AU623080B2 - Use of an agent with a high affinity for tumours - Google Patents

Abstract

The vehicle consists of tissue plasminogen activator or an essential part thereof and is used both for the diagnosis and therapy of malignant tumours.

Description

623080 COMMONWEALTH OF AUSTRALIA PATENT ACT 1952 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE CLASS INT. CIASS 9 rn.9 0 *999 9 9 9* 9 9 *9 0 9 4, ,~Q 0 9*9 4 9 9 9,~ 9 4, 9 9 *9 0 0 9 9 9 '9 0 9 Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority: Related Art-; NAME OF APPLICANT: BOEHRINGER INGELHEIM INTERNATIONAL GmbH ADDRESS OF APPLICANT: D-6507 Ingelheim am Rhein, Federal Republic of Germany.

4, .4 ft9 9 4> 4, 9 NAME(S) OF INVENTOR(S) ADDRESS FOR SERVICE: Sirkka-Liisa KARONEN Jan LINDGREN Hannu ARONEN DAVIES COLLISON, Patent Attorneys 1 Little Collins Street, Melbourne, 3000.

COMPLETE SPECIFICATION FOR THE INVENTION ENTITLED: "USE OF AN AGENT WITH A HIGH AFFINITY FOR TUMOURS" The following statement is a full description of this invention, including the best method of performing it known to us -1-

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1A The present: invention relates to an aqent comprising tissue plasminogen activator (tPA) or a substantial fragment thereof for use in the diagnostic and/or prognostic investigation of tumours, and in the treatment thereof.

In particular the present invention relates to the use of a biological agent comprising radioactively labelled tissue plasminogen activator (tPA) for the diagnosis, location and observation of malignant o tumours and to a method of scintigraphic display SD9 go, of malignant neoplasias using such an agent.

Early and reliable discovery of malignant neoplastic tissue and the resulting metastases is a major '°0oo criterion in the prognosis and prospect of recovery 0 from malignant tumours. In addition to computer tomography, immune scintigraphy has more recently played an important part in the diagnostic investigation of cancers. However, this last method, which makes use of radioactively labelled antibodies, has the 0o particular disadvantage that an antibody of this kind is capable of detecting only a very limited number of tumours of different tissues or only a specific type of tumour since, hitherto, no tumourspecific features were known and virtually every tumour has different surface antigens.

It is an objective of the present invention to provide an agent which could be tolerated by humans, which has a high affinity for tumour tissues and which could be used both diagnostically and/or prognostically and also therapeutically for a broad range of malignant tumours. As a solution to this 1; i ~us -ti~~LiYO- ~I C L L_---YII U -L6) t V 2 problem, it has surprisingly been found that radioactively labelled tPA or substantial fragments thereof can he used for the diaqnosis, location, discovery and measurement of maliqnant tumours with the result that it is not only possible to diaqnose tumours of different origins hut also to continuously monitor their rate of response to a particuaur therapy instituted as j result.

Therefore, in one aspect the invention provides a method for the diagnostic and/or prognostic investigation of one or more malignant tumours, said method comprising administering a targetting agent comprising tPA or a substantial fragment thereof, said tPA or fragment being labelled.

This method is faster and more sensitive and can he used with fewer restrictions than the methods of the prior art.

In a still further aspect the invention provides use of tPA, or a fragment thereof, for the preparation S, of an agent for the diagnostic and/or prognostic investigation of malignant tumours.

S, For immunological reasons, it is apparent to those skilled in the art that the agent according to the invention can be used species-specifically, for example human tPA can be used in humans and j species-specific animal tPA can be used in veterinary medicine.

S4 /cT i i i i- i r, i" 3 Human tPA consists of a polypeptide which occurs in single-chained and double-chained form. The published molecular weight of tPA ranges from 66,000 to 72,000 Da depending on its origin. The N-terminal part of the tPA molecule contains two structures which resemble or correspond to the "coils" found in plasminogen and prothrombin. Moreover, this part has a region which is homologous with the "finger-like" structure in fibronectin (Banyai, L. et al., FEBS Letters 163, 37-41, 1983; Pennica, o* D. et al., Nature 301, 214-221, 1983). The carboxyterminal part of the tPA molecule shows considerable :o o homology with other serine proteases (Strassburger, s W. et al., FEBS Letters 157, 219-223, 1983). In addition to these coils, the finger-like structure and the active region, tPA contains a growth factor domain which shows homology with epidermal growth factor. Thus, tPA appears to be made up of a number 0 .0 of different functional domains each of which shows S0 homology with other proteins (Erickson, L.A. et al., Clinics in Haematology 14, 513-530, 1985).

00 o 09 It is known that tPA has a high affinity to fibrin.

So Fibrin is the chief site of attack for plasmin o 0o in thrombolysis (Wallen, Activation of plasminogen with urokinase and tissue activator, in: Thrombosis and urokinase (Paoletti, Sherry, S. eds.) Academic Press, London, 91-102, 1977) and is a component of the stroma of some tumours (Dvorak, H.F. et al., Cancer Metastasis Reviews 2, 41-73, 1983).

On the other hand, the relative proportion of stroma and its composition varies considerably from tumour to tumour.

Malignant tumours often contain more plasminogen activity than the corresponding normal tissue, although there is considerable variation in this

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i i .11-^cI 1 I 4 activity between histologically similar tumours (Bigbee, W.L. et al., Biochim. et Biophys. Acta 540, 285-294, 1978; Booth, N.A. et al., Blood 61, 267-275, 1983). It was therefore all the more unexpected that the use of labelled tPA, in partiCtlae radioactivitely labelled tPA would open up a new method, based on a new principle, for detecting a large number of tumour types by autoradiography or scintigraphy, which could be used for oncological purposes.

04 0 There is some speculation that the plasminogen C 4activator plays a part in regulating extracellular oil proteolytic activities. This activity could be o responsible for the infiltration of malignant tumours i through the basal membrane (Gelister, J. S. K.

et al., B.M.J. 293, 728-731, 1986). On the other hand, there are indications that only the urokinaselike activator and not tPA has any proteolytic d 00 activity (Dan8, Urokinase-type plasminogen activator in cancer, XIV Annual meeting, International too Society for Oncodevelopmental Biology and Medicine, Helsinki 1986, Abstract 74).

SO, The resuits according to this invention show, surprisingly clearly, that radioactively labelled tPA can specifically accumulate in malignant tumours although the mechanism and conditions affecting this binding are, as yet, unexplained.

Thus, tPA according to the present invention is a useful vehicle and agent both for biochemical and pathological/diagnostic use and also for prognostic investigations of suspected or pre-existing malignant tumours. Moreover, the tPA which is to be used according to this invention may be labelled by known methods with known physiologically harmless or beneficial radioisotopes.

I I 5 Thus, in a yet further aspect the invention provides a process for the preparation of an agent for the diagnostic and/or prognostic investigation of tumours which comprises a. isolating or preparing tPA or a fragment thereof b. labelling said material, and a o c. converting said labelled material into a solution which is suitable for scintigraphy.

9 Oi Conveniently, the tPA is radioactively labelled Ssuch that the radioactivity dosage range delivered following administration is between 10 and 1000 MBq.

Particularly advantageous radioisotopes are those 131 123 of iodine 131 123I). Advantageously, the dosage of radioactivity to be used in the case 131 o oot of I is between 10 MBq and 500 MBq, more particularly between 40 MBq and 230 MBq and, in the case 123 of I, between 10 MBq and 1000 MBq, more particularly Bo 9m99m 111 ,o between 74 and 370 MBq. Moreover, 9 mTc or In or chelate-coupled metal ion isotopes may also be used for labelling.

Radioactively labelled tPA can thus be used for scintigraphy of the whole body or of part of the body after being injected into a patient systemically intravenously) or locally near a suspected tumour, e.g. by i.m. route). After administration to a patient, it is possible to determine the abovementioned diagnostic parameters of malignantly degenerate tissue, particularly tissue with a stroma component (esg. solid tumours). Radioactively S- 6 -6labelled tPA may also be used for the prognosis of a pre-existing tumour and its development before, during or after therapeutic treatment.

The term tPA as used herein refers to tPA prepared using known conventional cell cultures of animal or human origin or by the use of known genetic engineering techniques, i.e. by known methods of DNA recombination. As is well known, both prokaryotic and eukaryotic cells are suitable for this latter method.

o It is preferred to use tPA or fragments thereof which have been obtained by DNA recombination.

O For example, the method according to EP-A-117059, according to DE-0-3316297 or according to Collen, Nature 301, 214-221, 1983 may be used.

0 0 0The term tPA includes the plasminogen activator a (0 "a of the tissue type formed by genetically manipulated microorganisms or by correspondingly manipulated o.o or natural cell culture systems. It is known to the average person skilled in the art that natural allelic variations occur in individual species 0 0o and manifest themselves by one or more different amino acids or by different nucleotides or DNA sequences. Variations or mutations of this kind, which can also be produced by the known methods of DNA recombination or by controlled mutagenesis, as described for example in EP-A-93619 or EP-A- 199574, include simple or multiple substitutions, deletions, additions, insertions or inversions.

They also include tPA derivatives which contain the "coil" region characteristic of tPA and the serine protease region but are otherwise modified in one of the ways described above. According to the invention, tPA derivatives which show the r ~-c3or~~.aa~s 1 1 i 0 04.

0 00 0 G 04 0 0 0 4 9r 0 00 0a~c 0 0* 00 0 q 00* p 004 00 0 0 00 7 characteristic activity or affinity according to the invention or a higher activity or affinity than that of native tPA may also be used (e.g.

EP-A-93619, EP-A-199574). Thus, as used herein, the term tPA includes all tPA variants and derivatives.

Moreover, it is possible to use not only radioactive tPA as a whole molecule for the purposes described but also any parts or fragments thereof which will bind to malignant tissue or to the stroma of maliqnant tumours, or variants of or chimeric molecules of the known composition of tPA.

It has in fact been found, totally surprisingly, that not only the complete tPA molecule binds to tumours but also that a fragment isolated by HPLC with a size of approximately 10 kDa has a high affinity to tumour tissues. The radioactively labelled approximately 10 kDa fragments of tPA were even found to accumulate to a much greater extent in the tumour tissue than the native tPA molecule. It was also very surprising that this approximately 10 kDa fragment obviously binds to tumour sites which contain no or very little fibrin components. The evidence for this was obtained, for example, by double labelling with 1 1 1 In antifibrin and 1I-rtPA, showing that I-tPA binds in the peripheral part of the tumour but not in the necrotic, fibrin-containing centre of the tumour, the binding 111 site of In-antifibrin.

Therefore, even modified tPA molecules or fragments thereof are advantageous compared with proteolytically active tPA since although they have no proteolysis activity they do have a high affinity to tumour tissues.

I 8 The use of these "second generation" tPAs is also a part of this invention. Since the structure of tPA is known, it is possible by protein engineering to produce tPA derivatives which, for example, have several fingers and/or epidermal qrowth factor domains or which consist only of these domains or of the N-terminal.

In another aspect the invention provides an agent for treating one or more malignant tumours, comprising tPA, or a substantial fragment thereof, said tPA or fragment being labelled with a cytostatic or cytocidal active agent.

In still another aspect, the invention proves a method for treating one or more malignant tumours, said method comprising administering an agent, comprising tPA or a fragment thereof, said tPA or fragment being labelled.

In yet another aspect the invention provides use of tPA, a conjugate or a fragment thereof, for S' the preparation of an agent for the therapeutic treatment of malignant tumours.

2 In a yet still further aspect the invention provides a process for the preparation of an agent for the therapeutic treatment of tumours which comprises o! isolating or preparing tPA or a substantial fragment thereof "b.h radioactively labelling said material such that the radioactivity dosage delivered following administration is above 500 MBq, -9c. if desired conjugating the material obtained according to step a. or b. with a cytostatically or cytocidally active aqent and d. converting the radioactively labelled and/or conjugated material into a suitable acueous solution.

The expert is currently confronted with insuperable difficulties in the controlled and effective therapeuti o treatment of malignant tumours. Although in .recent ,9 times monoclonal antibodies against tumours have been developed, which also serve as vehicles in Os0 conjunction with cytostatic substances known a as immunotoxins Bjorn, Cancer Res.

>oo 46, 3262-3267, 1986), even these modern attempts at therapy have failed to solve all the problems.

The remaining problems are how to achieve the desired 0 00 0 0oO efficiency of binding to the cell surface, how to avoid effects of neutralisation Bjorn, loc. cit.), how the various types of tumour, I 000 particularly with a heterologous cell population, can be reached in toto, how to increase the low binding affinities of conjugated monoclonal antibodies 6 00 0' or immunotoxins and their specificity for the tumour cells.

It is also totally unclear whether these immunotoxins can be effective in solid tumours with a dense connective tissue component since the antibodies preferentially bind to surface antigens and do not reach cell masses located within the tumour or the centre of the tumour itself. Many of these unsolved problems are well known to those skilled in the art Vitetta, Science, 219, 644-650, 1983).

10 Due to the fact that the inventors of the present application have recognised the surprising affinity of tPA or more particularly one of the fragments thereof with tumours, the experts have been provided with an unexpectedly new opportunity of targeting their therapy on tumours. Accordingly, tPA or substantial parts thereof must be regarded for the purposes of the invention as a agent which is not only of use in the diagnostic and/or prognostic investigation of malignant tumours but also has e some therapeutic importance since tPA, coupled 9e 0 with a known cytostatic or cytocidal agent, i.e.

00 a synthetic or natural agent which is effective o* e against itumour cells ricin or other vegetable toxins, antibodies, hormones, translation inhibitors o ad or immune modulators or parts thereof) is able to transport this agent directly to the site of a tumour. In particular, tPA fragments with a high affinity for tumours are particularly suitable, Ai"0'1 such as the 10 kDa fragment of the tPA molecule, which can be isolated by HPLC. For the purpose of achieving this therapeutic use, anyone skilled in the art will be familiar with a number of known and well established methods enabling him for example 0a to produce proteins conjugated with toxins or haptens (W085/03508; Varga, in: Methods in Enzymology, 112, 259-269, 1985; Bacha, P. et al., J. Biol.

Chem. 258, 1565-1570, 1983; Gendloff, E.H. et al., J. Immunol. Methods 92, 15-20, 1986; Duncan, R.J.S.

1 et al., Anal. Biochem. 132, 68-73, 1983; King, T.P. et al., Biochemistry 25, 5774-5779, 1986; Myers, D.A. et al., Biochem. J. 227, 343, 1985 to name but a few). Procedures capable of conjugating tPA or substantial fraqments thereof with the toxins which act against tumout cells are thus within the specialist knowledge of those skilled in the art.

-I I LI" X-~rYlinr I I 11 Radioactively labelled tPA or substantial fragments thereof are also suitable. For example, 1 3 1 I-labelled t?A or substantial parts thereof can be used therapeutically above a radioactivity dosaqe of 500 MBa.

Slow-growing solid tumours, for example, do not respond well to conventional therapies because these therapies are directed against dividing cells; these tumours may, for example, be suitable candidateo for therapy with a conjugated tPA molecule or a conjugated part of tPA as the vehicle. In such a case the advantage would be that tPA remains bound to the tumour (accumulated thereon) for a relatively long time and the conjuqate acts as oa cytotoxic agent continuously and quasitopically, :obs targeted on the tumour tissue. Any progression or exacerbation in the tumour growth by the occurrence or recurrence of mitosis can therefore be suppressed by the therapeutic agent which is present at the o, site of the tumour.

O0 SThUs, this invention opens up new possibilities for the diagnosis and treatment of cancer.

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9 In One study, 11 male and 4 female patients ranging from 32 to 82 years old (average 58) were investigated 131 131l using I-labelled tPA. The 131I labelling was carried out by known methods. All the patients had malignant tumours or metastases which had been verified histologically or radiologically (by Xray computer tomography). The dosage of radioactivity used was within the range of 40 to 230 MBq (on average 165 MBq). After the uptake of the iodine radioisotope by the thyroid gland had been blocked by means of oral administration of sodium iodide and perchlorate (daily dose of 420 mg and 800 mg, respectively, for 10 days), the patients were given 'i; 12 an intravenous iniection of 1 3 1 1-tPA. The distribution of the radioactivity was investigated with a largefield-of-view gamma camera (crystal diameter at the top of the camera 40 cm or more) 4 and 24 hours after the injection and in three cases th/ ording with the gamma camera was repeated 2 to 5 after the injection. Computer analysis of the images obtained was carried out to determine the "target to non-target" ratio and to calculate the percentage uptake of the dosage in the target area.

a 0 4 SVThe scintigraphic results obtained from the patients with malignant tumours or with metastases t 131 0 using I-tPA are described in the Examples which a; follow. The relative intensity of the accumulation 044,.: of radioactivity was divided into the categories of "slight", "medium" or "strong", by subjective 131 assessment. In 10 cases an accumulation of I-tPA 4 <I was found. In three patients the dosage administered S« was less than 100 MBq; with negative results in the images. 12 patients were given a dosage exceeding 100 MBq. In this group, an accumulation of radioactive tPA in the pathologically altered tissue was found in 11 patients. In four patients, there was a cerebral concentration of radioactive tPA and in eight patients the concentration was found in extracerebral areas. Fiqures 1 and 2 show typical results.

Three patients showed both cerebral and extracerebral accumulation of 13I-tPA. In five patients no pathological concentration of 1 3 1 1-tPA was observed.

In this group, two adenocarcinoma, one hypernephroma, one prostate carcinoma and one carcinoma of the bladder had been diagnosed.

All the accumulations were visible 24 hours and, except in two cases, 4 hours after the injection of 131-tPA. The target-to-non-taret ratios 24 of I-tA The target-to-non-fcarget ratios 24 13 hours after the injection, which had been determined by computer analysis oF the imaqes used for this purpose, varied from less than 1.2:1 to 2.1:1.

In three of the patients investigated, an accumulation could still be detected five days after the injection 131 of I-tPA.

The blo-distribution of 1, 31 T-tPA is shown in Fiq. 3.

1.31 Three minutes after the injection of I-tPA, there is an accumulation in the liver of 59% and in the spleen of 96% of the maximum radioactivity in the organ in question. In the liver, the maximum 04 a radioac:ivitv was reached after 15 minutes and at in the spleen after 9 minutes. After the peak 6 had been reached, 20% of the radioactivity in the atolls t liver and IQ% of the radioactivity in the spleen were eliminated within the next 15 minutes. After 24 hours the radioactivity in the liver was 2% and in the spleen less than 1% of the maximum value.

Blood samples investigated by HPLC showed three 131 fractions of I-labelled tPA, which were designated I, II and III (Fig. The approximate molecular weights of the individual fractions which were ,determined by means of marker proteins are as follows: I=600 kDa, 11=80 kDa, III=10 kDa. The plasma radioactivity feil to the same extent as in the radioactively labelled protein component IT (80 kDa).

In front of this component II a larger complex I (600 kDa) was found which most probably represents a 1 31 I-tPA plasminogen activator inhibitor (PA) complex. The smallest component, which is the most interesting for the purposes of the invention as already described, namely component III with about 10 kDa had only a minimal cell-bound activity.

It constituted only 0.35% of the total blood radioactivity. It was found in blood platelets and 14 in the lymphocyte/monocyte fraction but not in the erythrocyte fraction. The following conclusions can be drawn from this: 1. 1 3 1 I-tPA is rapidly bound to its carrier proteins and broken down into a smaller component of about 10 kna, whilst the radioactivity found in the elution volume rapidly increases to the same extent as the radioactivity of the other components decreases (Fig. 2.

Most probably, the radioactivity accumulated by malignant tissue represents a 10 kDa metabolite Sna of 13 1 I-tPA which, compared with the whole tPA ona 0 molecule, is an ideal vehicle for the purposes I of this invention. No undesirable side effects 6a* V have been observed after administration of the radioactively labelled tPa.

a Legend relating to the drawings 131 Fig.l shows considerable accumulation of 1 I-tPA in a brain metastasis of a lung cancer.

131 SPFiq.2 shows considerable accumulation of I-tPA in a metastasis of lung cancer in the left upper arm.

p .a Fig.3 shows time/activity curves of liver, heart and spleen after intravenous injection of 131 11 -laballed tPA (190 MBg) in a male patient a=liver, b=heart, c=spleen Fig.4 shows gel filtration chromatography of plasma, minutes after injection of 1 3 1 I-tPA in patient 1 (Example I=600 kDa, II=80 kDa, kDa.

~LLII~IIICllllr_

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i f 15 shows the HPLC runs of plasma samples after the iniection of 131I-tPA in patient 1 (Example 1), taken after various time intervals.

a= after 15 minutes, b= after 30 minutes, c= after 60 minutes, d= after 150 minutes, e= after 240 minutes, f= after 24 hours.

The following Examples are intended to illustrate S* the invention without restricting it. The intensity 131 of the relative absorption of 131I-tPA (accumulation Sof 1 3 1 I-tPA in various malignant primary and secondary tumours) was subdivided, by means of the scintigrams, into the categories of weak medium and strong The individual data concerning the various parameters measured are given in Table e 1 which appears after the Examples.

Example 1: Female patient, 38 years old, with cancer in the S* right breast and metastatic pleural effusions into both lungs, a large metastasis in the liver and S two metastases in the right ovaryo Example 2: Male patient, 66 years old, with cancer of the prostate diagnosed three years earlier. The initial staging was T3 NX MO, and orchiectomy and electroresection of the prostate was performed. The patient had metastases in the left clavicular region and an irradiated 13 cm large tumour recurrency in the prostatic region.

Example 3: Male patient, 65 years old, in whom a small cell cancer of the lung had been detected six months earlier which was treated with chemotherapy. After the chemotherapy, scintigraphy was performed.

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16 At this time the patient had tumour recurrencies in the lungs and the mediastinum and brain metastasis.

Example 4: Male patient, 73 years old, who had been operated on for hypernephroma 11 years earlier. Lung metastases were observed 8 years later, including a pituitary lesion. Scintigraphy was performed after radiotherapy o to the pituitary region.

«n Example Male patient, 32 years old, with malignant melanoma of the skin which had been operated on four years earlier. At the time of scintigraphy, several subcutaneous and two cerebral metastases were detected.

0 0, The patient was given interferon and regional radiation therapy.

4 0* o, Example 6: 9 Male patient, 70 years old, with an adenocarcinoma of the left lung hilus detected nine months earlier o 4, and treated by radiotherapy. In CT there was infiltration of the mediastinum, metastatic lymph nodes in the retrosternal and pretracheal regions and brain metastasis.

Example 7: Female patient, 82 years old, with carcinoma of the bladder diagnosed 10 months earlier. Radiation therapy to the pelvic region was administered.

At the time of examination the patient had defuse lung metastases and liver metastases.

Example 8: Male patient, 69 years old, with small cell cancer of the lung which had been diagnosed 13 months earlier and treated by chemotherapy and radiotherapy L I i 17 of the mediastinum. In CT there were two cerebral metastases. The patient also had liver metastases verified by sonographic investigation.

Example 9: Male patient, 37 years old, in whom a neuroepithelioma had been removed from the skull 27 months earlier.

A new lesion was excised from the angle of the left jaw 23 months later. Two months before the o o scintigraphy there was a recurrency in the left o0* upper neck. The patient was given chemotherapy.

Example S° Male patient, 81 years old, with a tumour in the posterior part of the right pleura and pleural effusion. The tumour was 6 x 12 cm in size. Diagnosis o8 of malignant fibrotic histiocytoma was verified by sample biopsy. There was no therapy before scintigraphy.

Example 11: Male patient, 36 years old, with cancer of the stomach which had been treated by chemotherapy after an operation. Because of a stomach tumour, palliative gastrectomy had been carried out five months before the scintigraphy. At the time of scintigraphy, enlarged lymph nodes were found in the left clavicular region. In CT this patient had metastases in the liver, left thoracic wall and para-aortic lymph nodes.

Example 12: Female patient, 47 years old, in whom a 4 mm melanoma had been removed from the right foot 2 years and months earlier. Metastatic lymph nodes were found in the right inguinal region. Two metastatic lesions appeared in the left leg. These areas

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.Ir r 18 were irradiated. At the time of scintigraphy, several subcutaneous metastases including a 6 x 4 cm lesion in the left thoracic wall were found. The patient was given chemotherapy.

Example 13: Male patient, 48 years old, with sauamous cell cancer of the oesophagus and large lymph node metastases on both sides of the neck. The lymph node S metastases were treated by radiotherapy before scintigraphy. Active tumour tissue was found by ,t autopsy in the cervical lymph nodes.

SExample 14: Male patient, 63 years old, in whom a squamous cell lung tumour had been operated on 8 months earlier. A cerebral metastasis was verified by CT four months after the operation and the patient j was given radiotherapy. At the time of scintigraphy 4 the patient had metastases in the left upper arm and the thoracic wall.

Example j Female patient, 65 years old, with two malignant 1 tumours: cancer of the breast and cancer of the rectum, both operated on 4 years earlier. The patient was admitted to hospital because of lung metastases. These were probably of rectal origin since in CT residual tumours were detected in the rectal area.

Example 16: Administration of radioactive tPA a. 0.1 mg of tPA were radioactively labelled by the known iodogen method with 31 and an activity of 110 MBq (Fraker Speck, 19 Biochem. Biophys.Res.Comm. 80, 849-857, 1978) and taken up in blood-isotonic aqueous solution. After sterile filtration, this dose of radioactively labelled tPA was injected intravenously into patients suffering from malignant tumours (melanoma, adenocarcinoma of the colon or breast or epidermal lung cancer). External whole body scintigraphy was carried out with a gamma camera 1, 2, S% a 4 and 7 days after the injection.

a 00 S. b. 1.0 mg of tPA, radioactively labelled with o 131I with an activity of 185 MBq, was worked So up as described in Example 16a and administered in the same way to the same malignant tumours.

o Whole body scintiqc'aphy was carried out as Sdescribed in Example 16a.

Example 17: S '.131 Bio-distribution of I-tPA 131 0 0 190 MBq of I-tPA were injected intravenously into a 22 year old male patient with treated lowgrade astrozytoma. The radioactivity in the liver, spleen and heart was measured dynamically. Fig. 3 shows the time/activity curves obtained for the liver, heart and spleen.

Example 18: Identification of the approx. 10 kDa tPA fragment In a dosage of 3-5 mCi/mg of protein, 131I-labelled 131 recombinant tPA I-rtPA) was injected into patients and blood samples from 9 patients were tested.

Blood samples were taken before the intravenous injection and 15, 30, 60, 150, 240 min and 24 hours after the injections and placed in EDTA test tubes Cm i i 20 in known manner. The plasma and blood cells were then removed immediately by centrifuging. Blood platelets and the lymphocyte/monocyte fraction were separated by known methods using LETKOPREP

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test tubes. HPLC exclusion chromatography (gel filtration of the plasma) was carried out under the following conditions: 0 9 a 0 9 00 00 00 91 soo dol 0000- 4 0 009a O 91 0 04 Apparatus: UV Detector: Preliminary column: Column: Fraction collector: HPLC buffer: Flow rate: Wavelength: Gamma detector: Samples: Gel filtration: LKB 2150 HPLC Pump LKB 2152 controller LKB 2151, variable wavelength monitor Ultropac TSK SWP (7.5 x 75mm) LKB Ultropac TSK G300SW (7.5 x 300mm) LKB LKB 2212 Helirac 20 mmol/l L-arginine, 11 mmol/l phosphoric acid, 0.05% sodium azide, pH 7.3 0.75 ml/min, 1 minute fractions were collected 280 nm Beckman Model 170 Radioisotope Detector and Compugamma Model 1282, LKB Wallac The plasma samples were diluted 1:10 with HPLC buffer and filtered before injection onto the column Absorption of the eluate was recorded at 280 nm and the radioactivity was also measured using a gamma counter a a a a S C C C C a .at a a a a as.

a a a 0 0 a S 8 *0S 8 a a a a *00 0 0 @0 8 Table 1: Accumulation of 11I-tPA in various maliqnant primary and secondary tumours.

The intensity of the relative uptake of 131 I-tPA was graded, by means of the scintigrams obtained,, as weak medium and strong Example No.

(Patient) Ag e Sex Diagnosis 13 1 T-tpA Dose [MBq] Intensity (relative uyptake of 11I-tPA) Target-tonon-target ratio Time after injection of 1 31 I-tPA [h] Breast cancer Metastatic cancer of prostate Small cell cancer of the lung; with brain metastases Ilyperneph roma Malignant melanoma; with 2 metastases 37 1.81 negative negative 200 1.3 1.3 100 155 negative 4 24 4 24 48 2± 1.3 1.3 4 *0 P C P 4 PC~ C C C P S C C 0 0 CC C C C 4 4 0 C 4 0 0 C 0.0 0 0 C C C P 0 0 CO 4 Example No.

(Patient) Age Sex Diagnosis 1 31 -tPA Dose [MBq] Intensity Target-to- Time after (relative non-target injection of uptake of ratio 1 31 I-tPA [h] 13 -tPA) K4 Bronchial cancer; brain metastasis F Cancer of the bladder; diffuse lung metastases M Small cell lung cancer; brain metastases r4 Neuroepithelioma on the neck M Histocytoma of the pleura M Adenocarcinoma of the stomach; metastasis in the left thoracic wall 174 67 1+ 1+ negative 1.3 ,233 3± 181 192 185 1.2

K

1 3 1 TI-tPA D~ose [MBq] a C 0 CCC aO a a 0 a a a a a *0 a 9 0 C CC a 0CC 0 0 C aCt a a CC a Example No.

(Patient) Age Sex Diagnosis Intensity (relative uptake of 131 I-tPA) Target-tonon-target ratio Time after injection of 1 31 I-tPA [h] F Malignant melanoma metastasis in the left thoracic wall M Squamous cell cancer of the oesophagus; metastasis on the left side of the neck M Lunq cancer; metastasis in the upper left arm F Multiple lung metastasis; primary tumour unknown 229 1.3 185 1.7 185 1.4 2.1 1.2 215

Claims (25)

1. I 24 53039/000.313 Claims 1. A method for the diagnostic and/or prognostic investigation of one or more malignant tumours, said method comprising administering a targetting agent comprising tPA or a substantial fragment thereof, said tPA or fragment being labelled.

2. A method as claimed in claim 1, wherein said tPA or fragment thereof is of natural origin and has been isolated from a eukaryotic cell culture. 3, A method as claimed in claim 1, wherein said tPA or fragment thereof originates from transformed prokaryotic or eukaryotic cells.

4. A method as claimed in any one of claims 1 to 3, wherein said tPA or fragment thereof comprises at least So° one tumour-binding component and has substantially no proteolytic activity. 5, A method as claimed in any one of one of claims 1 to 4, wherein said tPA or fragment thereof is a derivative derived from a tPA-encoding polynucleotide by controlled mutagenesis and which has a characteristic activity or affinity to tomour tissues at least equal to that of the original tPA or fragment thereof. S

6. A method as claimed in any one of claims 1 to wherein said tPA or fragment thereof is a variant derived from an organism having multiple alleles or exhibiting tPA polymorphism.

7. A method as claimed in any one of claims 1 to 6, wherein said tPA is a tPA allelic variant, or a (7 derivative or a fragment thereof which has at least one finger-like structure and/or epidermal growth factor domain or consists only of at least one of these N- terminal parts or of combinations of these parts.

8. A method as claimed in any one of claims 1 to. 7, comprising a tPA fragment having a size of the order of kDa.

9. A method as claimed in claim 8, wherein the tPA fragment has a size of about 10 kDa and substantially does not bind to fibrin. A method as claimed in any one of claims 1 to 9, wherein said tPA or fra,,ment thereof, is radioactively labelled.

11. A method as claimed in claim 10 wherein said tPA or fragment therFeof is radioactively labelled With 1311, 12j 99 mTc and/or 111 In.

12. A method as claimed in claim- 10 or claim 11, wherein the dosage of radioactivity delivered following administration is between 10 and 1,000 M~q.

13. A method as claimed in any one of claims I to 12, wherein the agent is administered by a systemic route. 1,4. A method as claimed in. any one of claimo 1 to 12, wherein the agent administered locally into or onto the malignant tumourc. A method as claimed in claim 1 substantially as described herein.

16. A method for the treatment of one or more malignant turnours said method comprising administering an agent i' i -26- comprising tPA or a substantial fragment thereof, said tPA or fragment being labelled.

17. A method as claimed in claim 16, wherein said tPA or fragment thereof carries a natural or synthetic cystostatic or cytocidal agent.

18. A method as claimed in claim 16 or 17, wherein said tPA, conjugate, or fragment thereof, is radioactively labelled.

19. A method as claimed in claim 18, wherein the dosage of radioactivity delivered following administration is above 500 MBq, A method as claimed in any one of claims 18 to 19, wherein the agent is administered by a systemic route.

21. A method as claimed in any one of claims 16 to 19, wherein the agent is administered locally into or onto the malignant tumour.

22. A method as claimed in claim 16 substantially as described herein, 23, An agent comprising tPA or a substantial fragment thereof, said tPA or fragment thereof being labelled with a radiolabel sufficient to provide a dosage of radioactivity following administration of between and 1,000 MBq wherein the radioisotope used has a half-life of up to about 8 days; or a cytostatic or cytocidal active agent. 24, An agent as claimed in claim 23, wherein the tPA or fragment thereof is labelled at 110 to 185 MBq/mg of protein. An agent as claimed in claim 23 or 24 wherein the radio isotope is selected from t 31 f, oTc and 1 1 1n. A h 1} 920219,26 ®y I 27

26. An agent as claimed in claim 23, wherein the tPA or fragment thereof labelled with a cytostatic or cytocidal active agent is additionally labelled with a radiolabel.

27. An agent as claimed in claim 26 wherein the dosage of radioactivity delivered following administration is above 500 MBq.

28. An agent as claimed in any one of claims 23 to 27, comprising a labelled tPA fragment haing a size in the order of 10 kDa.

29. An agent as claimed in claim 28, wherein the tPA fragment has a size of about 10 kDa and substantially does not bind fibrin.

30. An agent as claimed in claim 23 substantially as hereinbefore described. 4 4 31, A process for the preparation of an agent for use in the method for the diagnostic and/or prognostic investigation of tumours as defined in any one of claims 1 to 15, which process comprises a. isolating or preparing tPA or a fragment thereof b, labelling said material such that the radioactivity dosage range delivered following administration is between 10 and 1000 MBq, and c, converting said labelled material into a solution which is suitable for scintigraphy.

32. A process as claimed in claim 31 substantially as described herein.

33. An agent for the diagnostic and for prognostic investigation of tumours prepared by the process as claimed in claim 31 and which is suitable for infusion or injection, 920219,27 -4 28

34. A process for the preparation of an agent for use in the method for the therapeutic treatment of a tumour as defined in any one of claims 16 to 22 which comprises a. isolating or preparing tPA or a substantial fragment thereof as a material to be labelled radioactively labelling said material such that the radioactively dosage delivered following administration is above 500 MBq, or (ii) conjugating the material obtained according to step a. with a cystostatically or cytocidally active agent and (iii) conjugating the material obtained according to step b(i) with a cytostatically or cytocidally active agent and c. converting the radioactively labelled and/or conjugated material into a suitable aqueous solution. A process as claimed in claim 34 substantially as described herein.

36. An agent for the therapeutic treatment of tumours prepared by the process as claimed in claim 34 which is suitable for infusion or injection. DATED this 19th day of February, 1992 BOEHRINGER INGELHEIM INTERNATIONAL GmbH by its Patent Attorneys DAVIES COLLISON CAVE 920219,28