NL8204108A - IMAGING TUMOR WITH RADIOACTIVELY MARKED MONOCLONAL ANTIBODIES. - Google Patents

Description

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Short designation: Imaging of tumor with radiolabeled monoclonal antibodies.

The invention relates to the detection of tumors. In another aspect, the invention relates to monoclonal antibodies labeled with radionuclides.

It has long been sought for a reliable technique for detecting tumors specifically at the site. Methods have been described for detecting, for example, carcinoembryonic antigen (CEA), which is related to human carcinoma, which circulates in the bloodstream. See U.S. patents 3,663,684 and 3,697,638. Obviously, these methods cannot be used to determine the location of the tumor. More recently, it has been proposed to use antibodies labeled with radioactive iodine isotopes to detect an antigenic agent related to a tumor. For example, U.S. Pat. No. 3,927,193 discloses a method using antibodies to CEA 125 131 labeled I and I. According to Patent 15, tests on male Syrian hamsters in which human seal cell carcinoma was introduced, by injection of labeled hijte anti-CEA, followed by examination of the animals' organs, demonstrated that the radioisotope was located in the tumor. It was therefore proposed that the location of a tumor in a human could be determined by in vivo administration of a parenteral solution of the antibody, followed by photo-scanning of the host.

The actual application of antibodies labeled with radioactive iodine has not met the expectations raised by the previous research. For example, Goldenberg et al., N. Eng.

J. Med., 298, 1384-1388 (1978) had some success in 1978 in detecting people with anti-CEA, which had been labeled with radioactive iodine. However, due to the residual background radioactivity, the reported limited success depended on the application of deduction methods. Mach et al. Are mentioned in N. Eng. J. Med., 303, 5-10 (1980) was even less successful and determined that only 0.1% of the administered dose was located in the tumor.

In selected cases, however, imaging of the tumor was still possible.

It is known that the affinity purification of heterologous antisera to obtain the antibodies used in the known tumor imaging methods usually results in the loss of high affinity antibody and that the antibodies obtained, which are a mixture of specific antibodies for different determinants on the antigen, are largely constituted by low affinity antibodies and antibodies which give nonspecific reactions. On the other hand, monoclonal anti-.

bodies are selected which show a high affinity for selected sites on the antigen and a low non-specific binding. Surprisingly, however, it has been found that monoclonal antibodies to tumor antigens, labeled by radioisotopes of iodine by known methods, still exhibit poor localization at the tumor site despite the fact that immunoreactivity can be demonstrated in vitro. This may be a consequence and presumably also a consequence of the loss of radioactive iodine by the labeled antibody. Therefore, there is still a need for a reliable method for detecting the correct site of a tumor in vivo.

According to the invention, a monoclonal antibody or antibody fragment for a tumor antigen directly with a metallic radionuclide or with a chelated radionuclide, for example, D-111-bound to DTPA, is labeled by conjugating a chelating agent to the antibody, followed by formation of a complex between the chelating agent and radionuclide. When the resulting labeled antibody or fragment 20 is injected into a host bearing a tumor, it is quickly and with great specificity located in the tumor itself. In some cases, localization in distant metastases can also occur, demonstrating tumor spread. The tumor site can be detected by photo-scanning methods. Mixtures of labeled monoclonal antibodies can also be used.

The object of the invention is therefore to obtain monoclonal antibodies to tumor-related antigens, which are labeled with metallic radionuclides.

Furthermore, the invention aims to provide an improved method for imaging tumors in an infected host.

The attainment of these and related objects will become apparent upon consideration of the accompanying drawing and the further description of the invention below.

Ill

Pig. 1 is a group of graphs showing the tissue distribution of In 35 and ^ ** 1 compared to the distribution of ^ Ga as a function of time after nude mice infected with human colon cancer were injected with anti-CEA, which is labeled with In and 125 67 I and Ga citrate.

Fig. 2 is a group of graphs depicting the amounts of In and I and Ga secreted by nude mice. 8204108 ί * ·, - 3 - ill 125 67 after injection of In and I-labeled anti-CEA and of Ga citrate.

Fig. 3 is a group of graphs showing distribution in tissues 111 125, ... ϊ 111 of In and I xn nude mice 48 hours after injection of the In and 125 111 125 5 i labeled anti-CEA compared to In citrate and I-labeled human albumin.

Fig. 4 is a group of graphs showing tumor / tissue ratios of 111 125 67 111 of. In and I compared to Ga after injection of In 125 67 and I labeled anti-CEA and of Ga citrate in nude mice, as a function of time.

As already mentioned, according to the invention, monoclonal antibodies to tumor-related antigens or fragments of these monoclonal bodies are labeled directly with metallic radionuclides or with chelated radionuclides. The monoclonal antibodies useful in the invention are products of the hydridome technology which is now well known. For example, reference can be made to the original work of Milstein and Kohler, reported in Nature, 256, 495-497 (1975).

Basically, the method boils down to injecting a mouse or other suitable animal with an immunogen, in this case with a tumor-related antigen, such as CEA. The immunized mouse is then sacrificed and cells are taken from the spleen and fused with myeloma cells to obtain hybrid cells called "hybridomas" which can be reproduced in vitro. The population of cell-producing antibodies is selectively grown and examined to isolate individual clones, each of which secretes a single antibody species specific for a particular region ("determinant") on the surface of the antigen molecule.

The individual clones can be further selected to find those which produce antibodies with the greatest affinity for the antigen and which exhibit low non-specific binding. The radiolabelled antibody (usually isolated from the ascites) is reacted with the metallic radionuclide or conjugated with an appropriate chelating agent, which is then used to bind the radionuclide. The chelating agent can be bound to the antibody using a wide variety of methods. Since the antibody is generally a polypeptide, one can use a chelating agent with a reactive group of the kind known to be capable of introducing a group to a protein substrate. Among the suitable reactive groups are listed those 8204108 <* - 4 - of formulas (1) to (12) of the formula sheet, wherein Ch represents the remainder of the chelating agent.

Suitable chelating agents include a wide variety of multi-tooth chelating agents, the ligands of which complex with metallic radionuclides. Among such agents are to be mentioned the polyamino carboxylic acids of formula (13), wherein x has the value 0 or represents an integer (x is preferably 0 or 1), y is the number 1 or 2 (y = 1 is preferred) and z is an integer having a value from 1-7 (z is preferably 1 or 7), while R is hydrogen or a group, through which the chelating agent is combined with an antibody, or a group, which affects the binding constant of the chelating agent for the radionuclide. Particularly suitable groups R include the group of formula (14) in which A represents the group by which the conjugation is effected. For example, A can be 15 N2 +, which is obtained by diazotization of the group -N ^, which itself can be obtained by reduction of a nitro group (-NO ^), which in turn can be obtained by nitriding the phenyl core.

The group A may also be a group of the formula (15), which is obtained by acetylation of the group -NH 2 by known methods, wherein L is a leaving group which is replaced during the conjugation, such as a halogen atom, for example Cl, Br or I (Br is preferred).

In other cases, R may be -CH ^ or -CI ^ CgH ^ OCE ^ a ^ H.

In the case of the polyaminocarboxylic acids, the conjugation can be accomplished via the carboxyl group itself by, for example, forming an acid anhydride as an intermediate, as reported by Krejcarek and Tucker, Biochemical and Biophysical Research Communications, 77, 581 (1977).

Among the polyaminocarboxylic acids, the polyaminoacetic acids are currently preferred. Specific examples of polyaminocarboxylic acids that can be used are ethylenediamine tetraacetic acid (EDTA) and derivatives thereof, such as diethylene triamine pentaacetic acid (DTPA), p-bromoacetamidophenyl (ethylenedinitrolotetraacetic acid) acid and 1- (p.aminophenyl) ethylenediaminetetraacetic acid diazonium salt is converted to allow conjugate. Among these chelating agents, DTPA is currently preferred.

Among the other suitable chelating agents are to be mentioned polyamino (alkylene phosphoric) acids, especially those of the formula (16), wherein x, y, z and R have the meanings given above. Preferably x has the value 0 or 1, y = 1 and z = 1. As specific polyamino (methylene phosphor) acids (y = 1), ethylene diamine tetra (methylene phosphoric) acid, 8204108, can be mentioned.

5-hexamethylenediaminetetra (methylene phosphoric) acid and diethylene triamine penta- (methylene phosphoric) acid which can be used in the invention.

Further chelating agents are the polyamines of the formula (17), wherein X, Z and R have the meanings given above.

Another class of chelating agents is that of the formula (18), in which R has the meaning given above, with the difference, however, that if R is not a group which allows conjugation, at least one of the phenol groups has a group is of the formula (19), wherein A has the above meaning.

The porphyrins are another particularly valuable class of chelating agents. Those suitable according to the invention include synthetic and naturally occurring porphyrins, including the porphyrins of the formula (20), wherein R has the above meaning and R 1 is hydrogen or a substituent, which is part of a naturally occurring porphyrin molecule. or a group introduced during synthesis, for example to allow conjugation or to affect the binding constant of the porphyrin. Preferred porphyrins are tetraphenylporphyrin (R = phenyl, R 1 = H) or tetrapyridyl porphyrin (R = 4-pyridyl, R 1 - H). One or more of the phenyl or pyridyl groups is substituted by -NH 2 or a group of the formula (15), wherein L has the meaning given above, or by other groups, to allow conjugation with the antibody. The substitution is usually in the para site for a phenyl group and in the 2 or 4 position of a pyridyl group.

Other chelating agents which can be used according to the invention are the crown ethers and their cryptand analogs of the general formulas (21) and (22), wherein R has the above meaning. Instead, the group used to allow conjugation can be incorporated directly into the crown ether or cryptand structure, as indicated in the formula (23), wherein A has the above meaning.

Derivatives of desferrioxamine B can also be used as a chelating agent. These have the formula (24), in which R has the above meaning.

Enterobactin derivatives are also suitable chelating agents. Preference is given to that of the formula (25), wherein A has the above meaning.

Carbocyclic analogues of enterobactin are also suitable. Listed below are compounds of the formula (26), wherein A has the above meaning.

8204108 V ΐ - 6 -

Other carbocyclic systems suitable as chelating agents are, for example, those of the formula (27) and the corresponding anilides wherein A has the above meaning.

It is understood by those skilled in the art that the ring represented for enterobactin and carbocyclic analogues may be further substituted by substituents which do not significantly degrade the ability of these compounds to bind metallic radionuclides and that substituents may be introduced provide end sites for conjugate to the antibody.

Yet another group of suitable chelating agents are the siloxanes of the formula (28), wherein R has the meaning given above / except that if R is not a group allowing conjugate, at least one of the catechol groups is a group of the formula (29), where A has the above meaning.

It will be understood that the above list of chelating agents is not exhaustive, but is merely illustrative of suitable chelating agents.

Direct labeling of antibodies with metallic radionuclides can be performed using known methods of introducing a metal ion to an antibody by attaching it to a site on the antibody itself. In some instances, the metallic radionuclide can be taken up by exposing the antibody, usually in solution, to the radionuclide in its proper oxidation state. For example, a currently preferred method involves direct metallation of an antibody with 103 Ru + 3 by contacting the antibody with a suitable ruthenium salt, for example, ruthenium trichloride, in a buffered solution. Suitable buffering agents include potassium acetate, sodium bicarbonate and trihydroxyethylamine (Tris).

Another suitable method involves direct metallation of the antibody with chromium. Chromium radionuclides can be introduced by mixing a chromate salt of the radionuclide, for example sodium or potassium chromate, with the antibody in the solution. ·

In other circumstances, the antibody may first be chemically modified to accept the radionuclide and then reacted with it. For example, disulfide groups in the antibody can be reduced, followed by the formation of a -S-M or -S-M-S group, where M is the radionuclide. For example, disulfide groups can be reduced using dithiothreitol, first forming a thiol group 8204108-7 which can be reacted with suitable metal ions, for example those of mercury, silver, zinc, lead or cadmium, in their oxide or salt form.

Radionuclides which can be used in accordance with the invention are those which form stable complexes with polyunsaturated chelating agents or, under suitable conditions, bind directly with the antibody.

The radionuclides are selected to have a half-life and other radiation properties that allow safe evacuation and introduction into humans. A large number of radionuclides have been studied for use in humans, which may be useful in the present invention. These can be grouped as follows:

The chelating agents of the porphyrin type (Formula 20), crown ether type (Formula 21) and cryptane type (Formula 22) are most suitable for binding radionuclides with an oxidation state of +2. Thus, 57 57 105 57 52, for example, 195 ml, Ni, Co, Ag, Cu and Mn form stable complexes in the oxidation mode +2.

The chelating agents of formulas (13), (16), (17), (18), (20) and (25) - (28) form stable complexes with radionuclides with an oxidation state of +3. As radionuclides which form stable complexes in the oxidation state 52 111 113m 99m 68 „67„ state +3 are to be mentioned Fe, In, In, tc, Ga, Ga, 20 16V 57co. «V 16 <W 146sd, 15V 95 ° kj, 103eu, 97ru, 9V l01m * h 201 and Tl. The chelating agents with catechol groups of the formula (25) - (28) are particularly suitable for use with radionuclides, which are highly mobile in vivo, especially radionuclides of iron, 52 * such as Fe.

As radionuclides, which can be directly bound to antibodies, ...,. 2Q3_ 197_ 105, 203 "97" 103D 99_.

are the following: Hg, Hg, Ag, Pb, Ru, Ru, Rh, 101m_. 48_

Rh and Cr.

The most suitable radionuclides are gamma emitters with an energy between 100 and 400 keV and with half-lives of about 5 hours to 5 days.

For reasons not currently fully understood, certain individual monoclonal antibodies to tumor antigens are resistant to direct metallation. Therefore, to record the radioisotope in this way, it is necessary to carefully choose specific antibodies. Therefore, it is presently preferred to bind a radionuclide to an antibody using the flexible approach, first combining a chelating agent with the antibody and then complexing with a radionuclide. In this regard, it is especially preferred to use In (T h = 2.8 days) as radionuclide in combination with DTPA as chelating agent.

8204108 - 8 -

The method of the invention can be used to detect tumors using monoclonal antibodies to tumor-related antigens in general. For example, in addition to CEA, melanoma-related antigens, α-fetoprotein, ferritin, human choriogonadotropin, zinc glycinate marker, and prostatic acid phosphatase (PAP) can be used as immunogens to produce the desired monoclonal antibodies, described above. , to stimulate. In actual use, the labeled antibody is injected into the patient in a suitable solution for parenteral administration. After sufficient time has elapsed, the patient is subjected to a photo-scan to detect any site of localized radiation indicating a tumor and / or one of its metastases.

The experiments below, performed with monoclonal 125 µl 15 antibody for CEA labeled with I and In, demonstrate the advantage of using monoclonal antibodies labeled with chelated radionuclides to image tumor according to the invention, as compared to methods based on iodine-labeled antibodies.

20 1. Preparation of radiolabeled anti-CEA

Anti-CEA monovlonal, available from Hybritech, Inc., La Jolla, Ca., 125 was labeled with I using the method of Bolton and Hunter, Biochem. J. 133, 529-39 (1973). The product was passed through a Sephadex G-25 column and the final material contained less than 0.5% free iodide. Binding of the radiolabeled monoclonal antibody was greater than 80% with equine anti-mouse antibody bound to sepharose and greater than 75% with sepharose bound CEA.

Ill

The same monoclonal anti-CEA was labeled with In using conjugated DTPA as a chelating agent according to the method of Krejcarek and Tucker, Biochemical and Biophysical Research Communications, 77, 581 (1977). The reaction product was purified by passing through a

Sephadex G-75 column. Antigen binding was comparable to that shown by the I-labeled antibody. The method used for labeling did not affect the affinity constant, Ka, for the antibody to unchelated antibody, as determined by tetration with CEA.

The in vitro stabilities of the labeled anti-CEA antibodies were determined by incubating a portion of the material at 37 ° C for 72 hours in a solution suitable for parenteral administration containing sterile 8204108 * * - 9 - normal saline , USP, and determined the free radionuclide by chromatography. The antibody solutions were spotted on Baker alumina IB strips and developed in 50% aqueous acetone. The protein-bound radionuclide remains at the origin while free radionuclide migrates. After the incubation time, the radioiodinated product showed less than 3% activity, which was present as free iodide. This was reduced by 50% by storage at 4 ° C. Under the same conditions, the In anti-CEA showed comparable stability.

10 2. Distribution studies in nude mice

Partition tissue studies of the radiolabeled mouse monoclonal anti-CEA were performed with nude mice bearing 0.5-1.0 g human colon tumors. The mice ranged in weight from 17 to 28 g (average about 23 g). The tumors were initiated by subcutaneous injection of a minced material containing CEA-producing colon adenocarcinoma human cells. Distribution studies were performed 3 weeks later. Two tests were performed.

In the former, 19 animals were divided into 4 groups of 4-6 animals and injected intravenously (IV) with 0.1 ml of sterile normal USP saline containing 0.5 microcurie I anti-CEA antibody 67 (about 0.3 yug antibody) and as control carrier-free Ga citrate, a known nonspecific imaging agent for human colon tumor. The injections were performed without anesthesia using a 100 µl Hamilton syringe to ensure accuracy.

Animals, with the dose partially infiltrating into the tail, were removed from the study. After the injection, the mice were placed in sterile metabolism cages, with a raised wire mesh floor to accommodate urine and faeces. Under the floor, sterile 4 x 4 gauze was filled in such a way that the animals could gnaw on it. The mice, which were kept in a clean room with sterile food and water ad libitum at 4.4 ° C, were sacrificed in groups at 4, 24, 48 and 72 hours after the injection of the tracer.

The mice were anesthetized with ether and blood samples were obtained by direct cardiac puncture. The mice were then sacrificed by neck dislocation. During the subsequent decomposition, the tumor, liver, kidneys, muscles, heart, lungs, thigh and spleen were removed, washed twice in water and once in 10% formalin, blotted dry with tissue paper and weighed on an analytical balance when wet. The intact stomach and intestines were also removed and counted. For the 8204108

+ V

- 10 - calculations of the total recordings by the organs were assumed to be blood, muscles and bones resp. 7%, 40% and 10% of the total body weight. The other organs were weighed as a whole. The radioactivity count was performed in an automatic gamma-tube counter ("well counter") whose vesters were set over the 125 67 27-35 keV photopeak of I and the 93 keV photopeak of Ga. The counter was programmed to stop working after 10 minutes or 1 million counts, in order to obtain statistical significance for the samples with the lowest activity levels, Corrections for background radiation and 10 reciprocal contribution of isotope activity between the settings of the window were performed by use prepared sources and solve simultaneous equations. The activity in the samples was then compared to standards of the injected material, the results expressed as percent injected dose per gram and 15 percent injected dose per organ. Tumor to tissue ratios were calculated from percent dose per gram data. Gut / urine and faecal activity was calculated as percent of the total injected dose.

In the second study, 37 nude mice bearing tumors grown from the same human colorectal cancer were divided into five groups of 7-8 animals. A group served as a control and was simultaneously injected IV with 1 microcurie of I-labeled human serum 111 albumin (0.01 mg HSA) and 3 microcurie carrier-free In citrate (0.01 mg citrate) and 48 hours after sacrificed the injection. In the remaining four groups, the animals received simultaneous injections of 3 microcuries of 111

In labeled anti-CEA (1.5 µg antibody) and 5 microcuries carrier-free 67

Ga citrate, The groups were resp. Sacrificed for 4, 24, 48 and 72 hours.

Tissue decomposition, preparation of the samples for radioactivity testing, counting methods and calculations were performed as described above. In the case of In, the spectrometer was set so that the 247 keV photopeak was measured using a 40 keV window (210-250 keV), 67 while Ga was counted with a 30 keV window overlapping the 93 keV photopeak. These settings resulted in less than 10% reciprocal contributions of radioactivity.

Ill 35 As shown in Figure 1, the concentrations of In anti-CEA located in the tumor increased over the entire time scale of the experiment. 23% of the injected dose had accumulated after 72 hours, and the results indicate that this level would have increased even more if attempts were made to take the samples later. In contrast, 8204108-11 - the blood level decreased during the measurement time. The liver showed rapid uptake after 4 hours, followed by a period of little change, then increasing to 72 hours. The other tissues showed little variation over the time course studied.

The level of Ga in the tumor remained fairly constant over the course of the run and after 72 hours was about 1/4 that of In 67 anti-CEA. Ga blood levels continued to decrease, while the concentration in the liver formed a pattern approximately similar to that of the Indium-labeled body. The concentration of Ga in the muscle decreased from 10 to 48 hours and went flat there, while the concentration in the bones remained the same throughout the period.

The amount of iodide in the tumor peaked at 4 hours and was initially slightly greater than the amounts of 11dium, after which it declined irregularly throughout the rest of the experiment and after 72 hours was only 1/10 of the amount of In. At that time, 67,125 twice as much ea appeared in the tumor as I. The counts of I in all tissues rapidly declined from their maxima after 4 hours and in some cases reached background level by 48 hours.

The excretion data (Fig. 2) show a steady increase of 111 In in the urine between 24 and 72 hours, reaching a maximum of 14% 111 of the injected dose. The concentration of In in the faeces remained fairly constant at 24 and 48 hours and then increased to 17% after 72 hours, while the amount in the intestines remained relatively constant over the entire time course, never exceeding 5% of the dose.

67 25 Ga's excretion pattern was somewhat similar to that of 111

In, however, showed more tracer in the gut and faeces than was observed for In and less in the urine.

125

Almost 60% of the radioactivity of I was excreted in the urine during the first 24 hours and after 48 hours, 75% was excreted.

125 30 Another 5% of I was lost in the faeces. Thus, after 72 hours, 125 about 80% of the originally injected I had been excreted by the animal. Chromatography of the I in the urine showed that the major part of it was free iodide, while a second species probably represented free I, complexed with something other than a protein.

Ill

Fig. 3 shows the data for In labeled anti-CEA after 48 hours of Hi 125 III and compares this with indium citrate and iHSA. The In citrate was used as a control for any Indium, which could be dissociated from the antibody, while the IHSA was used as an 8204108

* V

- 12 -

HI

nonspecific protein control. The In-labeled anti-CEA 111 showed a distribution pattern, which was very different from the In citrate, and concentrated more efficiently in the tumor by a factor of 10. The uptake of the two tracers by the other tissues was also different. The tumor-125 III concentration of IHSA was 20% that of In anti-CEA, but 125 125 was still 5% greater than that of I anti-CEA. The distribution of IHSA in the 111 non-tumor tissue is also very different from that of the In-labeled 125 anti-CEA product. It is particularly significant that IHSA, long regarded as a stable iodinated protein, exhibited nearly 60% excretion (43% in the urine) after 48 hours, which is remarkably consistent with the above finding. that anti-CEA was also found in urine. The fraction in the urine is thought to be free iodide.

Ill

The tumor / tissue ratios (Fig. 4) show that In anti-CEA 67 is superior to Ga in all tissues, except for blood, 67 for which it is approximately equal to Ga in that case. The 125 I anti-CEA ratios are apparently preferable to those exhibited by In and Ga in some 111 67 tissues, but they are misleading as they are due to low I levels in normal tissues rather than by localization in the tumor.

In view of the above tests, anti-CEA is clearly unsuitable as a tumor localizing agent. The rapid and intensive dehalogenation reduces the tracer concentration in the tumor and increases the background radiation of the blood, making it impossible to use it for imaging without advanced subtracting techniques. In fact, after 72 hours, the tumor contains double the amount of the nonspecific 67,125

Ga, calculated per gram, compared to I. Furthermore, the tumor / 67 blood ratios are better for Ga than for the iodinated antibody.

67

Considering that Ga is considered inadequate for imaging adenocarcinoma of the colon, the difficulties identified by Mach et al., N. Eng. J. Med., 303, 5-10 (1980) under 125

were easily explained. This using I

The data obtained unambiguously show that without good 131 131

subtraction techniques when using I very high doses of the I

labeled material should be administered to achieve the tumor concentrations necessary for efficient imaging. This would result in the patient receiving a high radiation dose because of the 131 physical half-life of 8 days and the 608 keV β component of I.

In contrast, the high percentage of the 111 injected dose of In anti-CEA and the more favorable physical properties 8204108 111 - 13 - of In the injection allow smaller amounts of radioactive pharmaceutical to be improved, while improving the image and radiation dose to the patient is minimized. The tumor to background ratios formed by In anti-CEA are well within the range required for imaging purposes due to the high degree of localization of radionuclide in the tumor. Furthermore, the data indicate that the tumor takes up radiolabeled material once incorporated or attached to other tissues.

Individual mice were photo-scanned after 10 4, 24, 48 and 72 hours after the injection of In-labeled anti-CEA using a Phagam® P Anger gamma camera. After 48 hours, the tumor was well defined with no need to resort to subtraction methods. After 72 hours, the definition of the tumor had improved even further.

The in vivo stability and thus the suitability for use in imaging a tumor of a directly labeled monoclonal antibody is demonstrated in the following experiment using a monoclonal anti-melanoma labeled with Ru compared to 125 with the same antibody, labeled I.

103 20 1. Preparation of Ru-labeled anti-melanoma 103

An aqueous solution of 220 µl RuCl (available from New England Nuclear, Inc.) was added to 100 µl of a saturated potassium acetate solution. The pH was adjusted to 5.3 with 70 µl 10 M NaOH.

This radionuclide solution was added to 500 µl of a 0.5 µg / µl mouse anti-melanoma antibody solution and the mixture was incubated at room temperature for 1 hour. The pH was adjusted to 7.2 by adding. 20 µl 10 M NaOH and the preparation was left at room temperature overnight. The unbound metal was separated from the antibody-bound metal by placing the reaction mixture on a column of Sephadex G-50 and eluting with common salt solution, which was adjusted to pH with phosphate

7.2 was buffered. The protein-containing fractions were collected and combined for in vivo studies in mice.

2. Distribution study in nude mice

Tissue distribution studies were conducted in nude BALB-C-103 mice using the Ru 125 labeled anti-melanoma prepared above and I-labeled anti-melanoma prepared in the same manner as described above for marking anti-CEA.

The mice were divided into two groups, which were injected intravenously 8204108 - 14 - 125 103 with resp. I-labeled and Ru-labeled antibody. The mice were sacrificed after 4, 24, 48, 72 and 96 hours, decomposed and then the radioactivity of the organs was determined as described above for the anti-CfeA monoclonal studies. The tissue distribution of I and Ru is shown in the table below.

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The data in the table show that the I-labeled anti-melanoma was rapidly de-halogenated, which was also the case with 125 103 the I-labeled anti-CEA, versus which the Ru-labeled anti-melanoma was stable. These observations are confirmed by the fact that the level of I in the blood 4 hours after the injection is very high and then drops to low, but constant levels after time courses of 24 - 96 hours. In contrast, the Ru content is drained from the blood relatively quickly and concentrates in the liver, as would be expected for a non-tumor infected host. Spleen and kidney levels in the mice injected with the Ru-labeled anti-melanoma are also consistent with in vivo stability.

It is known that CEA and some other tumor-related antigens are secreted or released by the tumor. In such cases, the occurrence of background radiation caused by the antigen circulating in the blood or other tissue associating with labeled antibody can be reduced by first administering undetected antibody which can combine with circulating antigen .

This tends to reduce the large uptake of labeled antibody by the liver. After an appropriate period of time, labeled antibody is added and the subject photo-scanned to determine the location of the localized radiation.

Although the above discussion has emphasized the use of a single monoclonal antibody labeled with a radionuclide, two or even more labeled monoclonal antibodies may be used for different antigen determinants within the scope of the invention. The different antibodies are chosen so as not to interfere with the binding of another antibody to the antigen. The use of a number of mutually non-interfering labeled antibodies can enhance the image of tumor, since the antigen has a higher capacity for labeled antibody. The use of a number of antibodies is particularly suitable for imaging a tumor, the associated antigen of which is present in a low concentration.

The above description of the invention pertains to presently preferred embodiments and, of course, other variations thereof may be apparent to those skilled in the art.

8204108

Claims (15)

A monoclonal antibody for a tumor-related antigen to which a metallic radionuclide is bound directly or via a chelating agent combined with the antibody. Monoclonal antibody according to claim 1, characterized in that the chelating agent is selected from polyaminocarboxylic acids, polyamino (alkylene phosphoric) acids, polyamines, porphyrins, crown ethers, cryptands, desferrioxamine B and derivatives thereof, and enterobactins and carbocyclic analogues thereof. Monoclonal antibody according to claim 2, characterized in that the chelating agent is a polyaminocarboxylic acid of formula (13), wherein x has the value 0 or represents an integer, y is the number 1 or 2, z is an integer with a value of 1 - 7 and R represents hydrogen or a group through which the chelating agent is conjugated to the antibody, or a group influencing the binding constant of the chelating agent for the radionuclide. Monoclonal antibody according to claim 2, characterized in that the polyamino (alkylene phosphoric) acid has the formula (16), wherein X has the value 0 or an integer, y is the number 1 or 2, z is an integer 20 with a value of 1 - 7 and R represents hydrogen or a group through which the chelating agent is conjugated to the antibody, or a group influencing the binding constant of the chelating agent for the radionuclide. Monoclonal antibody according to claims 3 and 4, characterized in that R represents hydrogen, a methyl group, a group -CE ^ C ^ E ^ OCS ^ CO ^ E or a group -CLH -A, wherein A represents a diazonium salt or a precursor thereof represents 6 or a group of the formula (15), wherein L is a group which is displaced during the conjugation. Monoclonal antibody according to claim 5, characterized in that 30 X = 0 and R represents a group of the formula -CgH ^ -NH-CO-GH ^ Br. Monoclonal antibody according to claim 5, characterized in that the chelating agent is a polyaminocarboxylic acid, in which X = 1 and R represents a group of the formula -CgH 1 -NH-CO-CH 2 Br. Monoclonal antibody according to claim 2, characterized in that the chelating agent is selected from: ethylenediamine tetraacetic acid, p-bromoacetamidodiphenyl (ethylenedinitrilotetraacetic acid), 1- (p-aminophenyl) ethylenediaminetetraacetic acid and diethylene triamine pentaacetic acid. Monoclonate antibody according to claims 1-8, characterized in that the radionuclide is a garoma radiator with a half-life of 5 hours 8204108 A * - 19 - up to 5 days and whose gamma emissions have an energy of 100 - 400 keV. 10. Monoclonal antibody according to claims 1-9, characterized in that the radionuclide is selected from 195mPt, 5 ^ Ni, ^ Co, ^ ^ Ag, ^ Cu, ^ Mn, Cl113m 99in · 67 68 _ 169 , - 166 ^ 67 146 _ 157 5dFe, In, In, ^ c, Q Ga, Ga, Yb, Tm, Tm, Gd, Dy, 95nr 103 97 99, 101me, 201 -, - 203 197 105 203 97 5 lib , Ru, Ru, Bh, Bh, Tl, Hg, Hg, Ag, Pb, Ru „48rv and cr. 11. Monoclonal antibody according to claims 1-10, characterized in that the tumor-related antigen is one of the following: carcinobryonic antigen, α-fetoprotein, ferritin, human choriogonado tropin, zinc glycinate marker, prostatic acid phosphatase and melanoma-related antigens. Preparation, characterized in that it is a solution of a monoclonal antibody for a tumor-related antigen according to claims 1-11, suitable for parenteral administration. 15 Preparation according to claim 12, characterized in that it comprises a mixture of two or more of these monoclonal antibodies. 14. A method of imaging a tumor, characterized in that: a) a human is administered a parenteral preparation containing a monoclonal antibody for a tumor-related antigen, the monoclonal antibody being a radionuclide that is bound to the antibody directly or through an antibody-conjugated chelating agent; and b) subject the patient to photo-scan to locate the antibody locating in the subject, as evidenced by the radiation from the radionuclide. Method according to claim 14, characterized in that a monoclonal antibody is used as defined in claims 1-13. 8204108