LU84441A1 - IMPROVEMENTS IN TUMOR DETECTION - Google Patents

Description

-¾ 2.

"Improvements in tumor detection" _

The present invention relates to the detection of tumors. In another aspect, it relates to monoclonal antibodies labeled with radionu-5 clides.

A reliable technique for site-specific detection of tumors has been sought for a long time. Methods have been described for detecting, for example, the carcinoembryonic antigen (CEA) associated with human cancer circulating in the blood stream. See U.S. Patents 3,663,684 and 3,697,638. These methods obviously cannot be used to determine the location of the tumor. More recently, it has been proposed to use antibiotics labeled with radioactive iodine isotopes to detect an antigenic substance associated with a tumor. Thus, the patent of the United States of America No. 3,927,193 describes a process using antibodies to CEA labeled with 125 131 20 I and I. According to this patent, in cExperiments carried out using male Syrian hamsters into which human ring cancer cells had been introduced in the form of seal rings, the injection of goat anti-CEA followed by the examination of the organs of the animals showed that the radioisotope s 'was ^^ f 3.

localized in the tumor. Therefore, it has been suggested that the location of a tumor in a human can be determined by in vivo administration of a parenteral solution of the antibody followed by photoscanning of the host.

The use carried out of the 3rd radioiodinated antibodies did not meet the forecasts of the work carried out previously. For example, Goldenberg et al., N.

* Eng. J. Med., 298, 1384-1388 (1978) obtained a certain. 10 successes in 1978 in sweeping human beings with radioiodi-tated anti-CEA. However, because of residual background radioactivity the reported limited success depended on the use of subtraction techniques.

Mach et al., N. Eng. J. Med., 303, 5-10 (1980) were even less successful and determined that only 0.1% of the administered dose was located in the tumor. However, in selected cases, the impression of the tumor was still possible.

It is well known that affinity purification of heterologous antisera to obtain the antibodies used in prior art tumor printing methods usually results in the loss of high affinity antibodies and that antibodies Obtained, being a mixture of antibodies specific for different determinants on the antigen, are mainly composed of low affinity antibodies and of antibodies which give non-specific reactions. Monoclonal antibodies, on the other hand, can be chosen so as to show a high affinity for selected sites on the antigen and a weak non-specific binding. However, it has surprisingly been found that monoclonal antibodies to antigens ............ 7 4.

iodine following well known procedures still show weak localization at the tumor site despite the fact that in vitro immunoreactivity can be shown. This can and probably should lead to the loss of radioactive iodine by the labeled antibody.

Therefore, it still appears necessary to develop a reliable technique for detecting in vivo the precise location of a tumor. According to the present invention, an antibody or a monoclonal antibody fragment vis-à-vis of a tumor antigen is labeled either directly with a metal radionuclide or with a chelate-bound radionuclide, for example in bound to DTPA, by bringing a chelating agent into conjugation with the antibody and then forming a complex between the chelating agent and the radionuclide. The resulting labeled antibody, or fragment thereof, when injected into a host carrying a tumor, localizes quickly and with high specificity in the tumor itself. In some cases, localization in distant metastases may occur as well as to indicate spreading of the tumor. The location of the tumor can be detected by photoscanning techniques. Mixtures of labeled monoclonal antibodies can also be used.

„It is therefore an object of the present invention to obtain monoclonal antibodies? vis-à-vis antigens associated with tumors labeled with metal radionuclides.

Yet another object of the invention is to

provide an improved method for printing or imaging tumors in an infected host. I

5.

Other details and particularities of the invention will emerge from the description below, given by way of nonlimiting example and with reference to the appended drawings, in which: FIG. 1 represents a group of graphs illustrating the distribution in 111In and 125I tissues compared to the distribution of ^? Ga as a function of time after injection in nude mice - infected with human colon cancer of 111 125 10 CEA labeled with χη and I as well as 67Ga citrate.

Figure 2 shows a group of graphs showing the amount of. In and of I thus 5 7 · · * that of Ga excreted by naked mice as a function of 111 15 of the time after injection of anti-CEA labeled with. χη and 125 57 au and citrate from ea.

FIG. 3 represents a group of graphs illustrating the distribution in tissues of In and 125 of -i in naked mice 48 hours after injection 111 125 * 20 d / anti-CEA labeled with. In and I as compared to human citrate albumin labeled with 111 _ 125_

In and at I.

FIG. 4 represents a group of graphs 111 125 illustrating the tumor / tissue ratios of In and Ί 67 ~ 25 compared to Ga, after injection of anti-CEA labeled 111. 125_ ^ _ .. ^ _ 67 „* · with In and I and with citrate of Ga in naked mice, as a function of time.

As has been clarified, according to the present invention, monoclonal antibodies or fragments thereof against antigens associated with tumors are labeled directly with metal radionuclides or with radionuclides linked to a chelate. Monoclonal antibodies that can be used in day / day / 6.

the invention are products of hybridoma technology which is currently well known. One can refer to this effect, for example, to the basic works of Milstein and Köhler reported in Nature, 256, 495-497 5 (1975). Basically, the method involves injecting a mouse or any other suitable animal with an immunogen, in this case an antigen associated with a tumor, such as CEA. The immunized mouse is then killed and the cells extracted from its spleen are combined with myeloma cells to give hybrid cells called "hybridomas", which can be reproduced in vitro. The population of antibody producing cells is selectively cultured and examined to isolate individual clones each of which secretes a single species of antibody specific to a particular region ("determinant") on the surface of the antigen molecule. The individual clones can again be examined to determine which ones produce antibodies with the highest affinity for the antigen and which show low non-specific binding. The antibody chosen for radioactive labeling (normally isolated from ascites) is reacted with the metal radionuclide or is conjugated with a suitable chelating agent which is then used to bind the radionuclide. The chelating agent can be linked to the antibody using a variety of processes. Generally speaking, can-? that the antibody is a polypeptide, one can use a chelating agent having a reactive group of the type known as being advantageous for introducing a fragment into a substrate of the protein type. Among the suitable reactive groups, the following groups may be mentioned: / 3 7.

Ο ”/ ^ Ν

Ch—- NCS Ch — C-N ^ J

5 ch — r - jr Ch SCUCl Λ / o 0 0 w

Ch — r-Ch C O N

10 \ S. // 0 F F '0

Ch- C —0- ^ ~ ^ -F Ch- N— S02-CH = CH2

^ F F

15 Ch-N j] Ch —N2 + (frcsn Ch-NH2)

K

0 0 II

Ch — NH- C- CH93r 'Ch- C- O- C — CH2CH (CH3) 20 0 0 o & Ch is the remainder of the chelating agent.

Suitable chelating agents are a large series of poly-toothed chelating agents whose ligands complex with metal radionuclides. Among these agents, there may be mentioned the polyaminocarboxylic acids of the formula: /

If 8.

K02C— (CK2) V / {C-V \ TC02H \ (CH2) C02K

5 \ I '\ / · "'

N— CH (CH ^ -rrrN— CH- (CH,? - "N

/! M i 2 η / R \ R · / \

. H02C-fCH2) y Y / X. (CH2) y C02H

10 '(1) in which x is equal to o or is an integer (preferably x = 0 or x = 1), y = 1 or 2 (preferably y = 1) and z is an integer from 1 to 7 (z is preferably equal to 1 or 7) and or R represents hydrogen or a group via which the chelating agent has come into conjugation with the antibody or a group which modifies the constant of binding of the chelating agent to the radionuclide. Among the groups R 20 of interest, mention may be made, in particular, of the group Ay in which A is the group through which the conjugation is carried out. For example, A can represent -N2, which is obtained by diazotization of the group -NH2, which itself can be obtained by reduction of a nitro group (-NO2) which, in turn, can be obtained by nitration of the phenyl ring. Group A can also be a group, obtained by -nh-c-ch2l

acetylation of the -NH2 group by known procedures, where L is a group which leaves displaced during conjugation, such as a halogen atom, for example Cl, Br / or I (Br being preferred). AJ

9.

In other cases, R can represent -CH3 or -CH2C6H4OCV ° 2H ·

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

Among the polyaminocarboxylic acids, polyaminoacetic acids are currently preferred.

Specific polyaminocarboxylic acids which can be used are ethylenediaminotetraacetic acid (EDTA) and its derivatives, such as diethyleneamino-pentaacetic acid (DTPA), p-bromoacetamidophenyl acid (ethylenedinitrilotetraacetic acid) and 1- (p-amino-phenyl) -ethylenediaminotetraacetic, the latter being converted into a diazonium salt to allow conjugation. Among these chelating agents, DTPA is currently preferred.

; Other interesting chelating agents include polyamino acids (alkylene phosphoric acids), in particular those of the formula: 0 0. ? not . "

25 HO-P (CH2) y (CH2Tv ~ P-OH (CH2 ^.? - 0K

\ / | . "\ /"

»CH-tCH2 ^ -N-CÏHG32H-N

° / 1 '\ l / v \

30. "i-p-Hcaj), Λ J (ch2 * T'- ° K

-1 day

OB

OH

•. (2) \ ^ 10.

in which x, y, z and R are as defined above. Preferably, x is equal to O or 1, y = 1 and z = 1. Specific polyamino (methylene phosphoric) acids (y = 1) which can be used in the context of the present invention are ethylenediaminotetra acid (methylene phosphoric) , hexamethylenediamino-tetra acid (methylene phosphoric) and diethylene-triaminopenta acid (methylene phosphoric).

Other chelating agents are the polya-10 mines of the formula:

V—: CH2 * TCH2 / CH2 ^ C! WOT1 ^ CHrCS2TSH

N-ÇîHCHjW N-ÇH— "η245-4ν 15 / R R / \ ·’

H2N — ÎCH2- * yCH2 \ / X

* <• t '· * (3) * 20 in which X, Z and R have the meaning given above.

Another class of chelating agents are those corresponding to the formula:

25 OH HO

Çj— C "2 CH2" 0 \ /

N— CH — CH -— N

o / l 2 ‘. \ o 30 "'/ R \

HO-P-CH, CK, P-OH

OH OH / in which R has the meaning given above, except that when R does not represent a group which allows conjugation, at least one of the phenol groups represents a group corresponding to the formula - ^ ^

5 ... 1 OH

in which A has the meaning given above ... - ...........

Another particularly interesting class of chelating agents is porphyrins.

: The porphyrins of interest in the context of the invention are synthetic porphyrins of natural origin, in particular the porphyrins of the formula: É W / Rl O h Λ 20 R., | R ^ / ~ Cf \

R R

25 1 l (5) in which R has the meaning given above 30 and R ^ represents hydrogen or a substituent forming part of a porphyrin molecule of natural origin or a group introduced during.

12.

synthesis, for example, to allow conjugation or to modify the binding constant of porphy-rine. Porphyrins of interest are tetra-phenylporphyrin (R = phenyl, R ^ = H) or tetra-5-pyridylporphyrin (R = 4-pyridyl, R ^ = H). One or more of the phenyl or pyridyl groups are substituted by ~ NH9 or C, where L has the meaning

Δ II

-NH-C-CH2L. r · - given previously, or by other groups to allow conjugation with the antibody. The substitution will normally be in the para position for a phenyl group and in the 2 position or in the 4 position for a pyridyl group.

Other chelating agents which can be used in the context of the invention are crown configuration ethers and their crypt-tandem analogs corresponding to the following general formulas:

R R

rr v;

n 0 N N

Rv / \> R R \ / ^ X ^ R

T I H E

and- 1 H H J ^ a. CT S.

"w ... w

RR · 'RR

30 (6) (7) 4 13.

in which R has the meaning given above. Alternatively, the group that is used to allow conjugation is directly incorporated into the ether structure of crown configuration or 5 of cryptand ", as illustrated by the following formula: λλ

10 VV

O

\ _ /. · · 15 in which A is as defined above.

Desferrioxamine B derivatives can also be used as chelating agent. These derivatives correspond to the formula:

O OH 0 O OH

· * "II II I

R-CH2-C-N — tCH2 ^ - nh-c— (οκ2 ^ ο-ν—

25 OH O - O

1 H -> 11 • E2N-ÉCH2hr-N-C— (CI ^^ C-NK- (CH2br— * / '9 30 / i 14.

in which R has the definition given above.

Enterobactin derivatives are also interesting chelating agents. The preferred derivatives are those corresponding to the formula: 5

OH OH

OH OH CH2-0- C— CH - NH— Λ — A

'10 ^ -C-NH-CH \ cH2

o = c I

I 0 I! 0-CH?: - CH- C = 0

2 I

15 NH

c = o

OH

20 ^^ OH

(9) in which A is as defined above.

Carbocyclic analogs of enterobactin are also of interest. Among these, mention may be made of the compounds of the formula: / / 15.

OH OH

° _y \

• OH OH CK? -CEjr — CH-— CH— NH— C - (/ A

s M 5 -i 1 Λ = -Λ

4 λ— C— HN-'CH CH

Yj I | 2

CH_ CH

| 2 I 2 10 · CH-CH —CH — CK2

NH

C = 0

i5 0H

OH

(10) 20 in which A is as defined above.

Other carbocyclic systems which can be used as chelating agents are those corresponding to the formula: /) / ï 16.

HO HO OH OH

5 CH, o

10 ... - I

o = c / ^ ΐγΌΚ

kci ^ -c-K

15 (11) and the corresponding anilides in which A has the definition given above.

Those skilled in the art will appreciate that the ring shown for enterobactin and carbocyclic analogs may be further substituted by substituents which do not materially affect their binding capacity with metallic radionuclides and that these substituents may be introduced with the aim of creating sites for conjugation with 11 antibodies.

Yet another group of interesting chelating agents are the siloxanes of the formula: / / 17.

OK OK 'CHR CKR · CKR OK OH

/ /) —- Si— O— Si— 0 - Si- ')> 5 2 3 I I I \ _y CH3 (CH2) 3 CH3 χΛγΟΗ

OH

10 (12) in which R is as defined above, except that when R does not represent a group which allows conjugation, at least one of the catechol groups represents a group corresponding to the formula —λ — A , in which A is as defined above- OH OH demment.

Those skilled in the art will understand that the foregoing list of chelating agents is not exhaustive, but rather gives examples of suitable chelating agents.

Direct labeling of antibodies with metal radionuclides can be accomplished using known methods for introducing a metal ion to an antibody by its attachment to a site on the antibody itself. In some cases, the metallic radionuclide can be incorporated by exposing the antibody, usually in solution, to the radionuclide in its appropriate oxidation state. For example, a currently preferred technique consists of direct mixing of an antibody with ^ ° ^ Ru + ^ by bringing ^ /

V

18.

the antibody with an appropriate ruthenium salt, for example ruthenium trichloride, in a buffered solution. Among the buffering agents that can be used are potassium acetate, sodium 5-bicarbonate and trihydroxyethylamine (Tris).

Another suitable technique 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 solution.

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

The radionuclides which can be used in the context of the invention are those which form stable complexes with poly-toothed chelating agents or which, under suitable conditions, combine directly with the antibody. The radionu-30 clides are chosen so as to have a half-life or other radiation characteristics which allow elimination and introduction without risk in / ¥ 19.

Human being. A large number of radionuclides which can be used in humans have been studied, which are of interest in the context of the present invention. These can be grouped as follows:

The chelating agents of porphyrin (formula 5), crown configuration ether (formula 6) s and cryptand (formula 7) are most advantageous for binding radionuclides having an oxidized state. . LO, 195m ^ 57__. 57 „105Ώ 67„ 10 tion +2. For example, Pt, Ni, Co, Ac, Cu - 52 and Mn form stable complexes in their +2 oxidation state.

The chelating agents of formulas 1 and 5 and 9-12 form stable complexes with radio-nuclides having a +3 oxidation state. Among the radionuclides which form stable complexes in the state, 3 1 -3 x. ·, ”. ., 52 11 113rrL 99m_ of oxidation +3 we will mention Fe, In, In, Te, 68 67 _ 169, - 57_ 167m 166m 146 „. 157 ^

Ga, Ga, ïb, Co, Tm, Tm, Gd, Dy, 95tri. 103 97 99 ^ IOIbl. ^ 20lm1

Nb, Ru, Ru, Rh, Rh and Tl. The chelating agents comprising catechol groups, formulas 9-12, prove to be particularly advantageous with radionuclides which are highly mobile in vivo, in particular iron radionuclides, such - 52 as Fe.

25 Radionuclides that can be directly. . . . . . ,. 203 „197„ 105 linked to the antibodies are the following: Hg, Hg, Ag, 203, 97 „103_ 99 10lm. . 48 „

Pb, Ru, Ru, R2, Rh and Cr.

The most suitable radionuclides are gamma emitters with an energy between 100 30 and 400 keV having half-lives of the order of 5 hours to 5 days.

For reasons that are not clear / 20.

Currently understood, some individual monoclonal antibodies to tumor antigens are resistant to direct metallation. Thus, to incorporate the radioisotope by this route, it is necessary to carefully choose the specific antibodies. Therefore, it is currently preferable to bind a radionuclide to an antibody using the most flexible method which is to first conjugate a chelating agent with the antibody and then form a complex with the radionuclide. In this regard, it is preferred to use In (T 1/2 = 2.8 days) as a radionuclide together with DTPA as a chelating agent.

The method of the present invention can be used generally for the detection of tumors using monoclonal antibodies corresponding to tumor associated antigens. In addition to CEA, antigens associated with melanomas, α-foeto-protein, ferritin, human choriogonadotropin, the marker formed by zinc glycinate, and prostatic acid phosphatase (PAP) can, for example, be used as immunogens to stimulate the production of the desired monoclonal antibodies, as described above. In the present use, the labeled antibody in an appropriate parenteral solution is injected into the patient. After a sufficient period of time, the patient is subjected to a light scan to detect any localized radiation site indicative of a tumor and / or one of its metastases.

The following experiments carried out with a monoclonal antibody corresponding to CEA labeled with du and ^ · * Ίη show the advantage of using / / 21.

monoclonal antibodies labeled with chelate-bound radionuclides for printing or imaging tumors according to the present invention compared to methods using iodine-labeled antibodies.

1. Preparation of radioactively labeled anti-CEA.

Monoclonal anti-CEA, delivered by Hybritech, 125

Inc., La Jolla, Ca #, is labeled with I in use; reading the method of Bolton and Hunter, Biochem. J., 10 133, 529-539 (1973). The product is passed through a Sephadex G-25 column and the final material contains less than 0.5% free iodide. The binding of radioactively labeled monoclonal antibody is greater than 80% with horse anti-mouse antibody bound to sepharose and greater than 75% with CEA bound to sepharose.

The same monoclonal anti-CEA is marked with „111. .

of In using DTPA conjugate as a chelating agent following the process of Krejcarek and Tucker, ”20 Biochemical and Biophysical Research Communications, 77, 581 (1977). The reaction product is purified by passing it through a column of Sephadex G-75. The antigen binding is comparable to that shown by 125 the I-labeled antibody. The labeling method does not change the affinity constant, Ka, for the antibody relative to the non-chelated antibody, such as determined by titration with CEA.

The in vitro stabilities of labeled anti-CEA antibodies are determined by incubating a portion of the material in a parenteral solution comprising sterile normal U.S.P. saline at 37 ° C for 72 hours and determining the amount of free radionuclide chromatographically. Antibody solutions ^ / 22.

are distributed in drops on alumina strips IB

Baker and developed in 50% aqueous acetone.

The protein-bound radionuclide remains at its starting point while the free radionuclide migrates. After the incubation period, the radioiodinated material shows less than 3% of activity present as free iodide.

Storage at 4 ° C reduces this value by 50%. Under 111 the same conditions, the anti-CEA In shows comparable stability.

10 2. Distribution studies in nude mice.

Tissue distribution studies of anti-CEA from radioactively labeled monoclonal mice are performed in nude mice carrying 0.5-1.0 g of human colon tumors. The weight of the 15 mice varies between 17 and 28 g (average of about 23 g). Tumors are induced by subcutaneous injection of a mince containing CEA-producing human colon adenocarcinoma cells. Distribution studies are undertaken three weeks 20 later. Two experiments are carried out. In the first, 19 animals are divided into four groups of 4-6 animals and injected intravenously (IV) with 0.1 ml of a U.S.P. solution. normal sterile saline containing 0.5 microcurie of anti-CEA 125 antibodies labeled with I (about 0.3 micrograms of antibody) and, 0 * 7 as a control, of citrate of ea without support, an agent impression of a known specific human colon tumor. The injections are performed without anesthesia using a 30,100 microliter Hamilton syringe to ensure accuracy. Animals in which the dose has partially infiltrated the tail are excluded from the study. After injection, / îf 23.

mice are placed in sterile metabolic cages with a raised wire mesh bottom for the collection of urine and faeces. Below the bottom, sterile 4x4 gauze was piled up so that it could not be eaten by animals. The mice, kept in a clean room at 4.5 ° C. with sterile food and unlimited water, are killed in groups 4, 24, 48 and 72 hours after the injection of the tracer.

The mice are anesthetized with ether and blood samples are obtained by direct cardiac puncture. The mice are then killed by a cervical dislocation. During subsequent dissection, the tumor, liver, kidneys, muscles, heart, lungs, femurs and spleen are removed, rinsed twice in water, once in formalin at 10%, wiped with blotting paper and weighed in the dry and wet state on an analytical balance. The intact stomach and intestines are also removed and subjected to; 20 a count. It is estimated that blood, muscle and bone account for 7%, 40% and 10% of total body weight *, respectively, for all organ removal calculations. The other organs are weighed in their entirety. The counting of the radioactivity is carried out in a channel counter for automatic gamma samples with windows adjusted to the photoelectric peak of 27-35 keV I and the photoelectric peak of 93 keV Ia / Ga. The counter is programmed to reject 1 million strokes in ten minutes to obtain statistical significance for samples with the lowest / lowest activity levels. Corrections relating to content and conr / f 24.

Reciprocal tribution of isotopic activity between window settings is achieved using prepared sources and solving simultaneous equations. The activity of the samples is then compared to standards of the material injected, the results being expressed as a function of the percentage of dose injected per gram and the percentage of dose injected per organ. The tumor / tissue ratios are calculated from the dose results in% / gram. The ac-10 activity in the intestines, urine and faeces is calculated as a percentage of the total injected dose.

In the second study, 37 nude mice carrying tumors developing from the same human colon cancer are divided into five groups of 7-8 animals. One group serves as a control and is injected simultaneously with the intravenous route 1 microcurie of albumin 125 of human serum labeled with I (0.01 mg of HSA) 111 and 3 microcuries of In citrate without support (0.01 mg citrate) and they are killed 48 hours after the injection.

• In the remaining four groups, animals receive simultaneous injections of 3 microcuries of in labeled anti-CEA 111 (1.5 micrograms of antibody) and 5 micrococuries of unsupported Ga citrate. The groups are killed respectively after 4, 24, 48 and 72 hours.

Tissue dissection, preparation of samples for radioactivity testing, counting techniques and calculations are performed as described above. In the case of In, the spectrometer is adjusted to count the photoelectric peak of 247 keV 30 using a 40 keV window (210-250 keV) tan- g η say that Ga is counted with a 30 keV window covering the photo-electric peak of 93 keV. These re- / 25.

glages lead to reciprocal contributions of radioactivity below 10%.

As shown in Figure 1, the concentrations of 111-labeled anti-CEA localized in the tumor are constantly increasing as a function of the time scale of the experiment. 23% of the injected dose accumulated after 72 hours and the results suggest that the percentage would have been even higher if the samples had been taken later. The percentage in the blood, on the contrary, decreases with time. The liver shows rapid absorption after 4 hours, followed by a period of minor change, and then increased to 72 hours. The other tissues showed only small variations over the period of time studied.

6 7

The content of 'Ga in the tumor remains rather constant during the experiment, being approximately 1/4 of the content of anti-CEA labeled with "^^ In after 72 0 η 20 hours. The content in the 'Ga blood drops steadily, while the concentration in the liver shows a general pattern somewhat similar to the antibody labeled with ^^ indium.The concentration in the muscles of 6 7 XGa falls during the first 48 hours and stabilizes - 25 then read, while the concentration in the bones remains practically the same throughout the experiment.

The quantity of iodide in the tumor removed after 4 hours, being initially slightly greater 111 than the quantities of indium, then decreases irregularly during the rest of the experiment, being only 1/10 of that of ^^ in after 72 hours. At this time, it appears in the tumor twice as much 6 ^ Ga as of /? 26.

125 125 I. The count rate of I in all tissues decreases rapidly from their maximum value reached after 4 hours, and in some cases reaches background levels after 48 hours.

5 The excretion results (Figure 2) show 111 a constant increase of In in the urine between the 24th and 72nd hours, reaching a maximum 111 of 14¾ of the injected dose. The concentration of In - in the faeces remains fairly constant between the 24th 10 and 48th hours, and then increases up to 17% after 72 hours, while the quantity in the intestine remains relatively constant over the entire period, never exceeding 5% of the dose.

0 η

The excretion pattern for xGa is somewhat similar to that for 1111, although it shows a greater amount of tracer in the intestines and faeces than that observed for the and a lesser amount in the urine.

125

Almost 60% of radioactivity due to I

i 20 are eliminated in the urine during the first 24 hours, and 75% after 48 hours. An additional 5% of 125 I is lost in the faeces. Thus, after 72,125 hours, approximately 80% of the I initially injected has 125

been excreted by the animal. Chromatography of the I

25 in the flour shows that most of it is in the form of free iodide with a second species 125 probably representing free I complexed with something other than a protein.

FIG. 3 gives the results relating to the anti-CEA labeled with ^^ In after 48 hours and the same as for indium citrate and IHSA. The ci-träte of In is used as a control for any quan- / 27.

possible amount of indium which may have dissociated 125 from the antibody, while the IHSA is used as non non-specific protein control. The anti-CEA marked with 1 111

In shows a very different 111 5 distribution diagram from that of In citrate, concentrating ten times more efficiently in the tumor. The attachments of the two tracers by the other fabrics are also 125 different. The concentrations of. IHSA in the tumor are 20% of the concentrations of 111 marked anti-CEA 111 at In, but still 50% higher than the concentrations of 125 anti-CEA labeled at I. The 125 distribution of IHSA in the tissue without tumor is also very different from that of product 111 of anti-CEA marked in. It is particularly significant that IHSA, long regarded as a stable iodine protein, shows almost 60% excretion (33% in urine) after 48 hours, which very well corroborates the fact above that '125 has also found anti-CEA labeled with I in' 20 urine. The urine fraction probably represents free iodide. The tumor / tissue ratios (Figure

4) show that the amount of 111ln labeled anti-CEA is greater than the amount of 'Ga in all tissues but that in the blood, the amount of anti-CEA

25 marked with 111In in this case is about the same as 6 7

• the amount of Ga. Reports concerning anti-CEA

125 labeled with I, although apparently preferable to those shown by ^^ In and ^ Ga in some tissues, are misleading since they occur from 125 30 low I contents in normal tissue rather than 'from a location in the tumor.

Based on previous experiences, it is / '/ 28.

125 clear that the I-labeled anti-CEA is not suitable for use as a tumor localization agent. The rapid and intense dehalogenation decreases the tracer concentration in the tumor and raises the background of radiation in the blood, preventing its use for printing or imaging without sophisticated subtraction techniques. In fact, at the 72nd hour the tumor contains a quantity twice as high in non-specific xGa as in 125 10 I. In addition, the tumor / blood ratios are better 67 for Ga than for iodized antibody. When considering the fact that zGa is considered to be unsuitable for printing or imaging the colon adenocarcinoma, the difficulties encountered by Mach et al. , N. Eng. J. Med. , 303, 5-10 (1980), are readily explained. These results obtained using 125 I unequivocally show that in the absence of subtraction techniques, if I was used, very large doses of the 20 I labeled material should be administered for achieve the necessary concentrations in the tumor in order to obtain an effective impression. This would imply that the patient received a high dose of radiation due to the physical half-life of 8 days and the 131 beta component of 608 keV from I.

On the other hand, the large percentage of the injected dose of anti-CEA labeled with 1: L1In acquired by the tumor, and the more favorable physical characteristics of the ^ · * Ίη allow the injection of smaller quantities of radioactive pharmaceutical product. active, while enhancing the image and using the radiation dose to the patient. The tumor / background radiation cony 111 29 relationships.

those labeled with In labeled anti-CEA are well within the range required for printing applications due to the large amount of radionuclide localizing in the tumor. In addition, the results suggest that the tumor acquires the radioactive label which formerly had been incorporated into or attached to other tissues.

Individual mice are photoscanned 4, 24, 48 and 72 hours after injection of In labeled anti-CEA using a Phogam H P Anger gamma camera. After 48 hours, the tumor is well delimited without the need to resort to subtraction techniques. Even after 72 hours, the demarcation of the tumor is even further improved.

The in vivo stability and therefore the ability to use a directly labeled monoclonal antibody for tumor printing are shown by the following experiments in the; 20 which uses a monoclonal anti-melanoma labeled 103. t at Ru compared to the same antibody labeled with, 125_ of I.

103 1. Preparation of Ru-labeled anti-melanoma. An aqueous solution of 220 μΐ of 103 25 RuClg (supplied by New England Nuclear, Inc.) is added to 100 μΐ of a saturated solution of potassium acetate.

The pH is adjusted to 5.3 with 70 μΐ of 10 M NaOH. This radionuclide solution is added to 500 μΐ of a solution of 0.5 μg / μl of anti-melanoma antibody from 30 mice and the mixture is incubated for 1 hour at room temperature. The pH is adjusted to 7.2 by the addition of 20 μΐ of 10 M NaOH and the preparation is stored - / / 30.

overnight at room temperature. The unbound metal is separated from the metal bound to the antibody by application of the reaction mixture to a column of Sephadex G-50 and by elution with phosphate buffered with saline at pH 7.2. The protein-containing fractions are collected and pooled for in vivo studies in mice.

2. Distribution studies on normal mice.

Tissue distribution studies are performed on normal BALB-C mice using 103 Ru-labeled anti-melanoma prepared according to the above procedure and 125-labeled anti-melanoma 125 prepared in the same manner as process 15 described above for labeling anti-CEA.

The mice are separated into two groups which are injected intravenously respectively with the antibody labeled with I and the anticoips labeled with ^^ Ru.

The mice are killed after 4, 24, 48, 72 and 96 hours, dissected and the radioactivity of the organs is determined as described previously for the studies with 125 monoclonal anti-CEA. The distribution of I and ^ ° ^ Ru in the tissues is given in the Table below.

t 31.

(D

ε ο g ra '0) S d)

iS G H

dJ ra O "sp η (D Md cm oo ld ο o Ό ra σπ o mP oo oh co oh o co oh t" r- h σ h ra ra yi ro cm "nc> N MH no" 5 <î ino ' îO On cm MH fl) OPN Ch S - N - ·. »» »» »» »» »» «P» gy \ K ΓΟΟ H * »p HO ΓΟΟ ΟΟ Ο Ο ΟΟ ΟΟ sP Ο Η 54 ε p G ) + [δ y 2 ϊ 1/1 m cm cm ο μρ Ο t "ho ^ idcti σ> ιη υο η ο οο cm cm ο σ in ν ο η cm η ιη ^ οη ιη ιη -» ρ cm σ • η G ΏΗ »σι coro CM Ο '(Ρ Ο 0) 0 Ο Ο HO ΟΟΗ σπ Η (P. 0 I ΓΟ ·» »» »» »» »» »» »» »» »» »LT) GO σ to · Η HO C0O ΟΟ ΟΟ ΟΟ C0O ΟΟ ΟΟ γο ο η -.m ω Ό iG G ΓΟ ^ ^ 12 00 η ro ion ο cm ο m cm γο * fi w „ΡΗ (Ν · ί ^ Η mo mil 03 0 in "COffl ΟΝ Ο 5 ^ 1 ιη ο" · ΗσΓ0'Ρθ) οιοσσο) θιηθΓ "ΐη œ HO) (DOP» »η -» »» »» »» »» ».» ».

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GO) PH -OOHOCMOHOCMOHOHOHCMCMH

o) S ^ ° ^ H hvd ^ ph oo oo oo σπ OO ho ho h / (üo o MG ro £ · S 12 o 00 »PHP '· HH cn σ o ma) σπ r» m cm cm in ^ hco oh r "co τρ ^ Ht ^ ho„ fl “hi σ h» • »ρσο] <- ΜΓ4ΗθΗθθΓΜθοθΗ '» ρ σ ro aa) ο ρ »» ρ »» »» »» »» »» »» »» ». p »9 \ Pi VOO ^ o HO - ^ PO OO COH OO OO oo r“ i uj P vü j_ | IBS “10 ^ ΟσΗοοο coocoh ^ p 4-1

Pb fl OM OO ['-O <3 * Mp P "-» pro (D CO OCO P- * CO sp O CO

K 2 1,3 ^ Ό <Νσ Γ0Ο oo Γ0Ο - "^ HOCOHOr ^„ HCO »pl ro> - -...... H -...... ^ p rS Φ ^ ^ H HOOOOOOOOOHCMOOOOOO h ts ra • PG ro 0.03 12 oo cn f- σ ro h p- ro r ~ m P? Ο "^ σ ^ ρ ^ σ oo cm ^ ^ po in ^ ri oo hh

2 ^ m y J * - I "-00 Ο · \ Ρ Hco roO OO o O C0H HCO CO

CN] o fl) OP CM »CM» »» »» .. »» .. .. .. (£> HH y '^' W HH ^ ^ ™ O 10 O OO 00 H oo oo oo 1 M h © H 'g ra ra in> »p OH ^ h ο ο σο h 4-1 P · fi ON Ι> α) <ΝΟΓΜΗΟΟσθΓ ^ ΗΜΡ <Χ> ΜΡΗ [^ · ΗΟ ro ra λ π h» »cmcohop-O ^ O -hhohcmoh

m S \ ° i HO * · · * - * '- - - CM «» »» »» »O

M® NoXH HH OO OO OO OO H Ol OO HO O ro H

PG fl)

D) (D GH

£ θ Γ '- ^ σσ oi> ^ o HCM HCO OOO HOO CMH HCM "^ p CM HO CN · = Ρ 00

H H m y I -C0 "O H ^ HCM OH OO HO O Γ 'O CO

ïï’rd ®> W CM CO N ^ vo COO r- o OO t '' H OO CMO Γ'-Ο H

vü Ό W (l) _l |

H K) CM H OOO O

/ ra mocM - »ρΓ'θΓ '^ οθ'» ρΗσθΓΜθο ^ οθΗθθ '»ρ σ ra λόη» h »οοΓΜΗσΗΓ ^ -Η • Lnroouor' -cm 5« j. \ o ,, ο-cm - »·» s s »-in - - -» »co · * r>

ΓΟ'Ο) HH 1-10 HO 0 ° OO CM O CM "» P H

5-1 g ra

2 ê IX

y S ix ix xh δ ΐχ ΐχ IX IX IX ra 2 Ρ * · · · · • -f '· · · G · 0) η 4-> 4-> -P -P -prai-p 4-10) 14 -1- ^ 4-) 1¾ ^ Η '© ·. . ·. 0). 0). G. 4-) · Ρ yg ο φ ®ma) pa) Ha) pa) oa) ma) rara

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α)

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P VD n I - Φ B

C U CP ^ H ^ & -P

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• r ~ i0rfiOCN H fi O

fi ÖJ Ί5 H H £ 0j • h η l '+1 in h n fi in φ H χ tfi' fi 13 fi fi M fin ns

Cu N fi O E O

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rfi tn P ι 'fin u fi O fi l> rOen O P \ P5 H Φ H en fi φ fin i p. Φ w in i en u fi £ fl ον Φ en Ρί Φ 1 PM CD 1! fi

P 00 1 s S

Φ ^ ô <HH - U Φ H IX in U Φ ^ Φ P fi nh Φ in fi ο Φ Φ CPH C0 fi .p in ό en p ι 'fi φ ηθ Φ o fi in φ H -PM \ PS H> 1 IM fi Φ O 0¾ ΗΌ) (D Min IO Er ”& rfi onh Φ 15 H eo (ts

Ό M I 'H

Φ <N ^ H ^ Φ in p h -P fi fi c φ fi -p in φ end Φ -p

in p ns O -P -P

• rl \ φ CP H CM fi en -p m mi - φ fi ip en o fi n en o en h Φ \ p $ cm 'Φ u

Φ u U Φ - M

H fienin IO û Φ / eeJ Φ O cm H Φ fi in fl ό h h M res fi - I "> in eeJ Φ ^ H cm in 13 'Φ H -P φ

M fi H

fi fi P m O & H $ M B fi »-J1 - ~» p o en υ Φ fi o \ φ fi

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The results in the Table show that 125 the anti-melanoma labeled with I is rapidly dehalogenated, just as occurs with the anti-CEA labeled with ^^ 1, while the anti-melanoma labeled with ^ ° ^ Ru is 5 stable. These observations are confirmed by the fact 125 that the content of I in the blood is very high 4 hours after the injection and drops to low but constant levels between the 24th and 96th hours. By 103, however, the amount of Ru is relatively quickly cleared from the blood and concentrated in the liver, as would have been expected in a host infected with a "non-tumor". The levels in the spleen and in the kidneys for mice injected with Ru-labeled anti-melanoma 103 are also compatible with in vivo stability.

It is well known that CEA and certain other tumor-associated antigens are secreted or spread by the tumor. In such cases, the incidence of background radiation increases because the antigen circulating in the blood and other tissues combined with the labeled antibody can be reduced by initially administering unlabeled antibody to combine with the circulating antigen. This would tend to reduce the high uptake of mar-25 antibodies in the liver. After an appropriate period, the labeled antibody is added and the subject is subjected to photoscanning to determine the localized radiation site.

Although the above demonstrates the use of a single monoclonal antibody labeled with a radionuclide, it is within the scope of the present invention to use two or even more than two anti-34.

monoclonal bodies marked for different antigenic determinants. The different antibodies are chosen so as not to disturb the binding to the antigen of another antibody. When using multiple labeled non-disruptive antibodies, the image of the tumor can be enhanced since the antigen will have greater capacity for the labeled antibody. The use of several antibodies is particularly advantageous for the printing or imaging of a tumor of which the associated antigen is present in a low concentration.

It should be understood that the present invention is in no way limited to the above embodiments and that many modifications may be made thereto without departing from the scope of this / patent. / Jf i

Claims (16)

35. 1. Monoclonal antibody corresponding to an antigen associated with a tumor to which a metal radionuclide is linked directly or via a chelating agent conjugated to the antibody. 2. Monoclonal antibody according to claim 1, characterized in that the chelating agent is chosen from polyaminocarboxylic acids, “polyamino (alkylene phosphoric acids), polyamines, porphyrins, crown configuration ethers, cryptands, desferrioxamine B and its derivatives, enterobactins and carbocyclic analogs thereof. 3. Monoclonal antibody as claimed in claim 2, characterized in that the chelating agent is a polyaminocarboxylic acid of the formula: HO9C (CH2) / (CH2'hrC02H \ lcs2 ^ v C ° 2K »· V. Il-LA / '/ i \ i. 1 \. A02C tCS2) y V (CK25y C02H 25 "in which x is equal to 0 tier, y is equal to 1 or 2, z is an integer from 1 to 7 and R represents hydrogen or a group via which the chelating agent is conjugated to the antibody or a group which modifies the binding constant of the chelating agent for the radionuclide. 4. Monoclonal antibody according to claim - / * f '36. cation 2, characterized in that the chelating agent is a polyamino acid (alkylene phosphoric) of the formula: 0 ·' - HO-F-rCH2) y. (CH27y? "° H OH ° / 1 i - · 10" / Ä \ ^ HO -? - ïCn2) v \. •! " ... OH · " . 'O II. (CH2t —-? - OH- Λ / - • -y \? J tCH27ÿ-ï-OH 20 OH in which X is 0 or is an integer, y is 1 or 2, z is an integer from 1 to 7 and R 25 represents hydrogen or a group via which the chelating agent is conjugated to the antibody or a group which modifies the binding constant of the chelating agent for the radionuclide. 5. Monoclonal antibody according to one or 2q of the other of claims 3 and 4, characterized in that R represents hydrogen, methyl, a group j-CH ^ CgH ^ OCH ^ CO ^ H or C ^ H ^ -A, where. A represents a salt of J 37. diazonium0 or a precursor thereof, or else a group -NH-C-CH ^ L, in which L represents a group displaced during conjugation. 6. Monoclonal antibody according to claim 5, characterized in that.X represents 0 and R re ~. π 5 has a group -CgH ^ -NH-C-C ^ Br. 7. Monoclonal antibody according to claim 5, characterized in that the chelating agent is a polyaminocarboxylic acid, X is equal to 1 and - V R represents a group -CgH ^ -NH-C-CE ^ Br. 8. Monoclonal antibody according to claim 2, characterized in that the chelating agent is chosen from the group comprising ethylene-diaminotetraacetic acid, p-bromoacetamidodiphenyl acid (ethylenedinitrilotetraacetic acid), 1- (p- aminophenyl) - y ethylenediaminotetraacetic and diethylenetriami-nopentaacetic acid. 9. Monoclonal antibody according to any one of claims 1 to 8, characterized in that the radionuclide is a gamma emitter having a half-life ranging from 5 hours to 5 days and whose gamma emissions have an energy of 100 to 400 keV. 10. Monoclonal antibody according to any one of claims 1 to 9, characterized in that the radionuclide is chosen from the following radionuclides. I95m_, 57 „. 57 „105 * 67„ 52__ 52_ 25 wings; Pt, Ni, Co, Ag, Cu, Mn, Fe, 111T 113m 99HU 67- 68_ 169, 166_ 167 „In, In, Te, Ga, Ga, yb, Tm, Tm, 146 15 7_ 95m 103„ 97 „99Ώ lOlrn, 201. Gd, Dy, Nb, Ru, Ru, Ru, Rh, Tl, 203 ^ 197 ^ 105 * 203n. 99B. , 48_ Hg, Hg, Ag, Pb, Rh and Cr. 11. Monoclonal antibody according to any of the claims of claims 1 to 10, characterized in that the antigen associated with the tumor is chosen from the group comprising the carcinoembryonic antigen, a- / r (V 38. foeto-protein, ferritin, human choriogonadotropin, the marker formed by zinc glycinate, prostatic acid phosphatase and the antigens associated with melanomas. 12. Composition comprising a solution for parenteral administration of a monoclonal antibody corresponding to an antigen associated with a tumor according to any one of claims 1 to 11. 13. The composition of claim 12, comprising a mixture of two or more of these monoclonal antibodies. 14. A method for printing or imaging a tumor comprising a) administering to a human subject a parenteral composition comprising a monoclonal antibody corresponding to an antigen associated with the tumor, the monoclonal antibody comprising a radionuclide linked directly or via a chelating agent conjugated to the antibody; and t 20 h) photoscanning of the subject to determine the site at which the antibody localizes in the subject as indicated by the emission of radiation by the radionuclide. 15. The method of claim 14, charac-terized in that the monoclonal antibody is as defined by any one of claims 1 to 13. 16. Monoclonal antibody, composition comprising it and method for printing a tumor, as described above. Drawings: ...... jfc ........ plates „_3.IL .. pages including ... ^ ......... guard pope j ...... pages ds description ....... ί ....... pages of claims ...... short description Luxembourg, le 2 6 OCT. 1982