Field of the invention The present invention relates to improvements in the diagnosis and treatment of peritoneal cancers, including ovarian cancer. More specifically, the invention relates to a kit, whereby the tumour to non-tumour ratio of cytotoxic agents in the treatment of disseminated carcinomas, in particular ovarian carcinomas, can be improved by reducing the concentration of a cytotoxic agent in the blood circu- lation after intraperitoneal administration of the same, a higher dosage and a more effective treatment regime beeing achieved without vital organs being exposed to high tox- icities. The invention also relates to the use of a compound comprising at least one affinity region for the preparation of a medicament for treatment of an intra- peritoneal tumour.

Background of the invention Ovarian cancer is the sixth most common cancer among women, excluding non-melanoma skin cancers. The American Cancer Society estimated that about 23,100 new cases of ovarian cancer had been diagnosed in the United States during 2000. Ovarian cancer accounts for 4% of all cancers in women. Ovarian cancer is also the fifth most common cause of cancer deaths among women, causing more deaths than any other cancer of the female reproductive system. It was estimated that there was about 14,000 deaths from ovarian cancer in the United States during 2000. If dia- gnosed, and treated while the cancer has not spread outside the ovary, the live-year survival rate is 95%. However, only 25% of all ovarian cancers are found at this early stage.

Thus, most women having an intraperitoneal cancer are diagnosed far too late. Late diagnosis results in a non-

effective treatment with no regression of the tumour. With the treatments used today, regression is obtained only in 40% of the patients.

The 5-year survival rate refers to the percent of patients who survive at least 5 years after their cancer is diagnosed. Of course, 5-year survival rates are based on patients diagnosed and initially treated more than 5 years ago. Improvements in treatment often result in a more favorable outlook for recently diagnosed patients.

There are many types of tumours that can start growing in the ovaries. Some are benign (non-cancerous) and never spread beyond the ovary. These patients can be cured by surgically removing one ovary or the part of an ovary containing the tumour. Other types of ovarian tumours are malignant (cancerous) and may spread to other parts of the body.

In general, ovarian tumours are named according to the kind of cells the tumour started from and whether the tumour is benign or cancerous. There are three main types of ovarian tumours. Epithelial tumours start from the cells that cover the outer surface of the ovary. Cancerous epithelial tumours are called carcinomas. Germ cell tumours start from the cells that produce the eggs (ova). Stromal tumours start from connective tissue cells that hold the ovary together and produce the female hormones, oestrogen and progesterone.

Epithelial ovarian carcinomas (EOC) accounts for 85%- 90% of ovarian cancers. The cells of EOC may have several forms that can be recognized under the microscope. In addition to their classification by cell type, EOCs are also given a grade and a stage. The grade is on a scale of 1, 2, or 3. Grade 1 EOC more closely resembles normal tissue 25 and tends to have a better prognosis. Grade 3 EOC less closely resembles normal tissues and usually has a

worse outlook. The tumour stage describes how far the tumour has spread from where it started in the ovary.

Effective therapeutic methods for the treatment of ovarian cancer have been subjected to intensive research.

Most women suffering from ovarian carcinomas die of loco- regional recurrence and peritoneal dissemination ; hence, regional therapy has been the main focus for some time.

Experience with intraperitoneal therapy of this tumour using conventional chemotherapy agents (Howel S. et al.

Intraperitoneal cisplatinum-based chemotherapy for ovarian carcinoma, Semin. Oncol. 1991,18 (Suppl 3), 5-10); radioactive colloids (Rosenhein N. B. et al. Radiocolloids in the treatment of ovarian cancer, Obstet. Gynecol. Surv.

1997, 34,708-20); immunoadjuvants (Bast R. C. et al.

Intraperitoneal immunotherapy of human ovarian carcinoma with Corynebacterium parvum. Cancer Res. 1983,43,1395- 1401); cytokines (Navoli M. et al. Intraperitoneal re- combinant alpha-2-interferon alternating with cisplatin as salvage therapy for minimal residual-disease ovarian cancer: a phase II study. J. Clin. Oncol. 1990,8 (6), 1036- 1041) have been reported.

Another area of investigation involves treatment with very high doses of anticancer drugs, and then"rescuing" the woman from the side effects with infusions of her own bone marrow stem cells or peripheral blood stem cells (immature blood cells that may be taken from the bone marrow or removed from the bloodstream by using a special filtering process). The bone marrow or peripheral blood stem cells are removed before a high dose of chemotherapy is administered and is returned to the woman (reinfused) after the high-dose treatment is complete. In that way, the side effect of suppressed blood cell production is overcome. This is an extremely high-risk, experimental procedure because, for the time, the woman is without her

normal supply of blood cells and is very vulnerable to infection.

Targeting biomolecules such as tumour specific monoclonal antibodies are widely used in the treatment of haematological cancer diseases and more recently in the treatment of disseminated solid tumours (Breitz H. B. et al. Radioimmunotherapy of Solid Tumours, in Radioimmuno- therapy of Cancer, eds. P. G. Abrams & A. R. Fritzberg, Marcel Dekker, Inc., New York, 2000, pp 265-306).

A number of tumour specific monoclonal antibodies and immunoconjugates, suitable for in vivo diagnosis and treatment of ovarian cancer, have been described in US patent No. 4, 958,009 (Anti-human ovarian cancer immuno- toxins and methods of use thereof), in US patent No.

5,817,313 (Monoclonal antibodies and conjugates thereof useful for the treatment of cancer), in US patent No.

5,804,187 (Modified antibodies with human milk fat globule specificity) and in US patent No. 5,650,291 (Monoclonal antibodies against an antigen associated with ovarian cervical and other tumours).

Over the past decade a number of clinical studies using intraperitoneal (i. p.) radioimmunotherapy in ovarian cancer have been reported. Various types of monoclonal antibodies including HMFG-1, HMFG-3, AUA-1 and HI7E2 have been labeled with I-131, Y-90 and Re-186, and injected into the peritoneal cavity through peritoneal dialysis catheter under local anaesthesia in volumes ranging from 1-2 liter of normal saline and a specific activity of 4-8 mCi/mg antibody.

The effective dose for i. p. 1-131 labeled antibodies is thought to be 150 mCi and the mean peak radioactivity in serum is at 44 hrs, corresponding to 26% of the total in- jected dose. Most of the radioactivity given (80%) is found as free iodine in the urine. However, when Y-90 labeled antibodies are used, the mean peak of radioactivity in

serum is 23%, and only 8.11% of the injected dose is re- leased in the urine after 72 hrs (Rosenblum M. G. et al.

Clinical pharmacology, metabolism and tissue distribution of Y-90-labeled monoclonal antibody B27.3 after intra- peritoneal administration. J. Natl. Cancer Inst. 1991 (83) 1629).

Although, toxic exposure to the peritoneum and its close surrounding is usually well tolerated and is 4-70- fold more advantageous than the i. v. route for targeting of peritoneal tumour sites (Ward, B. G. et al. Localization of radio iodine conjugated to the monoclonal antibody HMFG-2 in human ovarian carcinoma: assessment of intravenous and intraperitoneal routes of administration. Cancer Res. 1987 (47) 4719 ; Ward B. G. et al. Radiolabeled monoclonal anti- bodies in oncology. III Radioimmunotherapy. Nucl. Med.

Commun. 1991 (12) 333), a high percentage of the injected activity still localizes in normal tissues ; the dose- limiting organ being the bone marrow. Myelosuppression arises 4-6 weeks after the initial injection. Grade 3 platelet and granulocyte toxicity (according to the WHO standard) was observed at a total dose of 20 mCi with Y-90 labeled antibody and 160 mCi with 1-131 (Stewart J. S. et al. Intraperitoneal 1-131 and Y-90 labeled monoclonal antibodies for ovarian cancer: pharmacokinetics and normal tissue dosiometry. Int. J. Cancer 1988 (3 suppl.), 71; Stewart J. S. et al. Intraperitoneal yttrium-90 labeled monoclonal antibody in ovarian cancer. J. Clin. Oncol.

1990, (8), 1941; Maraveyas A. et al. Pharmacokinetics and toxicity of an yttrium-90-CITC-DTPA-HMFG-1 radioimmuno- conjugate for intraperitoneal radioimmunotherapy of ovarian cancer. Cancer 1993 (73) 1067), while grade 3 and 4 haem- atological toxicity was observed at a total dose of 150 mCi with Re-186 (Jacobs A. J, et al. A phase 1 trial of a rhenium 186-labeled monoclonal antibody administered

intraperitoneally in ovarian cancer carcinoma : toxicity and clinical response. Obstet. Gynecol. 1993 (82) 586).

From the review of the relevant clinical trials, it is clear that i. p. radioimmunotherapy is of benefit mainly to patients with small-volume disease. All studies per- formed agree that patients with lesions <2 cm have a prolonged disease-free period when compared with historical control group (Hird et al. Adjuvant therapy of ovarian cancer with radioactive monoclonal antibody. Br. J. Cancer 1993 (82) 586). In the same group of patients, tumour regression has been reported for a number of cases (Epenetos A. A. et al. Antibody guided irradiation of malignant lesions : three cases illustrating a new method of treatment. Lancet 1984,1441 ; Maraveyas A. et al. Phar- macokinetics and toxicity of an yttrium-90-CITC-DTPA-HMFG-1 radioimmunoconjugate for intraperitoneal radioimmunotherapy of ovarian cancer. Cancer 1993 (73) 1067) as well as decrease in tumour size (Jacobs A. J. et al. A phase 1 trial of a rhenium 186-labeled monoclonal antibody admin- istered intraperitoneally in ovarian cancer carcinoma: toxicity and clinical response. Obstet. Gynecol. 1993 (82) 586).

However, the results are rather poor in patients with nodules >2 cm in diameter subjected to radioactive doses at, or close to, the maximum tolerated dose, since too little of the radioactive antibodies are penetrating the tumourous tissue and the high-energy radiation reaches the limit of its distant killing effect (Ward B. et al. The treatment of intraperitoneal malignant disease with mono- clonal antibody guided 131-1 radiotherapy. Br. J. Cancer 1988 (58) 658). This group of patients number more than 50% of patients in need of treatment. Thus, the dose necessary to reach an effective therapy in this group of patients is hampered by the accumulation of radioactivity in the blood

circulation, leading to toxicity of normal organs, notably the bone marrow.

Most medical agents, and in particular larger molecules, administered into the peritoneal cavity are transported to the blood mainly through the lymphatic system, provided that the peritoneum remains intact. If these molecules are toxic to cells they may do considerable damage to body tissues outside the peritoneal cavity such as kidney, liver, lung and bone marrow, and may even be fatal. It is desirable to remove such materials from the blood as quickly as possible. Although the body has natural clearance mechanisms to remove exogenous molecules from the blood circulation, these systems are rather slow for in particular immunoglobulins ; leading to a high toxic exposure to the clearing organs such as liver and/or kidney.

Various means to clear the blood from cytotoxic tar- geting biomolecules (e. g. therapeutic or diagnostic mono- clonal antibodies) after i. v. administration have been reported (see review article by Schriber, G. J. & Kerr, D.

E., Current Medical Chemistry, 1995, Vol. 2, pp 616-629).

In the so-called avidin chase modality, avidin or streptavidin is administered systemically after adminis- tration of the therapeutic or diagnostic antibody to which biotin has been attached, at a time when a sufficient amount of the antibody has been accumulated in the tumour.

Avidin or streptavidin will associate with the antibodies and the so formed immunocomplex will clear from the blood circulation via the reticuloendothelial system (RES) and be cleared from the patient via the liver. These procedures will improve the clearance of biotinylated cytotoxic anti- bodies. An alternative approach to the same end, is the use of anti-idiotypic antibodies. However, all these methods rely on the liver or kidney for blood clearance and thereby expose either or both of these vital organs as well as the

urinary bladder to high dose of cytotoxicity. Another major drawback of the methods is the immunogenicity of these agents, particularly the streptavidin, which prevent re- petitive treatments once the immune response has been developed. Extracorporeal techniques for blood clearance are widely used in kidney dialysis, where toxic materials build up in the blood due to a lack of kidney function.

Other medical applications, whereby an extracorporeal apparatus can be used, include : removal of radioactive materials; removal of toxic levels of metals; removal of toxins produced from bacteria or viruses ; removal of toxic levels of drugs, and removal of whole cells (e. g. cancerous cells, specific haematopoietic cells-e. g. B, T, or NK cells) or removal of bacteria and viruses.

Various methods have been proposed to rapidly clear radiolabeled antibodies from blood circulation after the tumour has accumulated a sufficient quantity of immuno- conjugate to obtain a diagnosis or therapy. Some of the methods employed involve enhancement of the body's own clearing mechanism through the formation of immune complexes. Enhanced blood clearance of radiolabeled anti- bodies can be obtained by using molecules that bind to the therapeutic antibody, such as other monoclonal antibodies directed towards the therapeutic antibody (Klibanov et al., J. Nucl. Med. 29,1951-1956,1988; Marshall et al. Br. J.

Cancer 69,502-507,1994; Sharkey et al. Bioconjugate Chem.

8,595-604,1997), avidin/streptavidin (Sinitsyn et al., J.

Nucl. Med. 30,66-69,1989; Marshall et al., Br. J : Cancer, 71,18-24,1995), or glycosyl containing compounds which are removed by receptors on liver cells (Ashwell and Morell, Adv. Enzymol. 41,99-128,1974). Still other methods involve removing the circulating immunoconjugates through extracorporeal methods (see review article by Schriber, G. J. & Kerr, D. E., Current Medical Chemistry, 1995, Vol. 2, pp 616-629).

The extracorporeal techniques used to clear a medical agent from blood circulation are particularly attractive because the toxic material is rapidly removed from the body. Application of these methods in the context of im- munotherapy have been previously described (Henry Chemical Abstracts, 1991, Vol. 18, pp. 565; Hofheinze D. et al., Proc. Am. Assoc. Cancer. Res. 1987, Vol. 28, p 391; Lear J K, et al., Radiology 1991, Vol. 179, pp. 509-5 12; Johnson T. K. et al. Antibody Immunoconj. Radiopharm. 1991, Vol. 4, p 509; Dienhart D. G., et al. Antibody Immunoconj.

Radiopharm. 1991, Vol. 7,25 pp. 225; DeNardo G. L. et al.

J. Nucl. Med. 1993, Vol. 34, pp. 1020-1027 ; DeNardo S. J. et al. J. Nucl. Med. 1992, Vol. 33, pp. 862-863; DeNardo G.

L. J. Nucl. Med. 1992, Vol. 33, pp. 863-864; and US patent No. 5,474,772 (Method of treatment with medical agents).

To make the blood clearance more efficient and to enable processing of whole blood, rather than blood plasma as the above methods refer to, the medical agents (e. g. tumour specific monoclonal antibody carrying cell killing agents or radionuclides for tumour localization have been biotinylated and cleared by an avidin-based adsorbent on a column matrix. A number of publications provide data showing that this technique is both efficient and practical for the clearance of biotinylated and radionuclide labeled tumour specific antibodies (Norrgren K, et al. Antibody Immunoconj. Radiopharm. 1991, Vol. 4, pp 54; Norrgren K, et al., J. Nucl. Med. 1993, Vol. 34, pp. 448-454; Garkavij M, et al., Acta Oncologica 1996, Vol. 53, pp. 309-312; Garkavij M, et al., J. Nucl. Med. 1997, Vol. 38, pp. 895- 901. These techniques are also described in EP 0 567 514 (A method and a system for enhanced in vivo clearance of diagnostic and/or therapeutic agents by extracorporeal depletion, and the use of said agents for said purpose). A further development of this method with simultaneous labeling of biotin and radionuclides is described in a

patent application by S. Wilbur & B. E. B. Sandberg PCT/SE98/01345, Trifunctional reagent for the conjugation to a biomolecule.

Specific tissue or organ localization of a bio- molecule is a very important factor in its effective application. Lack of specific tissue localization is of particular important in the treatment with cytotoxic bio- molecules, where the desired effect is to kill certain types of cells such as in the treatment of cancer. In order to enhance the specificity, tumour specific monoclonal antibodies are used as a carrier (immunoconjugates) of various cytotoxic moieties used in prodrug protocols (Meyer et al., Bioconjugate Chem. 6,440-446 ; 1995 ; Houba et al., Bioconjugate Chem. 7,606-611,1996; Blakey et al., Cancer Res. 56,3287-3292,1996).

Although tumour-specific immunoconjugates are select- ively bound to tumour cells, an initial high concentration of the cell-toxic immunoconjugate in the peritoneal fluid is necessary to reach a sufficiently high concentration in the target tissue. While required for optimal therapy of the cancer, the high concentration of cytotoxic material in the peritoneal fluid will gradually increase the level of the cell toxic material in the blood and other non-tumour tissues, in most cases leading to tissue damage and/or lesion formation in sensitive and vital tissues like the bone marrow.

Even in cases where the bone marrow rescue is ef- fective, other sensitive organs like the liver, kidney, spleen, lung, etc. can be irreparably damaged. The most effective method for preventing tissue and bone marrow damage from toxic materials in blood is to dramatically decrease the amount of that toxic material in the blood. Of course, this must be accomplished in a manner that retains the therapeutic level of toxic material in the tissue being treated (e. g. tumour). Direct transport from the peritoneal

fluid to the blood circulation is not considered to be of major significance for most cytotoxic agents in comparison with the lymphatic transportation route. Hence, the concen- tration of cytotoxic agents in the peritoneal fluid is only to a small extent dependent on the concentration of the same medical agent the blood circulation.

Apart from the prolonged circulation time leading to undesired exposure of toxic immunoconjugate to healthy tissue, inadequate tumour tissue penetration and non- specific organ retention and metabolism contribute to a low therapeutic index ratio. Due to these problems, multi-step antibody-based radionuclide delivery approaches have been extensively investigated. The basic concept involves first the injection of a lesion specific targeting moiety, which apart from binding specifically to the lesion also has the feature of binding to a subsequently injected radioactive diagnostic agent or a therapeutic agent. By separating these two events one can allow the slow tissue penetrating non-radioactived non-cytotoxic antibody sufficient time to accumulate in the tumour mass, while the agent carrying the radionuclide/cytotoxin could be selected for more rapid tissue penetration. However, a prerequisite is that the former (and preferably also the later) can be cleared rapidly from the blood circulation.

The so called two and three-step approaches have recently been reviewed in the context of intraperitoneal administration in ovarian cancer therapy (Syrigos K. N. & Epenetos A. A. (2000) Intraperitoneal Radioimmunotherapy of Ovarian Cancer in Radioimmunotherapy of Cancer, eds. P. G.

Abrams & A. R. Fritzberg, Marcel Dekker, Inc., New York, pp. 315-316).

Summary of the invention The purpose of the invention is to produce a kit for controlled removal from a body fluid of a compound, or a part thereof, which previously has been given by means of

intraperionteal administration, the above-mentioned prob- lems being avoided. In order to achieve this purpose the invention has obtained the characterizing features of claims 1 and 25, respectively.

The intraperitoneal tumours are formed as small protuberances from peritoneum. More specifically, they are localized on the inside of the membrane, from which they mainly grow inwards, and they can be reached by adminis- trating a toxic drug to the peritoneal cavity. Thus, the inventive kit is especially adapted for treating early tumours intraperitoneally of as an alternative for systemic administration of an anti-tumour agent.

Not only the treatment but also the diagnosis of peritoneal cancers, including ovarian cancer, can be improved by using the kit according to the invention. Other carcinomas are also contemplated, where intraperitoneal administration of cytotoxic agents is applicable, either alone, or in combination with intravenous administration.

These include, but are not limited to, colon and/or rectal cancer.

Compounds, or parts thereof, used in the present invention are not to a larger extent able to diffuse or penetrate the peritoneum. In fact, in peritoneal dialysis the peritoneum is used as a semipermeable membrane.

However, the clearance of the peritoneal liquid takes place via the lymph system, and will subsequently enter the blood stream via the lymph nodes.

By using the inventive kit it is possible to reduce the level of cytotoxic agents or toxic immunoconjugates from the blood circulation after intraperitoneal injection of these compounds. Furthermore, by removing the circu- lating cytotoxic agent or immunoconjugate, it is possible both to decrease the toxic side effect on other organs and at the same time increase the therapeutic dose by means of a single administration or through the administration of

multiple doses, which results in an increased penetration of the tumour tissue by the cytotoxic agent or immunoconjugate. Thus, a cytotoxic targeting biomolecule (e. g. immunoconjugate) used for therapy of human ovarian cancer can be removed from the body fluid to improve its ratio of target-to-non-target concentration. An improved target-to non-target ratio provides a better therapeutic index. Furthermore, the bone marrow rescue can be avoided, which sometimes is used to circumvent potentially lethal effects. Such a rescue is both extremely costly and poses high risk for the patient.

According to the invention, the kit comprises (a) at least one compound comprising at least one affinity region; (b) a device for intraperitoneal administration of the at least one compound to a mammal; and (c) an apparatus to be connected to the mammal for extracorporeal removal of the at least one compound, or a part thereof, from a body fluid of the mammal by means of at least one receptor interacting with the at least one affinity region. The compound accord- ing to the invention can also be used for the preparation of a medicament for treatment of an intraperitoneal tumour.

For example, the compound can be a cytotoxic agent; a cytotoxic moiety conjugated to a targeting agent; and a cytotoxic moiety, the cytotoxic moiety being selected from the group consisting of a radionuclide, a chemotherapy drug, a synthetic or naturally occurring toxin, an immuno- suppressive, an immunostimulant, and a prodrug activating enzyme. If the biomolecule is the cytotoxic agent conjugated to a targeting agent, it is preferred that the affinity ligand be directly covalently bound to the cytotoxic agent. If the biomolecule is the targeting agent conjugated to the cytotoxic moiety, it is preferred that the affinity ligand be directly covalently or coordinately bound to the radionuclide or directly covalently bound to the chemotherapy drug, the synthetic or naturally occurring

toxin, the immunosuppressive, the immunostimulant, or the prodrug activating enzyme.

Thus, by using the kit according to the invention, the treatment of intraperitoneal cancers in mammals can be improved by: (a) administering the compound intraperi- toneally to said mammal; and (b) substantially reducing the level of the compound, or a part thereof, in a body fluid at suitable time intervals, whereby side effects associated with biomolecules or their cytotoxic fragments are reduced. The body fluid can be blood and the reduction of the compound, or its cytotoxic fragmentary part, in the circulation is achieved by passing the blood or a component thereof through an apparatus to be connected to the mammal for extracorporeal removal of the compound, or a part thereof, by means of at least one receptor interacting with the at least one affinity region of the compound. The blood component may be serum or plasma. The extracorporeal apparatus can comprise a solid support with a receptor bound thereto; and the biomolecule or cytotoxic fragment is conjugated to an affinity ligand with a high affinity to the receptor. Alternatively, the biomolecule or cytotoxic fragment has a high affinity to the receptor.

The part of the compound to be removed is a cytotoxic fragment, which can comprise a radionuclide or a toxic metabolite or structure derived from the cytotoxic agent, the chemotherapy drug, the synthetic or naturally occurring toxin, the immunosuppressant, the immunostimulant or the prodrug activating enzyme.

Thus, by using the inventive kit it is also possible to improve the treatment of intraperitoneal cancers in mammals by: (a) administering a conjugate of a biomolecule and an affinity ligand intraperitoneally to the mammal; and (b) substantially reducing the level of said biomolecule or cytotoxic fragment thereof in the blood circulation by passing at suitable time intervals the blood or a component

thereof through an extracorporeal apparatus comprising a solid support having a receptor bound thereto, the affinity ligand having a high affinity to the receptor, whereby the level of biomolecule or cytotoxic fragment in the circula- tion is reduced.

The inventive kit can also comprises more than one compound for intraperitoneal administration. These can be administrated together or in sequence, with or without a time lag, and with or without interacting with each other.

In addition, the imaging of peritoneal cancers in mammals can be improved by means of the inventive kit by: (a) administering a conjugate of a radionuclide and a tar- geting agent intraperitoneally to the mammal, the targeting agent having a high affinity for the cancer; and (b) sub- stantially reducing the level of radionuclide in the body fluid at suitable time intervals, whereby side effects associated with circulating radionuclide are reduced or whereby imaging contrast is improved. The reduction of radionuclide can be achieved by passing peritoneal liquid, blood, or a component thereof through an extracorporeal adsorption apparatus. The extracorporeal apparatus comprises a solid support with a receptor bound thereto, and the conjugate of radionuclide and targeting agent, or a cytotoxic fragment thereof, is further conjugated to an affinity ligand with a high affinity to the receptor.

Alternatively, the receptors in the extracorporeal may have a high affinity to the targeting agent. In a preferred embodiment, a radionuclide is directly covalently or coordinately bound to the affinity ligand, which is directly covalently bound to the targeting agent.

The invention can be illustrated with the simulation exemplified in Example 1.

As is explained in Example 1, clinical data from Maraveyas, A. et al. (Cancer 73: 1067-1075, 1994) relating to intraperitoneal administration of 90Y-HMFG-1 to patients

with ovarian cancer were utilized in the simulation. These data were utilized to simulate the effects of two extra- corporeal adsorptions with MitradepW (a blood filter having avidin immobilized to agarose particles) conducted at various time after administration of an antibody conjugate.

Each adsorption is assumed to remove 90 per cent of the circulating conjugate. It is also assumed that the rate of transport of conjugate from the intraperitoneal volume to blood or the biological half-life of the conjugate in blood is not influenced by the extracorporeal adsorptions. The results illustrated in Figure 1 illustrate how treatment of the blood at selected time intervals after administration can significantly or substantially reduce the level of biomolecule in the blood, thereby substantially reducing side effects associated with the circulating biomolecule and enhancing the target to non-target ratio for the biomolecule. This improved ratio can result in decreased side effects and/or improved contrast for imaging.

Although the present invention is described for application to human ovarian carcinoma it is also applic- able to other types of human cancer diseases where intra- peritoneal administration is deemed preferable. The subject procedure is also suitable for the treatment of other mammalian species. The clearance of the biomolecule from the blood is achieved by passing the patient's whole blood through an apparatus that specifically adsorbs the biomo- lecule. In the most preferred application, the biomolecule is labeled with biotin and the blood clearance is achieved by passing the blood on-line through an apparatus coated with avidin or streptavidin. Such an apparatus is described in EPO 5675 14 and exhibits the proper characteristics of the matrix and means of immobilizing the biotin-binding entity for processing of whole blood and obtaining excellent clearance in a reasonable time period. The biocompatibility of an agarose matrix containing immob-

ilized avidin for clinical use has been reported (Bosch, T. et al., Ex Vivo Biocompatibility of Avidin-Agarose: A New Device for Direct Adsorption of Biotinylated Antibodies from Human Whole Blood. Artificial Organs, 2000). It should be stressed that all extracorporeal applications where the patient's whole blood is processed would also be applicable for the processing of human plasma, although such an appar- atus would be both more cumbersome and less efficient.

Brief description of the figures In order to explain the invention in more detail illustrative embodiments thereof will be described below reference being made to the accompanying drawings in which Figure 1 illustrates a simulation of the effect of Mitradep° treatments on blood levels of a conjugate of Y- HMFG-1 and biotin.

Figure 2 shows the retention of antigen binding of HMFG-1 antibody labeled with ill In and biotin on an avidin agarose column.

Figure 3 illustrates the percent reduction in whole body (WB) radioactivity after intraperitoneal injection of In-HMFG-1 in rats.

Figure 4 illustrates the pharmacokinetics of bio- tinylated In-HMFG-1 following intraperitoneal injection in rats.

Figures 5A and 5B shows the radioactivity uptake in selected rat organs and tissues at selected times following intraperitoneal injection of 1llIn-HMFG-l.

Figure 6 illustrates the reduction in blood radio- activity following extracorporeal treatment at 12 and 18 hours after intraperitoneal injection (ECIA is extra- corporeal immunoadsorption).

Detailed description of the invention Preferably, the compound of the inventive kit com- prises at least one cytotoxic effector. In this connection

a"cytotoxic effector"means any compound which exhibits or results in a cytotoxic effect on a tumour cell. The cyto- toxic effector can be a chemotherapy drug, an immuno- suppressive drug, an immunostimulating drug, a synthetic toxin, a naturally occurring toxin, a radionuclide, or an enzyme.

As used herein, a"cytotoxic agent"includes all medical agents which, when administered intraperitoneally exert a cell toxic effect, are mainly transported to the blood circulation through the lymphatic route, and can be biotinylated or otherwise labeled with an affinity ligand without severely affecting the efficacy of the drug.

Preferably, the at least one affinity region of the compound in the inventive kit is an affinity ligand.

Cytotoxic agents to be used in the treatment of ovarian cancer include cisplatin, carboplatin or taxenes like docetaxel or paclitaxel or analogues or derivatives thereof, bleomycin, anthracyclines and derivatives thereof, alkylating agents like cyclophosphamide, etoposide, anti- estrogen drugs like tamoxifen, GnRH analogues, topo- isomerase I inhibitors like Topotecan, as well as naturally occurring toxins like doxorubicin and derivatives thereof.

According to the subject invention, a"targeting agent"is an agent which specifically binds to a tumour cell. Thus, the compound according to the invention may comprise, or compose, at least one targeting agent, which is a molecule having an adhesive region for a receptor or any cell surface structure on the tumour cell. Preferably, the targeting agent carries a cytotoxic moiety that, contrary to common cytotoxic agents, binds specifically to tumour cell with a high affinity and which could be administered intraperitoneally to a mammal or human being.

In a preferred application, the targeting agents are antibodies, which could be of different isotypes and could originate from any species. Of particular interest are the

monoclonal antibodies and derivatives thereof. The latter include fragments such as the F (ab') 21 F (ab'), F (ab) and the like. They also include genetically engineered hybrids or chemically synthesized peptides based on the specificity of the antigen binding region of one or several target specific monoclonal antibodies e. g. chimeric or humanized antibodies, single chain antibodies etc.

Especially preferred are bi-specific antibodies with high affinities for tumour cells as well as the cytostatic effector, e. g. cytostatic agents. These bi-specific antibodies can be two antibodies, each having a specific affinity, which have fused Fc regions. Alternatively, the bi-specific antibodies have different light chains of different affinity.

The compound according to the invention may also comprise, or compose, an immunoadhesin. Such a molecule is a genetically engineered, disulphide-linked homodimer which is structurally similar to an antibody. Immunoadhesins bind to their target with high affinity and specificity because the binding capacity of their adhesin domain is identical to that of the receptor or ligand of interest.

Likewise, the compound according to the invention may also comprise, or compose, a peptide of non-monoclonal origin, which specifically binds to a target cell, i. e. a tumour cell. Examples of suitable peptides are hormone analogues of somatostatin and melanocyte stimulating hormone. Alternatively, the compound may comprise, or compose, a carbohydrate that by means of lectin-carbo- hydrate interactions can target lectin domains of tumour cells.

Any of these targeting agents, such as antibodies or fragments or derivatives thereof, can be modified by the coupling of various number of polyethylene glycol chains in order to optimize the half-life in body fluid and the retention of the antibody or antibody fragments or deriv-

atives, in the tumour tissue. In a most preferred applica- tion, the antibodies or antibody derivatives should allow for the attachment of a sufficient number of affinity ligands, e. g., biotin residue, to be used for extra- corporeal removal through interaction with immobilized receptors, e. g., avidin, without significantly diminishing the binding properties of the targeting agent. The receptor can also be an antibody having affinity for the affinity region of the compound.

In order to enhance the specificity of the cytotoxic effector, tumour specific targeting agents or monoclonal antibodies are used as carriers (immunoconjugates) to carry cytotoxic agents (as defined above) and various other cyto- toxic moieties used in prodrug protocols. The conjugates of targeting agents and cytotoxic agents or other cytotoxic moieties are referred to herein as"targeting agent conjugates".

Preferably, the cytotoxic effector is a radionuclide such as a gamma-emitter, e. g. iodine-131 or metal ion conjugate, where the metal is selected from a beta-particle emitter, such as yttrium or rhenium. US. Patent No.

4,472,509, Gansow, et al., discloses the use of diethylene- triaminepentaacetic acid (DTPA) chelating agents for the binding of radio metals to monoclonal antibodies. The'509 patent is particularly directed to a purification technique for the removal of non-bonded and adventitiously bonded (non-chelated) metal from radiopharmaceuticals but is illustrative of art recognized protocols for preparation of radioisotopic pharmaceuticals.

According to such general procedures, an antibody specifically reactive with the target tissue associated antigen is reacted with a quantity of a selected bifunc- tional chelating agent having protein binding and metal binding functionalities to produce a chelator/antibody conjugate. In conjugating the antibodies with the chelators

an excess of chelating agent is reacted with the anti- bodies, the specific ratio being dependent upon the nature of the reagents and the desired number of chelating agents per antibody. It is a requirement that the radionuclides are bound by chelation (for metals) or covalent bonds in such a manner that they do not become separated from the biotinylation/radiolabeling compound under the conditions that the biomolecule conjugates is used (e. g. in patients).

Thus, the most stable chelates or covalent bonding arrangements are preferred. Examples of such binding or bonding moieties are: aryl halides and vinyl halides for radionuclides of halogens; N2S2 & and N3S chelates for Tc and Re radionuclides; amino-carboxy derivatives such as EDTA, DTPA, derivatives Me-DTPA and Cyclohexyl-DTPA, and cyclic amines such as NOTA (1, 4,7-triazacyclononane-1,4,7- triacetic acid), DOTA (1,4,7,10-tetraazacyclododecane- N, N', N", N-tetraacetic acid), TETA (1, 4,8,11-tetraaza- cyclotetradecane-N, N', N, N-tetraacetic acid), CITC-DTPA (-DTPA), SCNBz DOTA (isothiocyanatobenzyl 1,4,7,10-tetra- azacyclododecane=N, N', N", N" tetraacetic acid), and tri- ethylenetetraaminehexaacetic acid derivatives (Yuangfang and Chuanchu, Pure & Appl. Chem. 63,427.463,1991) for In, Y, Pb, Bi, Cu, Sm and Lu radionuclides.

Beta radiation emitters, which are useful as cyto- toxic moieties, include isotopes such as scandium-46, scandium-47, scandium-48, copper-67, gallium-72, gallium- 73, yttrium-90, ruthenium-97, palladium-100, rhodium-101, palladium-109, samarium-153, rhenium-186, rhenium-188, rhenium-189, gold-198, radium-212 and lead-212. The most useful gamma emitters are iodine-131 and indium-mll4. Other metal ions useful with the invention include alpha radi- ation emitting materials such as bismuth-212, bismuth-213, and At-211 as well as positron emitters such as gallium-68 and zirconium-89.

In another embodiment of the present invention, ra- dionuclide-labeled targeting agents are useful not only in treatment of peritoneal cancers, but also for imaging of such cancers.

In one embodiment at least one cytotoxic effector suitable for treatment of peritoneal cancer, such as but not limited to ovarian cancer, is administered intra- peritoneally to a mammal in need thereof. The cytotoxic effector can either have a direct cytotoxic effect on tumour cells or assist in the therapeutic therapy. Cyto- toxic effectors assisting in the therapeutic treatment can include but are not limited to cytotoxic effectors which can convert a prodrug to an active drug.

Such cytotoxic effectors, mostly enzymes, are chosen for their abilities to convert relatively non-cytotoxic prodrugs (drug precursors) into active cancer drugs. The drugs thus formed can then exert their cytotoxic effects.

For example, alkaline phosphatase, conjugated to monoclonal antibodies, convert etoposide phosphate into the clinically approved anticancer drug etoposide. Different carboxy- peptidases, for example conjugated to a monoclonal F (ab') 2 fragment, hydrolyze a-peptidyl methotrexate derivatives to methotrexate. Monoclonal antibody conjugates of penicillin- V amidase act on phenoxyacetamide derivatives of doxo- rubicin and melphalan, producing the cytotoxic drugs doxorubicin and melphalan, respectively. 5-Fluorocytocine is a substrate for cytocine deaminase conjugated to an anti-cancer monoclonal antibody, the anti-cancer drug 5- fluorouracil being produced.

The inventive kit for administration and extra- corporeal removal of compound (s), or part (s) thereof, from a body fluid of the mammal include the following compon- ents.

Intraperitoneal administration of medical agents can be achieved by utilizing various blood access devices,

such as various central vein catheters or various peri- toneal dialysis catheters. The implantation of these catheters can occur as a separate event or in connection with laparoscopy.

At a suitable time after an intraperitoneal adminis- tration of one or more compounds, cytotoxic molecules originating therefrom are removed from the body fluid by means of an apparatus for extracorporeal circulation, the body fluid being whole blood, plasma, or intraperitoneal liqiud.

When the cytotoxic molecules have reached the blood circulation, mainly through the lymph system, they are cleared from the blood system by such extracorporeal means.

To facilitate the extracorporeal depletion an apparatus for extracorporeal circulation of whole blood or plasma will be connected to the patient. This apparatus should provide conduits for transporting the blood to an adsorption device and conduits for returning the processed blood or plasma to the patient. In the case plasma is processed through the adsorption device, a plasma separation device is needed as well as means of mixing the concentrated blood with pro- cessed plasma. The later is normally achieved by leading the two components into an air-trap where the mixing occurs.

In the case where whole blood is processed an ordin- ary dialysis machine can constitute the base for such an apparatus. Dialysis machines are normally equipped with the necessary safe guards and monitoring devices to meet patient safety requirements as well as easy handling of the system. Hence, in a preferred embodiment whole blood is processed and a standard dialysis machine is utilized with only minor modifications of the hardware. However, such a machine requires a new program fitted to the new intended purpose.

In addition to the apparatus, special blood tubing lines suitable for the intended flow and distance from the patient and the machine are needed. These tubing lines could be made of any material compatible with blood or plasma and would include material used in ordinary tubings used in dialysis.

Blood access could be achieved through peripheral vein catheters or if higher blood flow is needed through central vein catheters such as but not limited to sub- clavian or femoral catheters.

Thus, according to the subject invention, peritoneal cancer cells can be treated with cytotoxic effectors (in- cluding cell-killing drugs) or conjugates of a targeting agent a cytotoxic effector. Such cytotoxic effector and targeting agent conjugates are referred to collectively herein as"biomolecules".

For affinity adsorbents, the matrix may be of various shapes and chemical compositions. It may, for example, constitute a column house filled with particulate polymers, the latter of natural origin or artificially made. The particles may be macroporous or their surface may be grafted, the latter in order to enlarge the surface area.

The particles may be spherical or granulated and be based on polysaccharides, ceramic material, glass, silica, plastic, or any combination of these or similar materials.

A combination of these could, for example, be solid par- ticles coated with a suitable polymer of natural origin or artificially made. Artificial membranes may also be used.

These may be flat sheet membranes made of cellulose, polyamide, polysulfone, polypropylene or other types of material which are sufficiently inert, biocompatible, non- toxic and to which the receptor could be immobilized either directly or after chemical modification of the membrane surface. Capillary membranes like the hollow fibers made from cellulose, polypropylene or other materials suitable

for this type of membranes may also be used. A preferred embodiment is a particulate material based on agarose and suitable for extracorporeal applications.

In a preferred embodiment the blood clearance is achieved by the use of a specific adsorption device. Such a device could utilize immobilized anti-species antibodies for the removal of therapeutic antibodies of, e. g., murine origin, or immobilized anti-idiotypic antibodies for removal of therapeutic antibodies regardless of the species origin. In a preferred application an affinity ligand is attached to the biomolecule and the adsorption device contains an immobilized receptor binding specifically to the affinity ligand. Any type of affinity ligand/immob- ilized receptor combinations can be used in this connec- tion, provided that the they do not significantly interfere with the binding affinity and selectively of the bio- molecule to the tumour, and provided that the affinity ligand-receptor interaction is not interfered with by blood or other body fluids or tissues being in contact with the biomolecule-affinity ligand conjugate and/or the receptor of the adsorption device.

The adsorbent device to which the receptor is immob- ilized may be of various shapes and chemical compositions.

It may for example constitute a column house filled with particulate polymers, the latter of natural origin or artificially made. The particles may be macroporous or their surface may be grafted, the latter in order to enlarge the surface area. The particles may be spherical or granulated and be based on polysaccharides, ceramic material, glass, silica, plastic, or any combination of these or a like material. A combination of these could for example be solid particles coated with a suitable polymer of natural origin or artificially made. Artificial membranes may also be used. These may be flat sheet membranes made of cellulose, polyamide, polysulfone,

polypropen or other types of material which are suffi- ciently inert, biocompatible, non-toxic and to which the receptor could be immobilized either directly or after chemical modification of the membrane surface. Capillary membranes like the hollow fibers made from cellulose, polypropen or other materials suitable for this type of membranes may also be used.

Thus, the receptor to which the affinity ligand has a high affinity may be immobilized to various types of solid supports. The coupling method of choice will depend on the nature of the receptor as well as the nature of the immuno- sorbent support matrix. For protein based receptors, functional groups such as hydroxyl-, amino-, carboxyl-or thiol-groups may be utilized. Glycoproteins may be coupled to the matrix via their glycoresidues. The solid support may also be activated to enable binding of the receptor by means in which the receptor forms linkages with the solid support through specific or non-specific reaction with the side-chains or the backbone structure of the receptor protein. The linkage between the solid support and the receptor may also be of non-covalent nature, where electro- static or hydrophobic forces are utilized. Apart from the biotin/avidin system other combinations or affinity ligand and corresponding receptors can be used within the scope of this invention. The following list is by no means complete and will merely serve as examples of additional combina- tions of affinity ligands and their receptors.

Antibody/antigen (haptens) e. g. anti-DNP antibodies/targeting molecules conjugated with DNP; Lectins/saccharide residues e. g. Lectin from Sambueus nigra/beta-D-gal (1-4)-D-glc ; Enzyme/enzyme inhibitors e. g. D-alanine carboxypeptidase from B. subtilis or E. coli/6-aminopenicillanic acid or p-aminobenzyl-

penicillin/e. g. dehydrofolate reductase/aminopterin or amethopterin; Protein/co-factors e. g. Intrinsic factor/vitamin B12 or cobalamin.

In the most preferred embodiment, the affinity ligand/immobilized receptor combination is biotin or biotin derivatives thereof and biotin binding molecules.

In particular, the affinity ligand can be biotin or derivatives thereof and the immobilized receptor can be avidin or streptavidin or any other biotin binding molecule. The affinity ligand pairs biotin/avidin and biotin/streptavidin are often used in other applications.

The very strong interaction (i. e. K = 10-10 M) of biotin with the proteins avidin and streptavidin provides a foundation for their use in a large number of applications, both for in vitro and in vivo uses. A further application of the invention is the simultaneous removal of several different biotinylated biomolecules through the same extra- corporeal procedure.

Modification of the biomolecule by attachment (con- jugation) of two separate moieties to a biomolecule is far from ideal. Conjugation to a particular reactive functional group (e. g. amine, sulfhydryl, aldehyde, ketone) precludes attachment of a second molecule to that group. Hence, modi- fication of larger biomolecules (e. g. proteins) in two subsequent steps can result in a heterogeneous population of modified biomolecules in which molecules that contain the second conjugated species may have less of the desired biological properties (i. e. tumour targeting) than those which do not contain the second conjugate. To circumvent these problems, the affinity ligand (e. g. biotin moiety) and an cytotoxic agent can be interconnected by means of a trifunctional cross-linking reagent to form a new type of reagent. With the use of this new class of reagents, an equal number of affinity ligands and radionuclide

binding/bonding moieties will be conjugated to the bio- molecule. With a combined affinity ligand and radiolabeling compound, site specific addition of both reagents can be made, and minimization of the number of conjugates to the biomolecule can be attained. A number of different types of trifunctional reagents suitable for labeling of biomolecules applicable in this invention are described in WO 00/02051, Wilbur & Sandberg.

As used herein"substantially reducing"the concen- tration or level of biomolecule or cytotoxic fragment in the blood means a concentration reduction of at least 25%, and in increasing preference, reductions of at least 50%, 60%, 70%, 80%, 90% and 95%.

In summary, the aim of the present invention is to provide a kit, whereby a patient's toxic exposure is attenuated. Such a toxic exposure can result in hemato- logical side effects, myelosuppression, as well as toxic effects on vital organs through which the toxified blood is passed such as liver, lung, heart, kidney, spleen, etc. The treatment regime can be separated into the following events: 1. Dose calculations dependent on the size of nodules and dissemination of the disease, preferentially based on laparoscopy or CT scan, according to methods known to those skilled in the art.

2. Intraperitoneal infusion of biomolecule or bio- molecule-affinity ligand conjugates, e. g., biotinylated immunoconjugate specific for ovarian carcinomas, by using methods known to those skilled in the art.

3. Systemic extracorporeal depletion of the biomo- lecule or biomolecule-affinity ligand conjugate, e. g., biotinylated immunoconjugate, or cytotoxic fragments thereof, from for example the blood circulation by passing the blood through a receptor (e. g., avidin) coated device on one or several occasions depending on the kinetics of

the uptake of the biomolecule-affinity ligand conjugate in the blood circulation and the total dose of the conjugate administered.

In comparison with intravenous injection, the clearance of toxic substances the intraperitoneal route will follow a pattern not previously described (c. f. Fig 1).

By using the kit according to the invention it is possible to increase the dose of anti-tumour agent with up to a factor of 5 by utilizing two consecutive treatments with Mitradep@. Additional Mitradep treatment will allow an even higher enhancement of the dose. This can be a tremendous advantage, especially in that bone marrow transplants can be avoided. The cost for a bone marrow transplant is SEK 2-300 000. Furthermore, patients having impaired or decreased bone marrow production resulting from medications will with advantage be treated with the in- ventive kit. With patients having severely damaged bone marrow due to radiation, chemotherapy, or other drugs, the inventive kit may not only be the choice when treating an intraperitoneal cancer, but also for diagnosing the same, since intraperitoneal administration is a safer choice. In this connection, small tumours (<2 mm) can be detected by means of imaging and suitable dosages can be calculated for individual patients. Thus, a cancer treatment with this kit is not only safer but also less expensive than previous treatments.

The following Examples illustrate the utility and advantages which can be obtained with the present inven- tion. Example 1 illustrates how extracorporeal treatment can reduce the cytotoxicity associated with intraperitoneal injections of radiolabeled antibody directed to cancerous tissue. Examples 2-5 illustrate the effectiveness of the subject invention in attenuating the patient's toxic exposure. Examples 2-5 illustrate that a radiolabeled anti-

ovarian cancer immunoconjugate can be biotinylated to a sufficient degree for extracorporeal depletion without significantly affecting the avidin-binding properties or the biodistribution in the blood and vital organs. Addi- tionally, Example 5 shows that the biotinylated immunocon- jugate can be efficiently cleared from the blood circu- lation.

EXAMPLES Example 1-Simulation of the Effect of Extracorporeal Adsorption on Blood Levels of Cytotoxic agent.

Figure 1 illustrates the percentage of injected radioactivity in the blood after intraperitoneal adminis- tration of 90Y-HMFG-1 to patients with ovarian cancer as reported by Maraveyas A. et al. (Cancer 73: 1067-1075, 1994). These data were utilized to simulate the effects of two extracorporeal adsorptions with Mitradep (a blood filter having avidin immobilized to agarose particles approved for human use in Sweden) conducted at various or suitable times after administration of the conjugate of antibody and biotin. The following equation, obtained by fourth degree polynomial regression, was utilized for calculations: y = 2*10 *x4-0. 0004*x + 0. 0104*x2 + 0.7863*x-0.1963 (R =0. 9994).

Each adsorption is assumed to remove 90 per cent of the circulating conjugate according to the specification of Mitradep@. It is also assumed that the rate of transport of conjugate from the intraperitoneal volume to blood or the biological half-life of the conjugate in blood is not influenced by the extracorporeal adsorptions.

When the areas under the curve (AUC) are calculated, the following results were obtained (ECAT is extracorporeal affinity treatment) : Adsorption Adsorption Reduction 1 Hrs 2 Hrs AUC (AUC without ECAT)/(AUC with ECAT) post-inj. post-inj. - - 2026 - 12 36 624 3.25 18 42 499 4.1 24 42 417 4.85

The calculations are under-estimations as data is available up to 90 hours only. The AUC is generally con- sidered as directly correlated to the myelotoxic side effects seen in treatment with the radioimmunoconjugate.

It appears from the foregoing data that the simulated extracorporeal treatment employing adsorptions at 24 and 42 hours could be more effective. Similar analyses conducted in vivo in animal models or in human clinical studies can determine and optimize the number and frequency of ad- sorption procedures necessary to enhance imaging contrast and/or reduce side effects. Suitable times for extra- corporeal treatment depend on the kinetic function (which describes the level of the cytotoxic agent in the blood circulation as a function of time after intraperitoneal infusion), the number of extracorporeal (e. g., Mitradep@) treatments, and the optimal time point of treatment (s). The blood kinetic curve is likely to be similar for all types of monoclonal antibodies and the shape is probably the same for smaller targeting molecules as well. Based on the blood kinetic curve, the optimal post-injection extracorporeal treatment time can be calculated by calculating the AUC (without ECAT)/AUC (with ECAT) ratio. The greater this ratio, the less the cytotoxic (radioactive) exposure.

Example 2-Radiolabeling and biotinylation of HMFG-LCITC- DTPA.

In this and subsequent examples, Indium-111 was used as a substitute for yttrium-90, because the former is a gamma-emitter and possesses less radiation hazard than yttrium-90.

Ten (10) Al of 1 M Sodium Acetate, pH 5.5, was added to a vial containing 22 MBq (40 Al) Indium Chloride to give a final acetate concentration of approximately 0.2 M.

900 Al HMFG-1-CITC-DTPA was added to the ill In solution.

(DTPA is diethylenetriamine pentaacetic acid). After incu- bation for 30 minutes in room temperature, 40 ßl of 50 mM disodium EDTA (ethylene diamine tetraacetic acid) in acetate buffer was added to quench the reaction. Utilizing gel filtration, the buffer was changed to 0.1 M NaHCO3, pH 8.4.

Four (4) gl of N-hydroxysuccinimide (NHS)-biotin (10 mg/ml DMSO) was added to a vial with 500 1 In-HMFG- conjugate (2.25 mg/ml), followed by addition of DMSO to give a final DMSO concentration of 10%. The mixture was incubated for 4 hours at room temperature. Low molecular weight components were removed by gel filtration. The quality of the radio conjugate was determined by TLC and HPLC.

The antigen-binding activity was analyzed after two separate biotinylation procedures conducted on non-radio- labeled HMFG-1-CITC-DTPA. 40 Ug of NHS-biotin was added per mg of antibody. As illustrated in Figure 2, more than 99% of the radioactivity bound to avidin agarose. The binding curves were not significantly different from the curve obtained with HMFG-1-CITC-DTPA.

Example 3-Pharmacokinetics of conjugates of Biotinylated In-HMFG-1 Rats of the Fl breeding of Brown Norway (BN) and Wistar Furth (WF) rats were injected intraperitoneally with approximately 150 Ag of biotinylated HMFG1 labeled with 5 MBq In. Whole body (WB) imaging was performed using a scintillation camera (General Electric 400T, GE, Milwaukee, WI, USA) equipped with a medium-energy collimator. Images were stored and analyzed with Nuclear MAC 2.7 software.

From the images, the total number of counts in the entire body were obtained. After radioactivity decay correction and background subtraction, the counts were used for the calculation of activity retention (%) in the body. See Figure 3.

To define the pharmacokinetics of biotinylated In- HMFG1, about 0.2 ml blood was obtained from the periorbital venous plexa on the following occasions: 5 min, 8,16,24, 48,72, and 96 hours after injection. The radioactivity was measured in an automatic NaI (T1) scintillation well counter and expressed in percent of injected activity per gram tissue (%/g) corrected for In decay. See Figure 4.

After peritoneal resorption, a fraction of bio- tinylated In-HMFG-1 antibody was transferred to the blood circulation reaching maximal activity concentration of 3%/g (about 65% of the injected activity) between 12h and 16 h post injection.

Example 4-Biodistribution of Conjugates to Organs and Tissues.

Dissections of organs and tissues of interest were performed after 8,24, 72, and 96 hours. The organs and tissues were removed, weighed, and measured for activity content. The radioactivity was measured in an automatic NaI (TI) scintillation well counter, and the counts were

corrected for decay. The distribution of the injected activity is shown in Figures 5A and 5B.

Example 5-Extracorporeal adsorption of Biotinylated In-HMFG-1.

The rats underwent arterial and venous catherization for extracorporeal affinity adsorption treatment. Blood was pumped from the arterial catheter through an adsorbent with avidin-agarose at a flow rate of 0.5 ml/min. During a 3- hours treatment approximately 3 blood volumes were processed.

In conclusion, radiolabeled anti-ovarian cancer immunoconjugate as exemplified with In labeled HMFG-1- CITC-DTPA can be biotinylated to a sufficient degree for extracorporeal depletion without significantly affecting the binding properties or the biodistribution in the blood and vital organs. It has also been shown that the same biotinylated immunoconjugate can efficiently be cleared from the blood circulation.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.