made to Fig.1 which shows the binding of 99m Tc l abel led aπti-Ly-2.1 with SπC l 2 reduction on RF/J and C57BL/6 thymocytes; amount of radioactivity incorporated as a f un c t i on of c e l l n umb er . I n t h e s ec ond a ss a y , two different MoAb, one directed against co l onic secretory epithelium (250-30.6) and the second, the aπti-Ly-2.1 MoAb, were l abe l l ed with TcNC l 4 ~ under identical coup l ing conditions and the two comp l exes 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-col on MoAb (250- 30.6). Again the specific MoAb complex 9 a 9 a m m Tc Ly-2.1 bound more efficiently than the TcN-anti-co 1 on complex as shown in Fig.3 with a specific ratio of 10. Fig.3 shows the specific binding of 99ln TcNCl • " laballed aπti-Ly-2.1 and anti-colon Mo-Ab on ITT(l) 75NS E3 target cel ls. Thus in these assays, it appeared that a stable bonding of 99m Tc to MoAb had been produced, so that only the binding of antibody to the appropriate target cell was detected. Stability £f ---l£N____£Ab . Aliquots of 99m Tc 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 99m TcNCl 4 , on RF/J and C57BL/6 thymocytes. _-_H££_L £_L __--T£ ££ £ £ !_£ £ £J2£ _.£_.!_Li.j__. £_L -——Ii__i-_l_lΔ__ ££H£l£2__l i At tnβ tim e when 99 Tc is obtained from a 99m Tc generator, there is appro imately 0.7 micro g 99m Tc/ml of 99 Tc 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 99 TcCl 4 " carrier to the 99m TcO -. - us ed for labelling. The addition of 2 micro g 99 Tc to the reaction mixture was thus equivalent to increasing the Tc 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 99 m T c - a n t i -L y - 2.1 containing added 99 Tc 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/5 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 9 " 9Tc as in Fig.2 aπd 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. Biodistributioπ: The in v i v o l oca l ization and b i o d i s t r i b u t i o n of 99m Tc-MoAb complexes was examined by injecting mice with 9 a 9 3 m m -Tc-MoAb aπd determining the relative amounts of radiolabel accumulated in the tumour or the tissue. These resu l ts were used to ca l cul ate the localization ratio derived as fol lows: tissue (cpm/g) / blood (cpm/g). In the first study, two groups of 16 BCF - mice bearing the E3 (Ly-2.1 + ) tumour (0.23 - 1.11 g) were injected i.v. with either 99m Tc-Ly-2.1 or 99 Tc-250-30.6 MoAb - each mouse received 115 micro Ci "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 M oAb 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 o o d . Th e n o n- s p ec ifi c a n t ib od y ( 250 - 30.6 ) t h e localization ratio of the liver, spleen and kidney were observed to be higher than that of the blood aπd 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 33 m Tc - y-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 radioactivi y in the tumour than in other tissues - except the l iv er. The 99 m Tc- M oAb comp l exes cou l d specifically localise to tumours iLn_ v_.V£. _-£_J_-£_L._._l_t _-_l_l A. Nuclear image obtained after the injection of 9 m T c - aπti-Ly-2.1 into a mouse bearing the E3 thymoma on the right thigh (as visualized). B. Nuclear image obtained after the injection of 99 Tc anti-Ly-2.1 into a mouse hosting a thymoma on its left anterior side and 1 25 I -an t i -co 1 on. C. 1 ' 5I 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 9m TcN- 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 wa.3 dissected and f ound tα have a localization ratio (tumour tα b lood a proximately 2.0). Radioactivity in this image is also concentrated in the central region of the mouse, indicative of signif icant distribution of antibody to large vascularized organs such as liver, lung and heart; a phenomenon that tends to obscure isualiza ion 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 99m Tc- MoAb this mouse was also injected, C, with a non-reactive MoAb (250-30.6) labelled with " * 2 ^I, 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 radioactivi 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. _U_L££_L !__;££ Immuπoscintigra hy , with the use of radio 1 abe 11 ed MoAb is a new method for the in ______£ detection of tumours and thus cancers of colon, breast aπd other tissues have been detected with some degree αf 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 r a d i o 1 a b e 11 e d 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. Ill e are adopting both approaches through the use of multiple MoAb aπd MoAb fragments (Fab or F(ab') 2 ). In this manuscript we report on the advantages of using TcN-MoAb to detect tumours. In this study a new method of coupling 99 TcNC1 4 to MoAb aπd the subsequent use of these complexes to detect tumours in vivo is described. As previously indicated, 3 Tc offers a number of advantages for r a d i o i s o t o p i c 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 scin t i gr aphic instrumentation. Tc0 4 ~ from a portable generator and must be reduced prior to coupling with MoAb. Many methods have been described for the reduction of per t echne t a t e ; these procedures generally lead to the reduction of p er t echne t a t e to the Tc (IV) or TC (V) oxidation state. At present, the most frequently used reducing agent for preparation of 99m Tc labelled compounds is SnCl 2 - 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 far binding sites. Indeed, in our hands p e r t e c h n e t a t e reduced with SnCl- readily bound to MoAb but such complexes showed no specificity when tested in our i^ ______££ binding assay or when tested Because αf the problems experienced with the 99m Tc labelling of MoAb with the use of SnCl 2 " we have used 9 " 9'm"Tc N C 1 4 A - 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 vjLy_£ by im unosc iπtigraphy . Tumours could be visualised as early a3 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 TcNCl ~ are clearly different to those made using SnCl- as the presence of the nitrido group attached to Tc modifies the chemical behaviour αf the Tc atom and makes it more favourable for co-ordination with certain ligaπds. Ligands which bind through sulfur atoms form more stable complexes with the TcN core than dα ligaπds binding through nitrogen. In our initial experiments, attempts were made to bind the TcN group directly to amino groups of the MoAb and while TcN- 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 l inkages 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 TCNC1 4 ~ was prepared in a stable dry form without the presence of any con amina ing metal ions and was successfully cαmplεxed 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 aπd activity of TcNCl - " comp l exes was determined In se v era l serological assays which invol ved 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 αf radio 1 abe 11 ed MoAb to target cells was demonstrated, the complexes were not non specifical ly "sticky" nor unstable with the release of 99m Tc to bind to other non reactive target cells. In vij_££ studies have shown that 99m TcNCl 4 ~ 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 _.π vitro properties. For example, it was possible to increase the amount αf a9m Tc bound to b 200 fold without affecting MoAb activity (text fig.5) . An important finding obtained in the study was to demonstrate that 9 TcN-MαAb complexes localized to tumours ill _.i_-£* 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 • in ection of a 1 ' _ ■ 5-! 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 99 TcNC1 4 ~ complex could be successfully coupled to MoAb, in a simple one step procedure that resulted in stable covaleπt complexes which retained MoAb activity. An alternative 99m Tc 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 99 Tc 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 label led MoAb. Our method eliminates the need f or DTPA coupled MoAb aπd the 99 T c C 1 4 " used f or label ling is free αf any heavy metal contamination, thus enabling high specific activity to be obtained. Thus TcNCl A " monoclonal antibody can be simply produced and have high activity for specifically localizing tumours both in vitro and in At present we consider the coupling method to be superior to other methods of coupling 99 m Tc to antibody and the i mm u n o s c i n t i g r a h i c findings to be superior to that obtained with r ad io 1 a b e 11 e d iodine. On this basis we are now conducting a chemica l trial to determine the clinical usefulness of the new reagent . TABLE 1 B i o d i s t r i b u t i o n and Localization of a Tc radiolabeled anti-Ly-2.1 in BCF. mice bearing the E3 thymoma and nude mica bearing COLO 205 tumor xenografts. Tissue Local iza ie Ratio E3/C0L0 205 Ratio Illillil 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 0.9B 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 _.£_! ££££_ Jt 1. HOGARTH, P.M., HENNING, M.M. aπd M C K E N Z I E , I.F.C. The a 11 oant ig enic phenotype of radiation induced thymomas in the mouse " . J. .C.I. 1982;69:619-626. 2. SEMPLE, T.U., QUINN, L.A., 111000 S , L.F. and MOORE, G.E. Tumour and Lymphoid cell lines from a patient with carcinoma o f t h e c o l o n f o r a c y to x i c i t y mo d e l . C a n c e r Res .1978 ; 38 :1345-1355. 3. HOGARTH, P . M . ED 111 A R D S , J., MCKENZIE, I.F.C., GODING, J.ω. and LIEU, F.Y. Monoclonal antibodies to murine Ly-2.1 surface antigen. Immunology 1982;46:1 5-1 4. 4. T H O M P S O N , C.H . , JO N ES , S .L. , P I HL , E. a n d C K E N ZI E , I . F . C. , Monoclonal antibodies to human colon aπd colorεctal carcinoma. Br. J. Cancer 1983;47:595-605, 5. PARISH, CR. aπd MCKENZIE, I.F.C. A sensitiv e rosetting method for detecting subpopu 1 at ions of Lymphocytes which react with a l l oantisera. J. Immuno l. M ethods 1978;20:173-183. 6. GREENWOOD, F.C. INNTER, III . M . and GLOVER, J.S. The preparation αf ' 31 I labelled human growth hormone αf high specific radioactivity. Biochem J. 1963; 114-123. 7. PAIK, CH. MURPHY, P.R. ECKELMAN, 111. C VOLKERT, 111. A . and REBA R.C 0 p t i i 1 i z a t i o n of the DTPA M i x e d- A πh y d r i d e Reaction with MoAb at Low Concentration. J. Nucl. Med. 1983;24: 932-936. 8. BONNYMAN, J. Effect of milking efficiency on 99 Tc con ten t of 9 9 m T c d e ri v e d f r om 9 9 πι T c g en e r at o r s. Int. J.Appl.radiat.Isot.1983;34:901-906. 9. DeLAND, F.H., KIM.E.E., C0RGAN R.L., CASPER, S., PRIMUS, F.J. SPREMULLI, E., ESTES, N. aπd GOLDENBERG, CM. Axil lary L y m p h o s c i π t i g r a p h y by R a d i o i m u π o d e t e c t i α π of a Carciπoe bryonic Antigen in Breast Cancer. J. N uc 1. M e .1979 ; 20 :1243-1250. P £t 8 UJe now describe the use of the above method to label a panel αf MoAb aπd these have been tested to show that the procedure established has general use, and can be used to label al l subclasses of MoAb with 99m Tc with retention of I mm u π o r e a c t i v 11 y ; the complexes formed can be used to localize tumors in vivo. !2_-I__SI_-k_L _!!___ _l_.I_i_._i__ _ii£_li R- " /J, CBA, AKR, C57BL/10(B10) , BALB/c, C57BL/6(B6), and ( C57 BL / 6 x B AL B / c ) F 1 (B6CF1 ) mice were bred in our colony. lH_!£_L £lii kinSLli Human tumor cell lines (CEM aπd Bordin - an EBV induced B cell line) were cultured in RPMI 1640 medium with L-glutamine. B 1115147 aπd several clonal variants (E3,D1) of the murine thymoma ITT(1)75NS (20) were cultured in DME with L-glutamine. The clonal variant I TT ( 1 ) 75NS.E3 (E3) was maintained by serial passage in ascitic fluid in (B6CF1) mice. For imaging experiments 10^ - 10 7 cells injected s u b c u t a n e o u s 1 y into B6CF1 mice and reached a size of 0.5-1.0 cm in diameter prior to experimentation. ϋl£n££i£n_li fin_i_l£_ii li 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 col lected aπd resuspended in phosphate buffered saline (PBS, pH 7.3): IgG antibodies were prepared by precipitation with 40? ammonium sulfate [ NH 4 (S0 4 ) 2 ] , f ol l owed 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 Pro t ein -A-S epharαse , washing with PBS aπd elutiπg with either 0.2 M glycine-HCl (pH 2.8) or citrate buf fers (pH 5.0, 4.0, 3.0) and neutralization with saturated Tris, after which antibodies were d ia l yzed against P8S ; o r ( i i ) ion-exchange chromatogra hy on 0 E A E - S e p h a c e 1 aπd with elutioπ using a linear gradient of 0.5 M NaCl In 0.01 M Tris buffer (pH 8.0). The purity αf the antibody (>90?) was confirmed either by high pressure l iquid chromatography (HPLC) or gel electrophoresis, and antibody activity assayed by a rαsettiπg test (29) or by the i m m u π o p e r o x i d a s e method on tissue sections (27). ,£l£_lI_Lii£n £l - 9 a 9-ml£ϋ__i ~ " £_. klL_>£iIin_l ° MoAb 9 9 9 9 m m TcNCl Λ ~ -as prepared as a dry salt residue (18). Sodium 4 azide (15-20 g) was added tα 1 ml sodium 9 a 9 a m m -Tc0 4 ~, produced from a fission product chromatogra hy generator, in 6-7 mis of concentrated hydrochloric acid (specific gravity, 1.18). The solution was refluxed for 5 minutes to destroy excess azide aπd then evaporated to dryness in a rotary evaporator. MoAb were labeled with 99m TcNCl 4 " by one of two procedures: (i) 100-200mug of MoAb (1 mg/ml) was directly added to the dried 99 TcNCl 4 ~ salt residue, or (ii) a modified version of the method previously described (19) where 20 mul of dithiothreitα 1 (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 aπd the column εluted with 0.1 M sodium acetate (pH 4.0). The protein fraction (1.5 mis) was added to the dry TcNCl 4 ~ residue, reacted for 2 minutes at room temperature prior to adjusting the pH to 7 with sodium hydroxide. Prior to use, the TcN- labeled MoAb was purified by passage over Sephadex G-25 (PD-10) and sterilised using a 0.22 mum membrane filter. __£__£i£3i££i _LH__i___-i_L.L In vitro cel l binding studies were performed on cultured tumor cell lines or mouse thymocytes (19). TcN- MoAb complexes were tested in one of two ways: (i) using one MoAb aπd two different target cells; or (ii) using two different MoAbs and one target cell line. The ability αf the MoAb tα bind to target cells was assessed after each step. In this assay cultured tumor cells or thymocytes (3x10 ) were incubated for 30 minutes on ice with one of the following: (i) untreated MoAb; (ii) DTT treated MoAb; or (iii) 99m TcN- 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 i m m u n o g 1 α b u 1 i π (' 5 I-SAM) for 30 minutes on ice. The cells were then washed 3 times with PBS (0.5? BSA) to remove unbound 125 I-SAM and the amount of 125 I-SAM bound determined. l_i!__in£!£inii3_-££_l_-._ Mice bearing E3 tumors were used in two studies. The first compared two identically labeled different MoAb aπd 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 (l2muCi/mug) of 99m TcN- labeled aπti-Ly-2.1 (specific MoAb) or anti-Ly-1.1 (nonspecific MoAb). Each animal was given an intraperitoπea 1 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 99m Tc photons. Data were stored in digital farm by a MDS modumed computer. RESULTS j__._ii£il££iin_l £_. ϋi£_i_L _.i_._L ---l£_l£i 4 - Af er r a d i o 1 ab e 1 i n g , unbound reaction products were removed by passage of the f inal reaction mixture through a gel permeation column of Sephadex G-25 (PD-10). The yield of TcN passing through the column was then a measure of the success of radio 1 abe 1 iπg and typical yields were 80-90?. (A typical example of the elutioπ profile is shown in Figure 6). It was noted that 98-99? of the radioactivity present in the protein f raction cou l d b e prec ipitated w ith tr ich 1 orace t ic acid (TCA). ϋn iX_Li_L 2-L — ~l£_l£iΔ~ ££J_.Ei£_i£_i _:£ _L____H__ 2££H££_. The methods of co plexiπg m TcNCl 4 ~ to MoAb were evaluated in serαlαgical assays to determine whether the labeling procedure used damaged or altered the binding or specificity of the MoAb. 99rπ TcNCl 4 ~ complexed directly tα amiπα groups of Ly-2.1 MoAb was tested in a binding assay using thymocytes from 2 strains of mice: RF/J ( L y - 2.1 + ) and B10 (Ly-2.1 -). 99m TcN-ant i -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 approximatel 1.2. In a second assay 99m TcNCl 4 " directly bound to 2 different MoAb: aπti-Ly-2.1 reactive with the E3 cell line, the other nonreactive (anti-cαlon carcinoma) produced a specific ratio (cpm aπti-Ly-2.1 bound / cpm anti-colon bound) of 2-3 (Figure 7b). The conclusion is that the 99 m TcN-MoAb complexes produced in this way were either unstable or "sticky" aπd on exposure to target cells the 99m TcN bound nonspecifically. 9 !_TcN£l 4 " ££_!£iex£d t£ £u 1 f h_/dry__. ££££ af jsr £££j_i£__ ££_i2£_ii£ni A s the f ormer labe l l ing method ga v e l aw specificity (previously due to nan-specific labelling) an alternative method of labeling was designed, utilizing the known ability αf 99m TcNCl 4 " to form a stable covaleπt linkage to sulfur atoms. MoAb wεre partial ly reduced with DTT to generate free sulfhydryl sites aπd mixed with the 99m TcNCl 4 " , leading to the formation of 99m TcN-MoAb. These c omp l ex e s were shown b y s odium d αd ec y l s u l f a te po 1 y aery 1 amide gel e 1 ec tr o pho r e sis (SDS-PAGE) to consist of intact IgG. The binding assay demonstrated 99m TcN-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 99m TcN-anti-Ly-3 than did the BID5147 (Ly-3~) cel l line (Figure 8a). Similarly two different MoAb, one directed against colonic secretary εpithεlium (250-30.6) and the other directed against Ly-2.1 , were l abe l ed with TcNCl 4 ~ under identical conditions aπd the complexes were testεd for their ability to bind to the muriπe thymoma E3 (Ly-2.1 + , Ca Colon Ab " ) (Figure 8b), when the reactive MoAb complex bound 6-8 times more eff iciently than did the 99 T c - a n t i - c o 1 o n complex. In separata experiments the 99 m- TcN-cαloπ MoAb, unable to bind to the E3 thymoma could bind reactive human tumor cell lines, Colo 397 aπd Colo 205 incorporating 90-250 times more radioactiv ity 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 αf 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 99 m T c - 1 a b e 1 e d I g G 2 a and IgM anti-Ly-2.1 (monomer) MoAb on reactive target cells (CBA, RF/J) compared to that f ound with nonreactive target cells (BALB/c, C57BL/6), whereas the IgG 1 and IgG 3 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 ( I g G 2 a ) and H129-19 (IgG., ) produced specificity ratios of 70-130 aπd 110-130 respectively (Table 2). f_£_ii£i_ ι _-£iina 2l laϋ _!£_>_> i££ni_>_!£££l _.*_•_. ---I_=_1£- 4~_L In contrast to the preceding results obtained by direct l abeling (using amiπo groups) the direct co plexiπg of TcNCl 4 " via amiπo groups with IgM class of MoAb produced 99m TcN-MoAb complexes that gave 10 times more specific binding than simi l ar comp l exes made with IgG M oAb. 99m TcNCl 4 ~ 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). Howεv er the partia l reduction procedure, comp lexiπg TcNCl 4 " through su lfur atoms, resulted in superior specificity ratios, aπd CBA thymocytes (Thy-1.2 + ) incorporated more than 90 times more T c - a n t i - T h y - 1.2 t h an n oπreac ti v e AK R thymocyte s ( Th y - 1.1 + ) . Th e i m un o p er o x i d a s e method was also used to assess the MoAb activity of two IgM MoAb, before and after labeling with 99m TcNCl 4 ~: (a) 3E1.2, which reacts strongly with membrane and cytoplasm of breast carcinoma and with the luminal membrane of normal breast aπd (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). ___221___l£_ ___L≤_i_____--_ 2_L ---__£NC 1 ~ labeled N£__bi. It was necessary to show that the partial reduction procedure used to l abel M oAb with 99 m TcNC l 4 " did not significant l y compromise the binding ability αf the MoAb to bind reactive target cells and this was demonstrated in three ways. First it was important to assess the immuπoreacti ity of the MoAb retained after labeling and so the percentage of binding of the radiolabeled 99m TcN-MoAb was determined. 99m TcN-anti-Ly- 2.1 was added to an increasing number of E3 cel ls (Ly-2 + ), and the amount of MoAb binding to the cel ls was determined (Figure 11 ). The degree of nonspecific binding was determined by running in parallel a πoπreactive 99m TcN-aπti- Ly-1.1 isotype control labeled under identical conditions. The reactive 99 m T c N -a n t i -L y - 2.1 achieved significantly higher binding tα E3 cells (60? in the plateau region) than the contro l 99m T c - a n 11 - L y - 1.1 (5?) (not shown). Second l y the binding of unmodified MoAb and 9 m TcN- 1 abe 1 ed MoAb was determined using the binding assay, where the amount of MoAb bound tα reactive cells was measured using a second antibody (anti-immunog 1 obu 1 in ) which was iodinated aπd reactive with the first. Increasing concentrations of unmodified aπti-Ly- 2.1 , DTT treated aπti-Ly-2.1 , 99m TcN-aπti-Ly-2.1 labeled to a high (100 muCi/mug) or low specific activity (12 muCi/mug) had equa l binding capacity on E3 cel ls (Figure 12a) , which suggests that this technique had not impaired the binding a c ti v ity o f the M oAb. Th is was a l so c onf irmed b y the rosettiπg assay, when the initial MoAb titer αf 1 :16,384 was unaltered after radio 1 abe 1 ing (Figure 12b). liH-iina 2l -"l£ϋ____n_'i-k_-_:___l _L£ _.£_!££ ££ϋ ii£££ __i___l _UJ-__£££π_: ££n£££_;£_l_-i£n£ 2. k___.___-l._. ^α c larify that the binding αf the TcN-MoAb complexes was primarily dependent on the concentration of reactive antigen binding sites on th e t arge t ce l l s , th e b in din g as sa y wa s us ed , wi th 3 different tumor cell lines E3, 01 aπd 8-15147, that differed in concentrations of Ly-2 present on the cel l surf ace. E3 aπd 01 are high aπd l ow Ly-2 + variants of thε ITT(1 )75NS cel l line, and B 1115147 being Ly-2 " was used as a control. The amount of binding was in proportion to the antigen density and 99 m T c -a n t i-L y -2.1 bound the E3 cel l l ine 8 times more antibody than the D1 cel l l ine aπd incorporated up to 10 Q times more radiolabel than did the nαπreactivs S ftf 5 47 cell line (Figure 13). I_i 2in£l The four different Ly-2.1 MoAb (IgG.. , IgG, , IgG 3 and IgM) were used in imaging experiments using B6CF1 mice bearing E3 tumor graf ts to determine which subc l ass best loca l ized the tumor in v iv o. In the f irst experiment mice with E3 tumor (0.82 cm in diameter) l ocated on one thigh were given intravenous injections of 99m TcN-aπti-Ly- 2.1 (IgG-. ) or 99 m T c - a π t i - L y - 1.1 (IgG 2 a ), the contro l a n t ib ody . S c in ti g rams im ages ob tain e d 28 h our s a f t er injection demonstrated the specific localization of the 99 m T c N - a n t i - L y - 2.1. Radioacti ity was concentrated in the central region of the mouse, indicative of the significant antibody distribution to vascularized organs such as the liver, lung aπd heart but the tumor was easily defined. Iilheπ y m TcN-anti-Ly-1.1 was used as a πonreactiv e 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 99m Tc -anti-Ly-2.1 (IgG 2 ), and all three tumors could be visualized. Howεver the high blood pool activity hindered visualization of the tumor close to the vascular organs such as the heart aπd 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 aπd 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 IgG. and IgG-. MoAb (data not shown). _iI_L_._i_i_ii_i_l T h e u s e o f 9 3 iB T c a s a r a d i o l a b e l f o r i munoiscint igraphy with MoAbs has been advocated as it is one of the most useful r a d i o n u c 1 i d e s because of its ideal nuclear properties ( T 1 / = 6 hr, energy 140keV, with no beta emmission). However little use has been made of this radioπuclide for r ad io 1 a b e 1 in g MoAb because of the complex chemistry involved in satisfactorily attaching it tα antibody. Several different methods have been used to complex 99 m T c to MoAb. The first relied on the conjugation of the metallic radioπuclide via bifunctional chelates, of proven success for 111 In in both experimental (5-8) aπd clinical application (9-11) but of little value for 99m Tc. The second approach involved the direct complexing of 99m Tc to MoAb to produce complexes with either amiπo 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 a m T c prior to coupling with MoAb, such as the production of colloid and the instability αf the 99m Tc-labeled MoAb complexes (16,19). Ui e now describe a simple one step method of radio 1 abe 1 ing MoAb with 99m TcNCl 4 ~ based on a substitution reaction and have complexed 99m TcNCl 4 ~ 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 TcNCl 4 ~ directly with amino groups of MoAb was characterized by low specificity ratios as the 99m TcN bound equally well to reactive aπd 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 preferεncε 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 99m TcNCl 4 ~ 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 pretiπning method necessary for the reduction of 99 Tc (16). The labeling efficiency of 99m Tc -1 abe1 ed 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 99m TcN 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 bifuπctioπal 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 99m T c -a n t i -Ly - 2.1 than the Ly-2L0lιl D1 cel l line (one has approximately B times the antigen density of the other) and 10 times more than the nonreactive Blιl5147 (Ly-2.1 " ) cell line (Figure 13). Finally, high specific activities were -achieved which al lowed the specific localization of labeled MoAb in the ap ro riatel reactive muriπe tumors. Specific localization by immunoscin t igr aph y of murine tumors was demonstrated with TcN-labeled MoAb by imaging studies aπd was illustrated in two ways. First, imaging studies showed that the E3 (Ly-2.1 + ) tumors were visible when mice were injected with reactive T c N - a n t i - L y - 2.1 whereas identical tumors could not be localized with nonreactive T cN -an t i -L y- 1.1 an isotype identical control. Second TcN-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 tα detect muriπe tumors aπd results indicate value in patients with cancer. TABLE 1 Characteristics αf murine monoclonal antibodies used for radiolabeling with 99m TcNCl 4 - Aπtibody Antigen Antibody Class Antibody purification (R β f) Specificity aπd Subclass anti-Ly-2.1 (21) Ly-2.1 ^a Protein A IgG 1 Protein A IgG 3 Protein A IgM( oπαmer) DEAE anti-Ly-3.l(22) Ly-3.1 IgG. DEAE aπti-Ly-1.l(23) Ly-1.1 l9G 2a Protein A aπti-Ly-15.2(24) Ly-15.2 X 9 G 2a DEAE anti-Thy-1.2 Thy-1.2 IgM(penta εr) NH 4 (S0 4 ) 2 H129-19(25) L3T4 DEAE HuLy-m9 human transferriπ IgG 1 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 99m TcN-labeled MoAb, prepared by the partial reduction αf MoAb with DTT aπd obtained from in vitro binding studies. Origin of Antibody Target ceill! 3 Specific Designation Reactive Nιoπrεactive Ratiol anti-Ly-2.1 IgM(moπomer) CBA BALB/c 10-15 IgG 3 CBA BALB/c 50-70 IgG- CBA BALB/c 30-50 IgG 2a RF/J C57BL/6 10-15 aπti-Ly-3.1 E3 thymoma BUJ5147 7-11 anti-Ly-1.1 CBA BALB/c 70-130 anti-Ly-15.2 RF/J BALB/c 50-60 aπti-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) 90-1 BO H129-19 E3 thymoma Btιi5147 110-130 1 Specific ratio = cpm bound reactive cells / cpm bound nonreactive cells. TABLE 3 Effect of conjugation with 99m TcNCl 4 - 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 99m TcN-labelεd -NH2 -SH 10-2 ++++ ++++ ++++ ++++ 10-3 +++ +++ +++ ++ 10-4 + + + + 1 Tissuεs wsre tested by immunoperoxidass and specificity graded: 0 negative, + = weak, ++ = moderate, +++ = strong, ++++ = very strong. REFERENCES 1. Hnatowich DJ, Davis Till, Grif f in Till, et a l : Radioactive labeling of antibody: a simple aπd effective method. Science 220:613-615, 1983. 2. Krejcarek GE, and Tucker KJ: Covalent attachment of chelatiπg groups to m a c r o m o 1 e c u 1 e s . Biochem. Biophys. Rεs. Co m. 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, McCal l MJ, Reardan DT, et a l: Conjugation of antibodies with bifunctioπal chelating agents: Iso th iocy ana t e and bromoace ta ide reagents, methods of analysis and subsequent addition of metal ions. Analytical Biochem 142:68-78, 1984. 5. Khaw BA, Strauss H 111 , Cahill SL, et al: Sequential imaging of I n dium-111 - 1 ab e 1 ed monoclonal antibody in human mammary tumors hosted in nude mice. J Nucl Med 25:592-603, 1984. 6. F awwaz R A , lilang TST , Estabrook A , et a l : Immuπoreacti v ity and b i od i s t r ib u t ion of Indium-111 - 1 abe 1 ed 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: R ad I o immun oimagiπg in malignant melanoma with In-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 In-labeled 791 T/36 monoclonal antibody for tumor i mu n o s c i n t i g r a ph y . Eur J Nucl Med 10:296-301 , 1985. 9. Rairweather DS, Bradwell AR, Dykes P 111 , et al: Improved tumor localization using indiuτn-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 0J, Kosciuczyk GC, Rusckowski M , et a l : Pharmacolciπetics of an indium-111 -labeled monoclonal antibody in cancer patients. J Nucl Med 26:849-858, 1985. 12. Pettit IJIA , Delaπd FH, Bennett SJ, et al: Improv ed protein labelling by stannous reduction of Pert eche t a e. J Nucl Med 21 :59-62, 1980. 13. Rhodes BA, Torvstad DA, Breslow K, et al: 99 m Tc- labeling and acceptance testing of radiolabeled antibodies and antibody fragments. In Burchiel S I, Rhodes BA, Friedman B. (Eds): Tumor Imaging: The R a d i o i m m u π o c h e m i c a 1 detection of cancer., New York, Massαn 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 99m Tc. Int J Nucl Med Biαl 12:3-8, 1985. 15. Rhodes BA, Zamora P0, Newe l l KD, et a l : T e ch π e t iu m- 99 m labeling of murine monoclonal antibody fragments. J Nucl Med 27:685-693, 1985. 16. Pettit 111 A , De land FH, Pepper GH , et a l : Characterization of tin-technetium colloid in technetium- labeled albumin preparations. J Nucl Med 19:387-392, 1978. 17. Pettit U A , Deland FH, Bennett SJ, et al : Improved protein l abe l ing by stannous tartrate reduction αf pert echnetate. J Nucl Med 21 :59-62, 1980. 18. Baldas J, and Bonπyman J: Substitution reactions of 99 m TcNC l 4 " - a route to a new class of 99 m Tc- radiαpharmaceuticals. Int J Appl Radiat Isσt 36:133-139, 1985. 19. Kanel los J, Pietersz GA, McKeπzie IFC, et al: Coupling αf the 99mTechπie t ium-ni t r ido group to monoclonal antibody and use of the complexεs f or the detection αf tumors in mice. J Natl Cancer Inst 77:431-439, 1986. 20. Smyth MJ, Pietersz GA, Classoπ BJ, et al: Specific targeting of ch l orambucil to tumors with the use of monoclonal antibodies. J Natl Cancer Inst 76:503-510, 1986. 21. Hogarth P M , Edwards J , McKenzie IFC , et a l : Monoclonal antibodies tα murine Ly-2.1 cell surface antigen. Immunology 46:135-144, 1982. 22. Murray BJ, Mercer III, McKenzie IFC, et al: The polypeptide structure aπd assembly of the L y - 2/3 he terod inters. I mmuπo g e πe t ic s 21:510-527, 1985. 23. Hogarth RM , Potter TA, Corne l l FN, et al : Monoclonal antibodies to murine cel l surface antigens. J Immunol 125:1618-1624, 1980. 24. Potter TA, Hogath PM, and McKenzie IFC: Ly-15: A new murine lymphocyte a 11 o a π t ig en i c 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 pheπotypical 1 y 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 su b p o p u 1 a11 oπ s of lymphocytes which react with a 11 oantisera. J Immunol Methods 20:173-183, 1978. FIGURE LEGENDS Figure 6 Purification of """TcNCl- " -labeled MoAb using Sephadex G25. The column was equilibrated with PBS aπd 0.5 ml fractions were collected. Figure 7 Binding of 99 m T c N C 1 4 " - 1 a b e 1 e d aπti-Ly-2.1 prepared by direct labeling of MoAb on RF/J (•) and BIO (o) thymocytes (Fig. 7a) or aπti-Ly-2.1 (•) and anti Ca. colon (o) on E3 thymoma cel l s (Fig. 7b). The amount of radioactivit bound as a function of antibody concentration is shown. Figure 8 Specific binding to E3 target cells (8a) anti-Ly-3 labeled with 99m TcNCl 4 " to DTT treated E3 (#) aπd Bill 5147 (O) target cells; or (8b) anti-Ly-2.1 (•) aπd anti- colon antibody (o) • Figure 9 Specific binding of DTT treated anti-colon antibody labeled with 99m TcNCl 4 " 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 99m TcNCl 4 " ( — . and the other prepared with DTT treated MoAb (- - - -) or CBA (•) aπd AKR (θ) thymocytes. Figure 11 Percentage binding of T c N C 1. ~ - 1 a b a 1 e d anti-Ly-2.1 (•) tα 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 cεll number is shown. Figure 12 Binding of aπti-Ly-2 to E3 target cells, using unmodified anti-Ly-2.1 (A); DTT treated aπti-Ly-2.1 99m (o), TcNCl Δ " labeled aπti-Ly-2.1 , high specific activity ( 100 muC i /m u g ) (•) aπd low specific activity (12muCi/mug) (■). (12a) detection using ^ I-sheep anti-mouse Ig; (12b) rosetting using sheep anti-mouse Ig. Figure 13 Binding of DTT treated aπti-Ly-2.1 labeled with 99m TcNCl 4 " to E3 (•), D1 (Δ) and B 1115147 (o) target cells. The amount of radioac ivity 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 99m Tc. 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 αf formula I Ab-Y-S-NTc(Hal) 3 Formula I wherein Hal is chlorine, bromine or iodine aπd 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 X Z n t Formula II -[NH-C-(CH) n ]_- wherein Z is H, alkyl, aryl, carboxy, halide, hydroxy or amino, X is NH, 0 or S aπd z is 0 or 1. 7. A conjugate as claimed in claim 5 and of formula Ab-S- Tc(Ha 1 ) 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(Ha 1 ) 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 aπd colon tumours. 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'), IgG., , I 9 G 2 a ' I 9G 2fa and IgG- j . 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 aπd reacting it with TcN (Ha 1 ) 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 h a v in g a n a n t i g e n b i n d i ng s i t e s u c c in i mi d y l p y r i d y 1 d i t h i o p r α p i o n a t e (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 aπd 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-ace t y 1 mεr c ap t o sue cinic anhydride (SAMSA) or SH introducing compounds to produce a side chain απ 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 aπd 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.

Inventors: KANELLOS JERRY, MCKENZIE IAN FARQUHAR CAMPBELL, PIETERSZ GEOFFREY ALLAN, KANELLOS, JERRY, MCKENZIE, IAN, FARQUHAR, CAMPBELL, PIETERSZ, GEOFFREY, ALLAN
Applicants: UNIV MELBOURNE, THE UNIVERSITY OF MELBOURNE
Application year: 1987
Main classification: C07F13
IPC codes: A61K51