CA2247308A1

CA2247308A1 - Modified cytokines for therapeutic use

Info

Publication number: CA2247308A1
Application number: CA002247308A
Authority: CA
Inventors: Micaela Pelagi; Giulia Casorati; Paolo Dellabona; Antonio Siccardi; Angelo Corti
Original assignee: Individual
Current assignee: Ospedale San Raffaele SRL
Priority date: 1996-02-27
Filing date: 1997-02-14
Publication date: 1997-09-04
Also published as: JP2000506835A; WO1997031655A2; AU1769497A; EP0885015A2; WO1997031655A3; ITMI960358A0; IT1282692B1; ITMI960358A1

Abstract

The object of the present invention is a new method and new products for the therapeutic use of cytokines. The method is based on the sequential administration of a component A (e.g. a biotinylated antibody) able to deliver onto a particular pathologic target a second component B (e.g. avidin), followed by a component able to bind the component B (component C) (e.g. biotin), conjugated with the cytokines. The method permits to increase the local concentration of the modified cytokines (C-cytokines) through the interaction with the "artificial" receptor B and to cause a local biologic response mediated by "natural" receptors.

Description

CA 02247308 l998-08-2~

MODIFIRn ~YTOKINR~ FOR T~RR~p~uTIc U~

The present invention refers to modified cytokine~
for therapeutic use. The immune system produces cytokines and other humoural factors in response to different inflammatory stimuli, to traumas, to viral and s bacteriological infections or to signals of cell degeneration, such as cancer. Although terms like "lymphokine", "monokines" and "cytokinesl' have been initially coined in order to distinguish products deriving from lymphocytes, monocytes and non-lymphoid cells, afterwards a sort of overlapping between these categories came out. Thus, the term "cytokines" is in current used as a synonymous of "lymphokines" and "monokines" and is hereinafter employed in this accepted meaning. A list of most of the cytokines ~nown in the art, as well as a list of their biological activities, is reported in Aggarwal B.~. and Pocsik E.
It is well known that dif~erent cytokines exert antitumoral, antiviral and antibacteric activity. On the basis of such activities, o~served both ln v;tro and i v vo in animal models, some cytokines have already been used therapeutically also in humans (De Vita et al., 1~95, in Biologic Therapy of Cancer, Lippincott Company, Phyladelphia). For example, such cytokines as interleukine-2 ~IL-2) and interferon a (IFNa) have shown positive antitumoral activity in patients with different types of tumors, such as kidney metastatic carcinoma, hairy cell leukemia, Kaposi sarcoma, melanoma, multiple mieloma, etc. Other cytokines like ~FN~, the Tumor Necrosis Factor ~TNF) q, TNF~, IL-1, 4, 6, 12, 15 and CA 02247308 l998-08-2~
W O 97/31655 PCT~EP97/00704 the Colony Stimulating Factors (~FSs) have shown a certain antitumoral acti~ity on some types of tumors and therefore are the object of further studies. Other cytokines have been used in the therapy of infective diseases (Aggarwal B.B. e Pocsik E.).
~n general, the therapeutic use of cytokines is strongly limited by their systemic toxicity. since this represents a crucial problem for their use in humans in therapeutically active amounts, many attempts have been made to develop new cytokines derivatives and new therapeutic strategies aimed at reducing the toxic effects of this class of biological effectors maintaining their therapeutic efficacy.
Tumor Necrosis Factor a (TNF) represents an emblematic case.
TNF is a cytokine, mainly secreted by macrophages, originally discovered for its capacity of inducing the hemorrhagic necrosis of some tumors (Carswell et al., 1975). Afterwards it has been demonstrated that TNF, besides exerting cytotoxic and cytostatic effects on different tumoral lines, can exert several other biological effects important for the regulation of the inflammatory and immune responses ~Beutler and Cerami, 1989;Fiers, 1991).
The idea is now confirmed that TNF can exert healthy or toxic effects for the organism by which is produced, as a function of its concentration, of its production site and of the time of persistence in the site of action. For example, the chronic exposition to low amounts of TNF can provoke cachexy while the acute hyper-production of TNF can cause serious vascular -CA 02247308 l998-08-2~

damages, shock and even death (Beutler and Cerami, 1989).
The potential antitumoral activity of TNF has been evaluated in various trials carried out on both animals and humans. Such trials have suggested that the antitumoral activity exerted in v vo by TNF mostly depends on its capacity of inducing damages to the tumor vascular system through direct effects on the endothelium and, in addition, through the activation of the inflammatory and immune responses (Sidhu and Bollon, 1993). On the contrary, less importance has been given to TNF-tumoral cells' direct cytotoxicity.
Although clinical trials (phase II) performed with TNF on different types of ~umors have not shown a remarkably antitumoral activity, encouraging data have been obtained by using TNF associated with other drugs ~IF~, IL-2, alkylating agentst Melphalan etc.). ~owever it was generally noted that various types of TNF
induced-toxic effects strongly limit the use of pharmacologically active amounts of the same (Spriggs and Yates, 1992). A fair success was instead obtained by using high amounts of TNF in regional therapy (e.g.
through perfusion of limbs of melanoma affected patients) so as to reduce the systemic toxic effects (Lienard et al., 1992).
Different alternative therapeutic TNF-based strategies are now under evaluation, aimed at increasing therapeutic efficacy of TN~ through an increase in the highest-tolerated amount and a reduction in the systemic toxic effects, obviously without jeopardize the antitumoral activity. It has been estimated that a CA 02247308 1998-08-2~
W O 97/31655 PCT~EP97/00704 reduction of about one order of magnitude of the amount necessary for exerting an antitumoral e~fect could result well tolerated.
This was tried through:
a) the development of fusion proteins which can deliver TNF into the tumor and increase the local concentration. For example, the fusion proteins consisting of TNF and tumor specific-antibodies have been produced (Hoogenboom et al., 1991);
b) the development of TNF mutants which maintain the antitumoral activity and have a reduced systemic toxicity. Accordingly, mutants able of selectively recognizing only one receptor (p55 or p75) have been already prepared (Loetscher H et al., 1993).
c) the use of anti-TNF antibodies able to reduce some toxic effects of TNF without compromising its antitumoral activity. Such antibodies have been already described in literature (Rathien et al.
1992).
d) the use of TNF derivatives with a higher half-life (for example TNF conjugated with polyethylene glycol).
The pharmacologic potentials of such strategies are now under evaluation.
Although the data obtained encourage the use of TNF
as an antitumoral agent, the problem of its systemic toxicity has not been solved yet.
Delivering systems for paramagnetic or radioisotopic cytotoxic agents employing the biotin-avidin interaction have been recently described (EP 251 494 and ~P 496 074).

CA 02247308 1998-08-2~

In particular, EP 251 494 describes a system for administering a diagnostic or therapeutic agent, which comprises: an antibody conjugated with avidin or streptavidin, an agent capable of complexing the conjugated antibody and a compound consisting of the diagnostic or therapeutic agent conjugated with biotin, which are administered seguentially and adeguately delayed, so as to allow the loca~ization of the therapeutic or diagnostic agent through the biotin-streptavidin interaction on the target cell recognizedby the antibody.
The described therapeutic or diagnostic agents comprise metallic chelates, in particular che7ates of radionuclides and low molecular weight antitumoral lS agents such as cis-platinum, doxorubicin, etc. In fact, it is expressly pointed out that the system is not suitable ~or compounds with molecular weights over 50,000 Daltons ~preferably not over 10,000 daltons), and necessarily cannot be applied to cytokines, such as TNF
(51,000).
EP 496 074 describes a method which provides the seguential administration of a biotinylated antibody, avidin or streptavidin and a biotinylated diagnostic or therapeutic agent. Also in this case, even though cytotoxic agents like ricin (a protein whose cytotoxic chain has a molecular weight of about 30,000 daltons) are mentioned, the application relative to radiolabelled compounds is mostly disclosed.
Wo 95/15979 discloses a method for localizing 3~ highly toxic agents (I) on cellular targets, based on the administration of a first conjugate ~C1) comprising CA 02247308 1998-08-2~

the ~pecific target molecule conjugated with a ligand (L) or an anti-ligand (AL) followed by the administration of a second conjugate (C2) consisting of the toxic agent (I) bound to an anti-ligand (AL) or to the ligand (L). In this case, even though cytokines are cited among the toxic agents (~), including TNF, as well as the avidin/biotin system for L/AL, it can be deduced that when cytokines are used, the administration of the ligand (L) or (AL) is highly preferred for dissociating the cytokine thus allowing the "free~' cytokine to exert the biological effects. This is likely due to the fact that the bound cytokine cannot react with its own receptors and exert efficaciouslY the desired biological effects. The necessary amount of L or AL is expected to be relatively high in order to compete for the binding on the cell surface, with possible consequent problems of toxicity. Moreover, when L/AL correspond to biotin/avidin, considering the high stability of such an interaction (the strongest non covalent known interaction, Kd=10-15 M) such binding is practically indissociable (3. Savage et al., avidin-biotin chemistry: a handbook. Pierce Biotec Company ~Rockford).
WO g5/15979 reports no specific experimental data which support the use of cytokines in the claimed method, but only generical citations that are not sufficient to provide the reproducible and practical teaching necessary for applying the method for localization to this class of substances that moreover do not act through a simple cytotoxic mechanism, but also through complex pro-inflammatorY~ immunostiulating, procoagulant and necrotizing mechanisms by which such W O 97/31655 PCT~EP97/~0704 substances can exert antitumoral effects without exerting direct cytotoxic effects, as on the contrary it is required by using the agents of the above-mentioned applications EP-251 494 and EP-49~ 074.
Now, contrary to the generic indications reported in WO 95/1597~, it has been surprisingly found that cytokines can be localized efficaciously in a biological active form only by means of a system in which the interaction between the coniugated cytokine and the target specific-conjugated component is not direct, as is the case of coniugation of a ligand/antiligand couple, but is rather mediated by a third component which can bind as a bridge between the target specific-component and the cytokine.
In accordance with the method of the present invention, the cytokine can also operate in a bound state and the administration of a member of the ligand/anti-ligand couple is not necessary in order to turn the cytokine in an active form. Such a result can be considered unexpected since it was unpredictable that the cytokines conjugated to a ligand could interact with the membrane receptors on the cell surfaces while interacting with the corresponding anti-ligand which in turn is involved in the interaction with the target specific component con~ugated to a suitable ligand.
Thus the invention provides pharmaceutical compositions in the form of combined preparations for sequential therapeutic use, comprising:
a) an anti-pathologic target compound conjugated to a ligand of an at least ternary ligand/anti-ligand/ligand system;

CA 02247308 1998-08-2~
W O 97/316~j PCTAEP97/00704 b ? an anti-ligand complementary to the ligand of compound a);
c) a cytokine conjugated to a ligand complementary to the anti-ligand b), with the proviso that the interaction ligand/anti-ligand/ligand is characterized by an affinity at least one order of magnitude higher than the affinity between the cytokine and its natural receptors.
Examples of compounds that can be conjugated to compound a) and to the cytokine, according to the invention, comprise haptens such as biotin and digoxigenin, while examples of "anti-ligand" compounds comprise anti-haptens antibodies ~for example, anti-biotin antibodies and anti-digoxigenin antibodies) or, when biotin is used as a ligand, avidin and its analogues (e.g. streptavidin, neutravidin).
Preferably, both compound a~ and the cytokine are conjugated to ~iotin, while avidin (or the analogue compound) is used as an anti-ligand.
The techniques used for the conjugation of the cytokines and of the antibodies are widely known and can be connected to chemical or to genetic engineering methodologies.
The anti-pathological target-compounds a) preferably are whole or fragmentary antibodies or monoclonal antibodies. Said antibodies have already been described and used widely, expecially in the case of antibodies directed against tumoral antigens.
Examples of cytokines or limphokines that can be used according to the invention comprise tumor necrosis factors, interferons, interleukins, colony stimulating CA 02247308 1998-08-2~
W O 97/31655 PCT~EP97/00704 factors (CSF~. It is also possible to use a biological response's modifier which can promote the local release of endogen cytokine, such as the lipopolysaccharide (LPS) or derivatives thereof, to obtain an increased local effect and lower systemic effects.
Moreover, as it is known that cytokines can sometimes exert additive or synergic effects, the strategy upon which the present invention is based can be performed in order to obtain synergic local effects and less effects at systemic level. The biologic therapy of cancer and the effects of the combination of different cytokines' are widely documented and well summarized by De Vita et al. 1995 (Biologic Therapy of ~ancer, Lippincott Company, Phyladelphia).
15Among cytokines the use of TNF is particularly preferred.
Preferably, the mutual interactions of the conjugates or their interactions with the artificial receptor will be characterized by affinity constants at 20least one order of magnitude higher than the af~inity constants of the cytokines' membrane receptors and by kinetic dissociation constants at least one order of magnitude lower than that of interaction of cytokine with its natural receptors.
25The anti-target compound or conjugated antibody can be locally administered or alternatively can be injected into the bloodstream, and can be reacted Ln v vo with the antigens or with the recognized cellular structures till the exceeding circulating compound or antibody is 30removed from the body, while a significant fraction remains bound to the pathological target. At this moment _ CA 02247308 l998-08-2~
W O 97/316~5 PCTAEP97/0~704 the anti-ligand b) can be administered, followed by the conjugated cytokine in such a concentration that a bond with the antibody or anti-target compound can be formed thus allowing the mound or the increased persistence of the cytokines in the target cells.
This can be achieved if the dissociation time of the modified cytokine from the "artificial" ~eceptor (represented by the anti-ligand) is at least one order o~ magnitude longer than the dissociation time from the "natural" receptors. As the examples relative to the applications of the invention demonstrate, it can be performed through a system consisting of a biotinylated antibody specific for a tumoral antigen, neutravidin, and biotin-TNF (bio-TNF). In this system the affinity 1~ between bio-TNF and the artificial receptor (avidin), equal to 10-15 M, and the affinity between bio-TNF and its natural receptors (TNF-R1 and TNF-R2), equal to 10-9 - 10-1~ M, are such that the mound and the increased persistence of TNF are favoured in the site where the 2~ artificial receptors are present (tumor).
In the preferred embodiments of the invention, a biotinylated monoclonal antibody specific for a tumoral antigen is brought into contact with the tumor through its intra- or para-lesional, intra-cavity (e.g. b7adder, peritoneal cavity), intra-artery (liver, central nervous system) administrations or at the systemic level, local or regional vascular perfusions (e.g. in the limb perfusional liquid, liver, breast), followed by analogue sequentlal administration of neutravidin or streptavidin and TNF-biotin.
Incubation times of some hours are preferably CA 02247308 1998-08-2~
W O 97/31655 PCT~EP97/00704 inserted between each administration to a~low the vascular system to eliminate the exceeding product thus obtaining a finer localization on the tumoral target.
Moreover, the use of TNF biotinylated at the amino terminal region (1-11 residues, VRSSSRTPSDK sequence) is preferred, so that the multivalent bond of TNF with its natural receptors is not hampered and its effects are not inactivated. AlternativelY, amino acids beariny groups which can be easily biotinylated through known techniques ~lysine, cysteine, tyrosine, hystidine, etc.
- see Savage et al., Avidin-biotin chemistry: a handbook, Pierce Biotec Company), can be inserted in the same region through genetic engineering-techniques, or glycosylation signals, so that specific biotinylation of carbohydrate residues is obtained, for instance with biotin-hydrazide or derivatives thereof. The biotinylation of TNF at the amino-terminal portion can be easily obtained as well by means of genetic engineering-techniques through the construction of conjugates of TNF and fragments of proteins directly biotinylated by the expression system. An example of such proteins is represented by acetyl-CoA carboxylase from E. Coli, and by its C-terminal domain bearing the biotinylation site.
A biotinylation of the alpha-amino groups is preferred in order to keep the structure of biotin-TNF
the closest to the structure of non-biotinylated TNF.
This can be done by using protocols for biotinylation based on the reaction of biotin-6-aminocaproyl-N-hydroxysuccinimide ester at a pH comprised between 5.5 and 7.5. Alternatively, it was observed that CA 02247308 1998-08-2~

biotinylated TNF can be obtained such that the capability of interacting with avidin and with membrane receptors is maintained, by "mixing'l the subunits from TNF for example biotinylated according to one of the above-described methods, and from non-biotinylated TNF.
Preferably, the mixing reaction is carried out incubating mixtures of biotinylated and non-biotinylated TNF in a ratio of 1:3 for 24-72 hours, at 4-C. Such forms of TNF conjugated at the N-terminal are new and represent a further aspect of the invention.
Kits containing suitable therapeutic materials can be prepared in order to make the commerce and the routinary use of the compositions of the invention easier. Preferably, a kit according to the present invention comprises:
- a vial containing 0.5 to 10 mg of biotinylated antibody;
- a vial containing 5 to 100 mg of avidin or streptavidin or neutravidini 20 - a vial containing 0.5 to 10 mg of biotinylated TNF
(or other biotinylated cytokine).
The method of the present invention offers some advantages compared to known methods, for example the advantages deriving from the direct conjugation of cytokines with antibodies:
1) for example, it does not re~uire the antibody affinity to be necessarily high, as it is possible that relatively high amounts of antibody and avidin are administered, followed by relatively lower amounts of biotin-TNF, and anyhow the tumor is efficiently labelled. On the other hand, this is CA 02247308 1998-08-2~
W O 97/31655 PCT~EP97/00704 not possible with conventional con3ugates antibody-TNF, for which the amount of antibody is invariably "linked" to TNF-amount.
2) The possibility of diffusion of the separated antibodies and TNF into the tumoral mass is presumably better than that obtainable with con3ugates antibody-TNF, due to their' lower molecular weight.

3) The tumor labelling with high affinity molecules like avidin and streptav~din (10-15 M) permits biotin-TNF amounts to be selected such that the interaction with the artificial receptor (avidin) is favoured thermodynamicallY and kinetically and the interaction with natural receptors at systemic level (10-9-10-1~ M) is hampered. In the case of antibody-TNF conjugates, it is extremely difficult to obtain antibodies able to bind the tumoral antigen with affinity in the order of 10-15 M.
The present invention is further described in the following examples.
Although the examples are particularly referred to the treatment of tumoral pathologies, it can be easily understood that the same strategy can be employed for the treatment of each other patholo~ies for which a local treatment with cytokines can be used.
R ~ Al'IPr. R
This example shows an application of the invention based on the system:
Pathological target: A : B : ~-cytokine where:
Pathological target = murine lymPhoma expressing the W O 97/31655 PCT~EP97/00704 murine antigen Thy 1.1 (RMA cells genetically engineered in order to express the allele Thyl.1 ~RMA Thy 1.1 C1.2);
A= biotinylated anti-Thy 1.1 monoclonal, antibody (mAb bio-19E12~.
B= neutravidin C-cytokine= biotin-TNF coniugate and where (:) represents a non-covalent interaction, while (-) represents a covalent bond.
MAte~i;31s - AH-BNHS (biotin 6-aminocaproyl-N-hYdroxysuccinimide ester) (SPA, B002-61) - SULFO-NHS-LC-Biotin (Pierce, 21335) _ Human TNFa (lx108 U/mg) (DRG080) - Lysine (Sigma cod. L5501) - Colture medium: sterile RPMI-1640 ~Gibco 31870-025) - Foetal Calf Serum (FCS) (PBI-Biological Industries, cod. 04-001-lA) - Foetal Calf Serum (FCS) (Sigma, F2442) - Geneticin (G418, Sigma cod. G9516) in 100 mM Hepes - Hepes (Sigma, H0887) - 200 mM Glutamine (Gibco cod. 04305030D) - 10,000 IU/ml Penicillin, 10,000 mg/ml streptomycin, 25 yg/ml Amphotericin B (Gibco cod. 15240-021) - NaC1 (BDH cod. 10241) - HCl 37~ (B~H cod. 10125) - 96-well flat-bottomed PVC plates for cell culture (Costar 3595) - 96-well round-bottomed plates ~PBI, 650180) - 2-mercaptoethanol (Merck cod. 12006) CA 02247308 l998-08-25 WO 97/316~S - PCT~EP97/00704 - Actinomycin D (Fluka 01815) - Thiazolyl blue (MTT) (Merck 11714) - PBS (NaCl 0.15 M, Na-phosphate 0.05 M, pH 7.3) - Neutravidin (Pierce cod. 31000) - Sodium azide (Baker, 9099) - mAb l9E12 Bx~m~le 1.1 Preparation of the biotinylated l9E12 mAb antibody (bio-lg~l2 ) 1000 ~l of a solution of the 19E12 mAb, 1 mg/ml in sodium bicarbonate pH 8.5, 34 ~l of a sulfur-NHS-LC-Bio-tin solution, 1 mg/ml (molar ratio mAb/biotin: 1/24) were pipetted into an Eppendorf tube. The mixture was incubated at room temperature (23/24~C) for 30 minutes.
After incubation the mixture was dialyzed overnight against 2 litres of PBS at 4~C and kept at +4~C.
~ le 1.~
Preparation of biotin-TNF (bio-TNF) In this chapter some examples of the protocols used for the preparation of biotin-TNF conjugates are repor-ted.
Example 1.2.1 (preparation of bio-TNF cod. ~B) 60 ~l of a solution of TNF (0.5 mg/ml) in bidistil-led water, 6 ~l of 1 M Na-carbonate pH 6~8, 6 ~l of AH-BNHS, 3 mg/ml in DMSO (molar ratio TNF/Biotin: 1~66~, were pipetted into an Eppendorf tube. The mixture was incubated at room temperature (23J24~C) for 3 hours and mixed with 7.5 ~l of a 1 M lysine solution. After a fur-ther 1 hour incubation at room temperature 240 ~l of the solution RPMI, 10~ FCS, 2 mM glutamine, 100 IU/ml peni-cillin, 100 ug/ml streptomycin, 250 ng/ml amphotericin W O g7/31655 ~CTAEP97/00704 B, were added to the mixture. The mixture was then dialyzed overnight against 2 litres of 0.9~ NaCl at 4-C
(3 changes) and kept at -20~C.
~ xample 1.2.2 (preparation of bio-TNP cod.~C, cod.~D, cod.#E, cod.#F, cod.#G) The bio-TNF cod.#C product is prepared according to example 1.1.1 except for the use of a conjugatioh ~uffer at p~ 7.8.
Comparative solutions of bio-TNF (cod.#D, cod.#E, cod.#F, cod.#G) are also prepared using different incu-bation buffers, various TNF/biotin molar ratios (see ta-ble 1).
Rx~m~e 1 3 Determination of the biologic activity of biotinylated TNF on RMA Thy 1.1 C 1.2 cells.
This experiment is aimed at determining the speci-fic biologic activity of different ~io-TNF preparations obtained in different conjugation's conditions (see example 1.1), through a cytotoxicity test on RMA Thy 1.1 Cl.2 cells.
Procedure:
To each well of a 96-well flat-bottomed plate (Costar 3595) are added:
a) 60000 RMA Thy 1.1 Cl.2 cells (Mycoplasma free) in 50 ~l of RPMI, 5~ FCS, 2 mM glutamine, 100 IUfml penicillin, 100 ~g/ml streptomycine, 250 ngfml am-photericin B, 50 nM 2-mercaptoethanol, 500 ~g/ml G418 (Complete-RPMI);
b) 50 ~l of the standard TNF solution or of the solu-tion of the sample in complete-RPMI at the desired concentration;

W O 97131655 PCT~EP97/00704 c) 10 ~1 of 330 ng/ml Actinomicyn D in Complete-RPMI.
Then the plate was incubated for 24 hours at 37DC, 5% C02. Furthermore, 10 ~1 of a thiazolyl blue solution (MTT), 5 mg/ml in PBS, were added to each well. After further 4 hour incubation at 37-C, 5% C02, 100 ~1 of ly-sis solution (33% (v/v) N,N-dimetylformamide), 20% (p/v) sodium dodecylsulfate, in water, brought to p~ 4.7 with glacial acetic acid) were added to each well. The solu-tions were mixed in the wells with a multi-channel pi-pette and incubated for 24 h at 37DC. The absorbance of each well was then read by a multi-channel microplate reader at ~70 and 650 nm (reference).
The cytotoxic activity was calculated by interpola-tion of the absorbances on a calibration curve obtained with non-biotinylated TNF.

W O 97/31655 PCT~EP97/00704 ~eslllts Table 1 Bio-TNF biologic activity, measured through cytotoxicity test on RMA cells.
-______ ____ Biotinylation reaction cod. TNF~biotinbio~logic activ.a) (#) (molar ratio) (pH) ~U/mg~
TNF A 0 (non biotinyl.) - 1.0 x 108 bio-TNF B 1~66 6.8 5 x 107 bio-TNF C 1/66 7.8 2.5 x ~07 bio-TNF D 1/66 8.8 3.1 x 107 bio-TNF E 1~138 8.8 3.9 x 106 bio-TNF F 1/275 8.8 3.0 x 105 bio-TNF G 1/550 8.8 1.0 x 105 ___________________________ _______ a) Measured using a calibration curve obtained with non-biotinylated TNF (cod.#A) ~xample 1.4 Comparison of the binding and of the association and dissociation kinetics of bio-TNF and TNF from cells pre-treated with anti~odies and neutravidin The experiment is aimed at demonstrating that the 25 constitution of artificial receptors on tumoral cells through the biotinylated antibodies and neutravidin pre-targeting system remarkably increases the total amount of bio-TNF which can bind to the cells compared to the maximum amount which can bind to natural receptors.
Moreover, this experiment is aimed at evaluating the association and dissociation times of bio-TNF with W O 97/31655 PCT~EP97/00704 natural and artificial receptors (neutravidin) from cells pre-treated with antibodies and neutravidin.
Proced~lre The experiment was carried out treating MRA thy l.1 Cl.2 cells as described in examples l.5 and l.6. Total bound TNF was detected using an indirect method based on the use of anti-TNF rabbit polyclonal antibo~ies and rabbit fluoresceinated goat anti-igG antibodies and FACS
analysis.
Associat1on kinetic 50,000 cells in 50 ~l PBS/FCS 2~ were seeded in the wells of a round bottomed plate, mixed with l ~l of bio-l9El2 mAb, 0.5 mg/ml in PBS/FCS 2~ (antibody's final concentration lO ~g/ml), and incubated for lO min on ice.
The cells were washed two times by adding Z00 ~l/well of PBS/FCS 2~ and spinning for 2 min at 1300 rpm. The cells were resuspended by vortexing and mixed with 50 ~l/well of 2~ PBS/FCS and with l ~1 of 2.5 mg/ml neutravidin in PBS/FCS 2~ (neutravidin's final concentration 50 mg/ml). After lO min incubation on ice, cells were washed two times again with 2~ PBS/FCS, as above.
50 ~l of PBS/FCS 2%, l ~l of TNF or bio-TNF at a concentration of 22.2 ~g~ml (final concentration of TNF
and bio-TNF 450 ng/ml) were then added to each well, and samples were incubated for l h on ice.
After a further washing with 2~ PBS/FCS, 50 ~l of rabbit anti-TNF polyclonal serum tGenzyme cod#IP300) l:lO00 in PBS/FCS 2~ were added to each well, and incubated for lO min on ice.

CA 02247308 1998-08-2~
W O 9713165~ PCT/EP97/00704 ZO
Cells were washed two times by addition of 2~
PBS/FCS (200 ~l/well) and centrifugation. Afterwards 50 ~l of goat anti-rabbit immunoglobulin anti-serum conjugated with fluorescein (goat anti-rabbit-FITC) in PBS/FCS 2% were added to each well and incubated for 10 min on ice.
After the final washing with 2~ PBS/FCS samples were resuspended in 200 ~1 of 2% PBS/FCS and analysed with FACS.
Dissoci~tion k;netic 100,000 cells in 50 ~l 2~ PBS/FCS were seeded in 8 wells of a round bottomed plate, mixed with 1 ~l of bio-l9E12 mAb, 0.5 mg/ml in 2% PBS/FCS (antibody's final concentration 10 mg/ml), and incubated for 10 min on ice.
Ce~ls were washed two times by adding 200 ~l/well of 2% PBS~FCS and centri~uged for 2 min at 1300 rpm.
Cells were resuspended by vortexing and mixed with 50 ~l/well of 2% PBS/FCS and with 1 ~l neutravidin 2.5 mg/ml in 2% PBS/FCS (final neutravidin concentration 50 ~g/ml). After 10 min incubation on ice, cells were washed two times again with 2~ PBS/FCS, as above.
50 ~l of 2% PBS/FCS, 1 ~l of TNF or bio-TNF at a concentration of 22.2 ~g/ml (final concentration of TNF
and bio-TNF 450 ng/ml), were then added to each well and the samples were incubated for 1 h on ice.
The 8 samples were washed with 200 ~l of 2% PBS/FCS
by centrifugation (twice). Cells were resuspended in 50 ~l of RPMI, 5~ FCS, 2 mM glutammine, 100 I~/ml penicillin, 100 ~g/ml streptom~cin, 250 ng/ml am~hotericin B, and at different moments, fixed with CA 02247308 1998-08-2~
W O 97/316~5 PCTfEP97/00704 0.25~ paraformaldehyde for 1 h at 4-C.
After a further washing with 2% PBS!FC~, 50 ~l of ra~bit anti-TNF polyclonal serum ~Genzyme cod#IP300) 1:1000 in PBS/FCS 2% were added to each well, and incubated for 10 min on ice.
Cells were w~shed two times again through the addition of 2% PBS/FCS (200 ~l/well) and centrif~gation.
Afterwards 50 ~1 of goat anti-rabbit i~munoglobulin anti-serum conjugated with fluorescein (goat anti--iO rabbit-FITC) 1/12000 in 2~ PBS/FCS and incubated for 10 min in ice.
After the final washing with 2% PBS/FCS the samples were resuspended in 200 ~1 of 2~ PBS/FCS and analysed with FACS.
Resll~ts The neutravidin pre-targeting on the cells provokes an at least 10-20 times higher increase in the bio-TNF
binding to the cells, compared to the binding obtainable with the sole natural receptors (determined without neutravidin or using non-biotinylated TNF). The best increase was observed with bio-TNF cod.~B (biotinylated at pH 6.8) (data not shown).
Moreover, as it can be noted in figure 1 ~lower panel), the time of persistence of bio-TNF on RMA-cells pre-treated with antibodies and avidin is about 30 times higher (t1/2=7 h) then the persistence of the TNF on the sa~e cells (tl/2=0.22 h).
It can be concluded that the binding demonstrate that the exploitation of the antibody's and neutravidin's pre-targeting strategy in accordance with the invention, allows the maximum amount of bio-TNF

CA 02247308 1998-08-2~

bound to tumoral cells to be increased 10-20 times and such as amount to persist on the cell membrane about 30 times longer.
R~mDle 1 5 ~omparison of TNF and bio-TNF ci~otoxicities on tumoral cell~ pre-treated with biotinylated antibodies and neutravidin, n v;tro, in the presence of actino~ycin D
The example is aimed at demonstrating the ability of a biotinilated antibody/avidin system to deliver biotin-TNF on the cell surface in the bilogical active form and to increase the in vitro citotoxicity of the same, compared to that obtainable simply using the natural receptors.
For this aim RMA Thy 1.1 Cl 2 cells, the human monoclonal anti-Thy 1 antibody, biotinylated (mAb bio-l9E12), neutravidin, and bio-TNF cod~B have been used.
The experiment was conducted by se~uentially incubating and washing the cells with a~ mAb bio-19E12, b) neutravidin, c) bio-TNF.
As it is ~nown that the TNF binding to natural receptors induces the synthesis of protective factors, such as Mn superoxidedismuthase, with the aim of inhibiting the production of such factors and of increasing the cytotoxic activity of TNF, the cytotoxicity test was carried out in the presence of a transcription inhibitor (actinomycin D).
Procedllre 1,000,000 cells in 50 ~l of 2~ PBS/FCS and 1 ~1 of a solution of 0.5 mg/ml mAb bio-19E12 were added to each well of a 96-well round-bottomed plate (~ostar 3595).
The suspension was incubated for 10 min on ice. Cells CA 02247308 1998-08-2~
W O 97/31655 PCT~EP97/007~4 . 23 were washed two times by adding 200 ~l/well of 2 PBS/FCS and spinning for 2 min at 1,300 rpm. Cells were resuspended by vortexing and mixed with 50 ~l/well of 2%
PBS/FCS and with 1 ~l of 0.5 mg/ml neutravidin in 2~
PBS/FCS. After 10 min incubation on ice, cells were washed two times again with 2% PBS/FCS, as above.
50 ~1 of 2~ PBS/FCS, 1 ~l TNF or bio-TNF at the desired concentration were then added to each well and incubated for 15 min on ice. After further washing with 2~ PBS/FCS, cells of each well were resuspended in 1.5 ml of RPMI, 5% FCS, 2 mM glutamine, 100 IU/ml penicillin, 100 ~g/ml streptomycin, 250 ng/ml amphotericin B, 50 nM 2-mecaptoethanol, 500 ~g/ml G418 (complete-RPMI).
Cells were then seeded on 96-well flat-bottomed plates (60,000 cells/100 ~l/well), mixed with 10 ~l/well of a solution of 330 ng/ml actinomycin D in complete-RPMI, and incubated for 24 h at 37~C, 5% C02.
The cell viability was determinated as described in the example 1.3.
Reslllts The results of the experiment conducted in the presence of actinomycin D are reported in fig. 2. It can be seen that, while in the absence of neutravidin the amount of TNF or bio-TNF necessary for killing 50~ of the cells (LD50) (which can be detected from the 50 lowering of the absorbance) is higher than 100,000 U/ml, the LD50 of bio-TNF in the presence of neutravidin is about 2,000-3,000 U/ml. Furthermore, while the cytotoxicity of TNF is not influenced by the presence or by the absence of neutravidin, the cytotoxicity of bio-CA 02247308 l998-08-25 W O 97/31655 PCT~EP97/00704 TNF as it would be expected is strongly dependent on the presence of neutravidin. This shows that: a) TNF and bio-TNF are weakly cytotoxic for RMA cells in the absence of neutravidin, b~ the presence of "artificial"
neutravidin receptors on RMA cell membranes strongly increases the bio-TNF cytotoxicity, c) bio-TNF bound to the membrane through an antibody-biotin-avidin t'bridge"
can interact with its own natural receptors and trigger cytotoxic effects.
In an experiment carried out in the absence of actinomycin D, both TNF and bio-TNF have shown no cytotoxic activity at all (data not shown).
~.x~le 1.6 Comparison of the i~ ~I~Q tumorigenicity ~mouse) of RMA
Thy 1.1 c12 cells pre-treated with biotinylated antibodies, ne~travidin and with TNF or bio-TNF, in the absence of Actinomycin D
This experiment is aimed at demonstrating the reduced in v vo tumorigenicity of cells pre-treated with bio-TNF bound to the cellular surface through the antibody-biotin-neutravidin system (artificial receptor), compared to cells pre-treated with TNF or with bio-TNF exclusively bound to the natural receptors.
The adopted model is based on the subcutaneous administration of RMA Thy 1.1 Cl.2 cells pre-treated in vitro. The experiment was carried out using 4 groups of female C57 BL6 mice (5 mice for each group) and measuring the diameter of the tumoral mass at different days. Mice were treated with pre-treated cells as shown in table 2.
It is important to remember that the amounts of TNF

W O 97/31655 PCT~EP97/00704 and bio-TNF used in this study (respectively 50,000 U/ml and 10,000 U/ml) are such as to determine partial cytotoxic effects only in the presence of actinomycin D, as demonstrated in the example 1.5 (fig. 2~. In the present experiment cells were treated in the total absence of actinomycin D, i.e. in conditions according to wich both TNF and bio-TNF are not cytotoxic.
Table 2 Experimental scheme for the treatment of RMA Thyl.1 Cl.2 cells injected into the mice.
Group mice mAb bio-19E12 Neutrav. TNF ~io-TNF
~#) ~N.) ~lOyg/ml) (lO~g/ml) (50000U/ml) (lOOOU/ml) 1 5 - - _ _ 2 5 +
3 5 + + +

4 5 ~ ~ _ +
________________ ______________________________ Proced-7re:
180,000 cells in 50 yl 2~ PBSJFCS were seeded in four wells of a round bottomed plate, mixed with 1 ~l of bio-19E12 mAb, 0.5 mg/ml in 2% PBS/FCS, and incubated for 10 min on ice.
Cells were washed two times by adding 200 yl/well of 2% PBS/FCS and spinning 2 min at 1,300 rpm. Cells were resuspended by vortexing and mixed with 50 yl/well of 2% PBS/FCS and with 1 ~1 of neutravidin 0.5 mg/ml in 2% PBS/FCS. After 10 min incubation on ice, cells were washed two times again with 2% PBS/FCS, as above.
Then, 50 yl of 2~ PBS/FCS, 1 yl of TNF or bio-TNF
at a respective concentration of 50,000 and 10,000 U/ml, CA 02247308 1998-08-2~
W O 97/31655 PCT~E~97/00704 were added to each we~l, and incubated for 15 min on ice.
After further washing with 2~ PBS/FCS, cells from each well were resuspended in 200 ~l PBS, transferred to Eppendorf tubes, and mixed with 1.2 ml PBS. By the aid of syringes for insulin, 3~,000 cells/200 ~l/mouse were injected subcutaneously at the inguinal level. The mean diameter (lateral and longitudinal) of the tumoral mass was determined for different times using a calibre.
Results:
The results are reported in table 3. It can be noted that the cell treatment with bio-19E12/neu-travidin/bio-TNF mAb (group 4), determined a reduction of the RMA Thyl.1 ~1.2 cell tumorigenicity in mice, compared to mice treated the same way but without bio-TNF (group 2). Differently the pre-treatment with bio-l9E12/neutravidin/TNF mAb ~group 3) did not produce a significative reduction of the tumorigenicity compared to the control group (group 2). In a further experiment carried out with 50,000 UJml bio-TNF, instead of 10,~00, the volume of the tumoral mass after 17 days was lower than the detection limit (15 mm3) (data not shown).
These results indicate that bio-TNF bound to the cell membrane through the antibody/biotin/neutravidin "bridge" can trigger such effects that the RMA Thy 1.1 Cl.2 cell in v vo tumorigenicity is reduced.
Furthermore it is interestingly observed that although the amount of bio-TNF used for the treatment of the RMA cells was such to determine the cellular lysis, in the presence of actinomycin D (see the example 1.5), i.e. in such conditions that TNF cannot exert direct W O 97/31655 PCTrEP97100704 cytotoxic effects, nevertheless the treatment provoked a reduction 5 times higher of the volume of the tumoral mass, after 17-19 days from the injection. This sugqests that the reduced tumori~enicity is due to the host's indirect effects on the tumoral cells (inf-ammatory, immune effects, etc.).

CA 02247308 l998-08-25 W O 97/31655 PCT~EP97/00704 Table 3 Diameter and volume of the tumoral masses in four groups of mice treated accordin~ to the scheme reported in table 2 at different days from the injection ______________ Diametera) Volume (mm) (mm3) ___________________________ Group Mice day day day day day (N.)11 14 15 19 19 Group 1 1 - 5 7 12 mean 3.6 5 7.5 10.3 lZ31 +SD +0.64+1.63 +2.1 +2. 3 +194 Group 2 1 6 9 11 15 mean 6 8.4 10 14.8 16g6 +SD +0.0+0.5 +1.0 +0.44 +50 Group 3 1 6 8 9. 5 15 mean 67. 6+0.54 9.7 15 1766 +SD +0. 7+0.5 +1.09 +O.o +0.0 WO 97/316S5 PCT~EP97/00704 Table 3 (continue) Diameter and volume of the tumoral masses in four groups of mice treated according to the scheme reported in table 2 at different days from the in3ection _______________ Diametera)Volume (mm) (mm3) Group Mice day day day day day (N.)11 14 15 lg 19 Group 4 1 3 3 4.5 7.5 2 3 3 5.5 9 4 - - ascite ascite mean 3 3.75 5.5 8.6 322 fSDiO.0 +0.95 +0.88 +0.75+ 30 _________ _________ . ____________ a) mean of longitudinal and lateral diameters.
~x~mple 2 This example shows an application of the invention based on the system:
Pathological target: A: B: C-cyto~ine release-inducer where:
Pathological target= murine lymphoma expressing the human Thyl.l antigen (RMA Thy 1.1 Cl.2 cells);
A= biotinylated human anti-Thy 1 monoclonal antibody (bio-19E12 mAb).
B= neutravidin C-cyto~ine release-inducer= biotin-lipopolysaccharide conjugate (LPS) W O 97~31655 PCT~EP97/00704 and where (:) represents a non-covalent interaction, while (-) represents a covalent bond.
Ex~mDl e ~ 1 Preparation of LPS-biotin ~ter;~l~
Lipopolysaccharide from Salmonella minesota (LPS): Sigma cod. L2137 lot. 101H4029 Biotinamidocaproylhydrazide: Sigma B-3770 Tris base: BDH cod. 10235 Sodium-M-periodate: Sigma S-1878 Sodium-acetate: BOM cod. 10235 Sodium-azide: J.T. Baker cod.9099 ~01 l~t; or~
- "Stop solution": 0.1 M Tris-~Cl pH 7.5 (in water) - Labelling solution": 100 mM Na-acetate, 0.02~ Na-azide, pH 5.5.
Metho~
100 ~l LPS (0.8 mg/ml in labelling solution) were mixed with 50 ~l Na-M-periodate (30 mM in labelling solution). The mixture was incubated at room temperature in the dark for 30 min. After incubation the mixture was dialyzed against distilled water (4 changes of 1 h/each).
50 ~l biotin-hydrazide (0.5 mg/ml in labelling solution) were added to the sample and the mixture was incubated for 1 h at room ~emperature.
50 ml of "stop solution" were added to the sample.
The mixture was dialyzed against distilled water (4 changes of 1 h/each) and kept at -20'C.

E;~mpl e ~ . ~
Control of the preparation of LPS-biotin M~teri~ls Sodium chloride ~NaCl), BDH cod. 10241.
(NaH2PO4*H2O): BDH, cod. 102454R.
Sodium hydroxide (NaOH): BDH, cod. 10252.
BSA, bovin albumin: Sigma A4503.
Tween 20 (polyoxyethylene(20)sorbitanmonolaurate: BDH
lot. ZA1645815.
Streptavidin: SPA ~Società prodotti antibiotici) SAS1-610.
STV-HRP: Sigma S 5512.
OPD tablet, 5 mg: Sigma P 6912.
H2SO4: BDH 10276-5G
H202: Carlo Erba A90201i404.
So~ llt; Orlfi - PBS: 0.15 M NaCl, 0.05 M Na-phosphate pH 7.3.
- PBS-3~ BSA
- PBS-0.5% BSA-0.05% Tween 20 - PBS-0.05% Tween - OPD: 2 tablets in 15 ml distilled water and 20 ml H202 .
1096 H2S~4-Method The preparation of LPS-biotin was incubated in PVC
microplate (Becton Dickinson cod. 3912) with 10 ~g/ml streptavidin in PBS, 100 ~l/well, 1 h at 37~C. The plate was washed three times with PBS, then it was blocked with PBS-3~ BSA, 200 ~l/well for 2h at room temperature.
Serial dilutions (1:2) o~ biotin-LPS in PBS-0.5~
BSA-0.05~ Tween were prepared. The plate was washed W O 97/31655 - PCT~EP97/00704 three times again with PBS.
100 ~l/well sample were added, incubating 1 h at 37-C.
The plate was washed three times with PBS. 100 ~l streptavidin-HRP 1:2000 in 0.5% PBS/BSA / 0.0~ Tween were added to each well. After 1 h incubation at 37'C, the plate was washed three times with 0.05~ P~S-Tween.
100 ~l of OPD solution were added to each well.
The reaction was blocked with 100 yl of 10~ H2SO4.
Reslllts Measured optical density Biotin-~PS (~g/ml) Optical density (4~2 nm3 0-3 1.150 0.1 1.107 15 0.03 0.986 .01 0.670 ' Rx~l e ~.3 Comparison of the i~ v vo tumorigenicity (in mouse) of RMA Thy 1.1 Cl.2 cells pre-treated with biotinylated antibodies, neutravidin and with LPS or LPS-biotin This experiment is aimed at demonstrating the reduced in v vo tumorigenicity of cells pre-treated with bio LPS bound to the cellular surface through the antibody-~iotin-neutravidin system ~artificial recep-tor), compared to cells pre-treated with LPS.
The employed model is based on the s.c.
administration of RMA Thy 1.1 Cl.2 cells pre-treated in vitro. The experiment was carried out using 3 groups of C57 BL6 female mice (5 mice each) and measuring the diameter of the tumoral mass at different days. Mice were treated with pre-treated cells as indicated in CA 02247308 1998-08-2~
W O 97/316~ PCT~EP97/00704 table 4.
Table 4 Experimental scheme for the treatment of the ~MA Thy 1 1 Cl.2 cells injected into mice.
______________ ~roup mice mAb bio-19E12 Neutrav. LPS bio-LPS
~#) (N.) (10 ~gJml~ (10 ~g/ml) (2 ~g/ml) (2 ~g/ml) 1 5 + +
2 5 + + +
3 5 + +
__________________ ______ ________ Procedllre The experimental procedure is identical to that reported in the example 1.6 except for the use of LPS or biotin-LPS respectively in place of TNF and biotin-TNF.
Reslllts The results are reported in table 5. As it can be noted, the treatment of the cells with bio-19E12/neutravidin/bio-LPS mAb (group 3) determined the reduction of the tumorigenicity of the RMA Thy 1.1 Cl.2 cells in mice, compared to the mice treated the same way but without bio-LPS (group 1) or with LPS (group 2).
The results indicate that the bio-LPS bound to the cellular membrane through an antibody-biotin/neutravidin "bridge" can trigger effects such that the tumorigenicity of the RMA Thy 1.1 C1.2 cells is reduced ;Ln v vo.
RMA Thy 1.1 non-treated fresh cells were injected into the other flank of the two mice survived after the thirtieth day to verify any immune responses triggered by the previous treatment. As it can be noted in tab~e W O 97/31655 PCT~EP97/00704 5, the two mice survived also after the second injection. Since the injected cells were not treated to the first injection, these results prove that the first treatment induced an immunologic memory.
Table 5 Per cent survival among the three groups of mice treated according to the scheme reported in table 4 after some days from the injection ________________________________________________________ Day after the first injections Group 10 18 19 Z0 21 22 25 30a) 40 45 60 1 100 60 0 0 0 0 0 0 0 0 o a) Second injection: non-treated fresh RMA Thy 1.1 cells were administered to the two survived mice.
Ri hl i oar~Dhv Aggarwal B.B. and Pocsik E. Cytokines from clone to Clinic. Arch. Biochem. Biophys. 292, 33S-359 (1992).
Beutler, B., and A. Cerami. (1989). The biology of cachectin/TNF - a primary mediator of the host response. Ann. Rev. Immunol. 7: 625.
Carswell, E.A., L.J. Old, R.L. Kassel, S. Greene, N.
~iore, and B. Williamson. 1975. An endotoxin-induced serum factor that causes necrosis of tumors. Proc. Natl.
Acad. Sci. U.S.A. 72:3666.
Corti A., Bagnasco L., Cassani C. (1994) Identification of an epitope of TNF receptor type 1 (p55) recognized by a TNF antagonist monoclonal antibody. Lymph. Cytokine Res. 13, 183-190.
Fiers W. (1991). TNF: Characterization at the molecular and cellular and i~ v vo levels. FEBS Lett. 285, 199-212.
~oogenboom, HR., Volkaert G., and Rausw C.M. (1991) Construction and expression of antibody-TNF fusion proteins. Molecular Immunology 28: 1027-1037.
Lienard, ~., Ewalenko, P., Delmotte J.J, Renar, N., ~ejeune, F. (1992) High-dose recombinant tumor necrosis ~actor in combination with interferon gamma and Melphalan in isolation perfusion of the limbs for melanoma and sarcoma. J. Clin Oncol. 10:52-60.
Loetscher H., Steueber D., Banner D., Mackay F., Lesslauer W. (1993) Human TNF alpha mutants with exclusive specificity for the 55 k~a or 75-kDa TNF
receptors. J. Biol. Chem. 26~3:26350-26357.
Rathien DA, Furphy L.J. and Aston R. ( 1992) Selective enhancement of the tumor necrosis activity of TNFalpha with monoclonal antibody Br. J. Cancer 39:266-273.
Paganelli G., P. Magnani, F. Zito, E. Villa, F. Sudati, L. Lopalco, C. Rossetti, M. Malcovati, F. Chiolerio, E.
Seccamani, A.G. Siccardi and F. Fazio. (1991). Three-step monoclonal antibodies tumor targeting in CEA-positive patients. Cancer Res. 51:5960-5966 Paganelli G., C. Belloni, P. Magnani, F. Zito, A.
Pasini, I. Sassi, M. Meroni, M. Mariani, M. Vignali, A.G. Siccardi and F. Fazio. ~1992). Two-step tumour targeting in ovarian cancer patients using biotinylated monoclonal antibodies and radioactive streptavidin. Eur.

J. Nucl. Med. 19:322-329.
Paganelli G., A.G. Siccardi, M. Malcovati, G.A.
Scassellati and F. Fazio. (1993). Biotinylated monoclonal antibodies: their potential for diagnosis and therapy of cancer. Curr. Op. Ther. Pat. 3: 1465-1474.
Sidhu R S. and A.P. Bollon (1993) Tumor Necrosis Factor activities and cancer theraphy- a perspective Pharmac.
Ther. 57, 79-128.
Spriggs ~R., Yates SW ~1992) Cancer chemotheraphy.
~xperiences with TNF administration in humans. In Tumor Necrosis Factor: The Molecules and their emerging roles in medicine, ed. B. Beutler Raven Press, Ltd, New York, pp 283-406.

Claims

1. Pharmaceutical compositions in form of combined preparations for sequential therapeutic use comprising:
a) an anti-pathologic target compound conjugated with a ligand of an at least ternary ligand/anti-ligand/ligand system;
b) an anti-ligand complementary to the ligand of the compound a);
c) a TNF conjugated at the amino-terminus with a ligand complementary to the anti-ligand b), with the proviso that the ligand/anti-ligand/ligand interaction is characterized by an affinity at least one order of magnitude higher than the affinity between the cytokine and its natural receptors.

2. Compositions according to claim 1 wherein biotin is the ligand and avidin, streptavidin or neutravidin are the anti-ligand.

3. Compositions according to claims 1 or 2, wherein both the compound a) and the TNF are conjugated with biotin and avidin, streptavidin or neutravidin are the component b).

4. Compositions according to any of the previous claims, wherein an antibody is the anti-pathologic target.

5. Compositions according to claim 4, wherein the antibody is a monoclonal antibody.

6. Composition according to claims 4 or 5, wherein the antibody is an antibody directed against tumoral antigens.

7. Tumoral necrosis factor conjugated with a ligand at the amino terminal.

8. Tumoral necrosis factor according to claim 7, wherein the ligand is conjugated with the .alpha.-amino group of the residue 1.

9. Tumoral necrosis factor according to claims 7 or 8, wherein biotin is the ligand.

10. Tumoral necrosis factor according to claims 7 or 8, wherein a protein or a fragment of biotinylated protein is the ligand.

11. Process for the preparation of the tumoral necrosis factor of claims 7-10 which comprise the reaction between the tumoral necrosis factor with biotin-6-aminocaproyl-N-hydroxysuccinimide at a pH between 5.5 and 7.5.

12. Biotinylated tumoral necrosis factor obtainable by mixing the subunits from the biotinylated and the non-biotinylated tumoral necrosis factor in a ratio 1:3 for 24-72 h at 4°C.