ITMI960358A1 - CYTOKINES MODIFIED FOR THERAPY USE - Google Patents

Abstract

The compounds of formula I: (FORMULA I) where Ra, Rb, B and R are as defined in the description, exhibit antagonistic properties of osteoclastic hyperreactivity.

Description

Description of the industrial invention entitled: "MODIFIED CYTOKINES FOR THE USE OF THERAPY"

The present invention relates to modified cytokines for use in therapy. The immune system produces cytokines and other humoral factors in response to various inflammatory stimuli, trauma, bacterial and viral infections, or signs of cellular degeneration, such as cancer. Although terms such as "lymphokines", "monokines" and "cytokines" were originally coined to distinguish products derived from lymphocytes, monocytes and non-lymphoid cells, it was later discovered that some overlap exists between these categories. Therefore, the term "cytokines" is currently used as a synonym of "lymphokines" and "monokines" and according to this meaning it is also used hereinafter. A list of most of the known state-of-the-art cytokines and their biological activities is reported in Aggarwal B.B and Pocsik E.

It is well known that various cytokines are endowed with anticancer, anti-viral and anti-bacterial activities. On the basis of these activities, observed both in vitro and in vivo in animal models, some cytokines have already been used in humans for therapeutic purposes (De Vita et. Al., 1995, in Biologie Theraphy of Cancer, Lippincott Company, Phyladelphia ). For example, some cytokines such as interleukin-2 (IL-2) and interferon a (IFNa) have shown good anticancer activity in patients with various types of fear, such as metastatic renal cell carcinoma, the "hairy celi "leukemia, Kaposi's sarcoma, melanoma, multiple myeloma, etc. Other cytokines such as IFNP, Tumor Necrosis Factor (TNF) a, TNF0, IL-1, -4, -6, -12, 15 and Colony Stimulating Factors (CFSs) have shown some anti-tumor activity on some types of cancer and are therefore the subject of further studies. Other cytokines have been used in the therapy of infectious diseases (Aggarwal B.B and Focsik E.)

In general, the therapeutic use of cytokines is severely limited by their systemic toxicity. Since this constitutes a crucial problem for their use in humans at therapeutically active doses, various attempts have been made to develop new cytokine derivatives and new therapeutic strategies aimed at reducing the toxic effects of this class of biological effectors while maintaining their therapeutic efficacy. .

An emblematic case is constituted by the Tumor Necrosis Factor a (TNF). TNF is a cytokine, secreted mainly by macrophages, originally discovered for its ability to induce hemorrhagic necrosis of some tumors (Carswell et al., 1975). Subsequently it was demonstrated that TNF, in addition to exerting cytotoxic and cytostatic effects on various tumor lines, is able to exert numerous other biological effects important for the regulation of the inflammatory and immune response (Beutler and Cerami, 1989; Fiers, 1991) .

The idea that TNF can exert beneficial or toxic effects for the organism that produces it is now well established, depending on its concentration, the production site and the time spent in the site of action. For example, chronic low-dose exposure to TNF can lead to cachexia while acute TNF overproduction can lead to severe vascular damage, shock and even death (Beutler and Cerami, 1989).

The potential anti-tumor activity of TNF has been evaluated through various studies conducted both on animal models and on humans. These studies have indicated that the anti-tumor activity exerted by TNF in vivo depends mainly on its ability to induce damage to the vascular system of the tumor through direct effects on the endothelium and, furthermore, on the activation of the inflammatory and immune response (Sidhu and Bollon, 1993). A lesser importance was instead attributed to its direct cytotoxicity on tumor cells.

Although clinical studies (phase II) conducted with TNF on different types of tumors did not indicate a significant anti-tumor activity, encouraging data were obtained using TNF in combination with other drugs (IFNgamma, IL-2, alkylating agents, Melphalan etc. .). However, in general it has been seen that toxic effects of various kinds, induced by TNF, constitute a strong limitation to the use of pharmacologically active doses (Spriggs and Yates, 1992). On the other hand, some success has been obtained through the use of high doses of TNF in regional therapies (e.g. by perfusion of the limbs of patients with melanoma) in order to reduce systemic toxic effects (Lienard et al., 1992).

Various alternative therapeutic strategies, always based on the use of INF, are currently being evaluated, with the aim of increasing the therapeutic efficacy of TNF through an increase in the maximum tolerated dose and a reduction of systemic toxic effects, without of course compromising anti-tumor activity.It has been estimated that a reduction of about an order of magnitude in the dose necessary to exert an anti-tumor effect could be well tolerated.

This was attempted through:

a) the development of fusion proteins that can carry TNF to the tumor and increase the local concentration. For example, fusion proteins between TNF and tumor specific antibodies have been produced (Hoogenboom et al., 1991);

b) the development of TNF mutants that maintain antitumor activity and have reduced systemic toxicity. In this sense, mutants able to selectively recognize only one receptor (p55 or p75) have already been produced (Loetscher H et al., 1993).

c) the use of anti-TNF antibodies able to reduce some toxic effects of TNF without compromising its anti-tumor activity. An antibody of this type has already been described in the literature (Rathien et al. 1992).

d) the use of TNF derivatives with a longer "half life" (for example TNF conjugated with polyethylene glycol).

The pharmacological potential of these strategies is being evaluated.

Although the data obtained are encouraging towards the use of TNF as an anti-tumor agent, the problem of its systemic toxicity has not actually been solved yet.

SCHIO have recently been described (EP 251494 and EP 496074) of the delivery systems of cytotoxic, paramagnetic or radioisotopic agents that exploit the biotin-avidin interaction.

In particular, EP 251494 describes a system for the administration of a diagnostic or therapeutic agent which comprises: an antibody conjugated to avidin or streptavidin, an agent capable of complexing the conjugated antibody and a compound consisting of the diagnostic or therapeutic agent conjugated to biotin, which are administered sequentially, suitably spaced, so as to allow the localization of the therapeutic or diagnostic agent, exploiting the biotin-streptavidin interaction, on the target cell recognized by the antibody.

The diagnostic or therapeutic agents described include metal chelates, in particular radionuclide chelates and low molecular weight anti-tumor agents such as cis-platinum, doxorubicin, etc. In fact, it is explicitly stated that the system is not suitable for compounds with molecular weight higher than 50,000 daltons (preferably not higher than 10,000 daltons) which effectively excludes the applicability to cytokines, such as TNF (51,000).

EP 496074 describes a method which provides for the sequential administration of a biotinylated antibody, avidin or streptavidin and a biotinylated diagnostic or therapeutic agent. Also in this case, while generally mentioning cytotoxic agents including ricòna (a protein whose cytotoxic chain has a molecular weight of about 30,000 daltons), the application to radiolabelled compounds is mostly described.

W0 95/15979 describes a method for localizing highly toxic agents (I) on cellular targets, based on the administration of a first conjugate (Cl) comprising the target-specific molecule conjugated to 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, while mentioning among the toxic agents (I) also cytokines, including TNF, and the biotin / avidin system as L / AL, it can be inferred that, in the case of the use of cytokines, the administration of the ligand is highly preferable. (L) or (AL) to dissociate the cytokine and thus allow the "free" cytokine to exert biological effects. This is intuitively due to the fact that the "bound" cytokine is unable to interact with its own receptors and effectively exercise desired biological effects. It is foreseeable that the quantity of L or AL required will be relatively high in order to compete in the binding on the cell surface, with consequent possible toxicity problems. Furthermore, in the case in which the L / AL pair consists of biotin / avidin, considering the high stability of this interaction (the strongest known non-covalent interaction, Kd = 10-15 M), this bond is practically indissociable (D. Savage et. Al., Avidin-biotin chemistry: a handbook. Pierce Biotec Company WO 95/15979 also does not report any specific experimental data supporting the use of cytokines in the claimed method, but only generic citations that are not sufficient to provide teachings reproducible and concrete for the application of the localization method to this class of substances which act moreover not simply through a cytotoxic mechanism, but through complex pro-inflammatory, immunostimulating, procoagulant and necrotizing mechanisms by means of which they are able to exert anticancer effects even without exerting direct cytotoxic effects, as instead required by the agents used in the aforementioned applications EP-251 494 and EP-496074.

It has now unexpectedly been found, contrary to the generic indications reported in WO 95/15979, that cytokines can be effectively localized in biologically active form only by resorting to a system in which the interaction between the conjugated cytokine and the target-specific conjugated component is not it is direct as in the case of conjugation to a ligand / anti-ligand pair, but is rather mediated by a third component capable of bridging the target-specific component and the cytokine.

According to the method of the present invention, the cytokine can also perform its action in bound form and it is not necessary to administer an element of the ligand / anti-ligand pair to release the cytokine in active form. This result is to be considered unexpected since it was not foreseeable that the cytokines conjugated to a ligand, although engaged in the interaction with the corresponding antiligand in turn engaged with the target-specific containing conjugated to a suitable ligand, would still be able to interact with the membrane receptors present on target cells.

The invention therefore provides pharmaceutical compositions in the form of associated preparations for sequential use in therapy which include:

a) a pathological anti-target compound conjugated to a ligand of an at least ternary ligand / anti-ligand / ligand system;

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 condition that the ligand / anti-ligand / ligand interaction is characterized by an affinity at least one order of magnitude higher than the affinity between cytokine and i its natural receptors.

Examples of compounds that can be conjugated to compound a) and to cytokine, according to the invention, include haptens such as biotin and digoxygenin, while examples of "anti-ligand" compounds include antiaptene antibodies (for example, anti-biotin antibodies and antidigoxigenin antibodies) or , in the case of the use of biotin as a ligand, avidin and its analogues (eg streptavidin, neutravidin).

Preferably, both the compound a) and the cytokine are conjugated to biotin, while the avidin (or analogous compound) is used as antiligand.

Conjugation techniques to cytokines and antibodies are widely known and can use chemical or genetic engineering methods.

The anti-pathological target compounds a) are preferably antibodies or monoclonal antibodies in whole or fragmented form. Said antibodies have already been extensively described and used, especially in the case of antibodies directed against tumor antigens.

Examples of cytokines or lymphokines usable according to the invention include tumor necrosis factors, interferons, interleukin, colony stimulating factors (CSF). It is also possible to use a biological response modifier capable of promoting the local release of endogenous cytokine, such as lipopolysaccharide (LPS) or its derivative, so as to obtain a greater local effect and less systemic effects.

Furthermore, since it is well known that cytokines can sometimes exert additive or synergistic effects, it is possible to exploit the strategy underlying the present invention to obtain local synergistic effects and lower systemic effects. Biological cancer therapy and the effects of combining different cytokines are well documented and well summarized by De Vita et al.

1995 (Biologie Theraphy of Cancer, Lippincott Company, Phyladelphia).

The use of TNF as a cytokine is particularly preferred. Preferably, the interactions of the conjugates with each other or with the artificial receptor will be characterized by affinity constants at least one order of magnitude higher than the affinity constant of the membrane receptors of the cytokines and by kinetic dissociation constants at least one order of magnitude lower than those of the interaction of the cytokine with its natural receptors.

The anti-target compound or conjugated antibody can be administered locally or injected into the bloodstream, and allowed to react in vivo with recognized antigens or cell structures until such time as the circulating excess of compound or antibody is removed from the bloodstream. organism, while a significant fraction remains in form bound to the pathological target At this point, it is possible to administer the anti-ligand b) followed by the conjugated cytokine at a concentration such that a bond can be formed with the antibody or anti target and allow an accumulation, or increased permanence of the cytokine on the target cells.

For this to happen it is necessary that the dissociation rods of the cytokine modified by the "artificial" receptor (constituted by the anti-ligand) are at least one order of magnitude longer than the dissociation rods from the "natural" receptors. As demonstrated in the application examples of the invention, this can be achieved with a system consisting of a biotinylated antibody specific for a tumor antigen, neutravidine, and biotin-TNF (bio-TNF). In this system the affinity between bio-TNF and the artificial receptor (avidin), equal to 10 M, and the affinity between bio-TNF and its natural receptors (TNF-R1 and TNF-R2), equal to 10 <- 9> -10- ^ K, are such as to favor an accumulation and an increased permanence of TNF in the site where the artificial receptors (tumor) are present.

In the preferred forms of the invention, a biotinylated monoclonal antibody specific for a tumor antigen is brought into contact with the tumor through systemic, intralesional or para-lesional administration, intracavitary instillations (e.g. bladder, peritoneal cavity), intra-arterial infusion ( liver, central nervous system), loco-regional vascular perfusions (e.g. in the perfusion fluid of the limbs, liver, lung), followed by analogous sequential administrations of neutravidin or streptavidin and TNF-biotin.

Between one administration and the other it is preferable to insert incubation times of the order of a few hours in order to allow the circulatory system to eliminate excess product and thus have a finer localization on the tumor target.

Furthermore, it is preferable to use biotinylated TNF in the amino-terminal region (residues 1-11, sequence VRSSSRTPSDK), so as not to prevent the multivalent binding with its natural receptors and therefore not to inactivate the effects of TNF. Alternatively, it is possible to insert in the same region, through genetic engineering techniques, amino acids containing groups that can be easily biotinylated by known techniques (lysines, cysteines, tyrosines, histidines, etc.) (see Savage et al., Avidin-biotin chemistry: a handbook , Pierce Biotec Company) or glycosylation signals, in order to obtain specific biotinylation on carbohydrate residues, for example with biotin hydrazide or with its derivatives. The biotinylation of TNF in the aminoterminal portion can also be easily obtained through the construction, by means of genetic engineering techniques, of TNF conjugates with proteins or protein fragments directly biotinylated by the expression system. An example of such proteins is E. coli vinegar 1-CoA carboxylase, and its C-terminal domain containing the biotinylation site.

In order to keep the structure of biotin-TNF as close as possible to that of non-biotinylated TNF, biotinylation at the level of the alpha-amino groups is preferable. This can be achieved through the use of biotinylation protocols based on the reaction of biotin-6-aminocaproyl-N-hydroxysuccinimide ester at pH between 5.5 and 7.5. Alternatively, it has been observed that it is possible to obtain biotinylated TNF in a form that maintains the ability to interact with avidin and membrane receptors by "mixing" subunits between biotinylated TNF, for example by one of the methods described above, and TNF not biotinylated. Preferably, the mixing reaction is obtained by incubating mixtures of biotinylated TNF and non-biotinylated TNF in the ratio 1: 3 for 24-72 hours, at 4 ° C. Such forms of TNF conjugated at the N-terminus are new and constitute a further aspect of the invention.

In order to facilitate the commercialization and routine use of the compositions of the invention, it is possible to prepare kits containing therapeutic grade materials. 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 neutravidin;

a vial containing 0.5 to 10 mg of biotinylated TNF (or other biotinylated cytokine).

The method of the present invention has various advantages over known methods, for example those based on the direct conjugation of cytokines to antibodies:

1) for example, it does not require the affinity of the antibody to be necessarily high, as it is possible to administer relatively high doses of antibody and avidin and still have an efficient labeling of the tumor, followed by relatively lower doses of biotin-TNF. However, this is not possible with conventional antibody-TNF conjugates, for which the antibody dose is invariably "linked" to the TNF dose.

2) The diffusibility of separate antibodies and TNFs in the tumor mass is presumably better than that obtainable with antibody-TNF conjugates, due to their lower molecular weight.

3) The labeling of tumors with high affinity molecules such as avidin and streptavidin (10-M) allows to select doses of biotin-TNF such as to thermodynamically and kinetically favor the interaction with the artificial receptor (avidin) and disfavour the interaction with natural receptors at the systemic level (10 ~ ^ _10-10 In the case of antibody-TNF conjugates, it is extremely difficult to obtain antibodies able to bind tumor antigen with an affinity of the order of M.

The present invention is further described in the following examples.

Although these examples refer in particular to the treatment of tumor pathologies, it is easy to understand that the same strategy can be applied to the treatment of any other pathology that is curable with a localized cytokine treatment.

In this example an application of the invention based on the system is demonstrated:

Pathological target: A: B 'c-cytokine

where is it:

Pathological target - murine lymphoma expressing 1 * murine Thy 1.1 antigen (RMA cells genetically engineered to express Thyl.l alial (RMA Thy 1.1 C1.2);

A = anti-Thy 1.1 monoclonal antibody, biotinylated (mAb bio-19E12). B = neutravidine

C-cytokine = biotin-TNF conjugate

and where

(:) represents a non-covalent interaction, while

(-) represents a covalent bond.

Materials

AH-ENHS (biotin 6-aminocaproyl-N-hydroxysuccinimide ester) (SPA, B002-61)

SULFO-NHS-LC-Biotin (Pierce, 21335)

TNF to human (1x108 U / mg) (DGR080)

Lysine (Sigma code L5501)

Culture medium: sterile RPMI-1640 (Gibco 31870-025)

Foetal Cali Serum (FCS) (PBI-Biological Industries, code 04-001-1A) Foetal Cali Serum (FCS) (Sigma, F2442)

Geneticin (G418, Sigma code G9516) in Hepes 100 mM

Hepes (Sigma, H0887)

Glutamine 200 mM (Gibco code 04305030D)

Penicillin 10,000 IU / ml, Streptomycin 10,000 mg / ml, Amphotericin B 25 pg / ml (Gibco code 15240-021)

NaCl (BDH code 10241)

HC1 37% (BDH code 10125)

96 Well FVC Plates for Flat Bottom Cell Culture (Costar 3595)

96-Well Round Bottom Plates (PBI, 650180)

2-mercaptoethanol (Merck code 12006)

Actinomycin D (Fluka 01815)

Thiazolyl blue (ΜΤΓ) (Merck 11714)

PBS (0.15 M NaCl, 0.05 M Na-phosphate, pH 7.3)

Neutravidine (Pierce code 31000)

Sodium azide (Baker, 9099)

mAb 19E12

Example 1.1

Preparation of biotinylated mAb 19E12 antibody (bio-19KL2)

1000 µl of a solution of mAb 19E12, 1 mg / ml in sodium bicarbonate pH 8.5, 34 µl of a solution of Sulfo-NHS-LC-Biotin, 1 mg / ml (molar ratio mAb / biotin: 1/24). The mixture was incubated at room temperature (23/24 * C) for 30 minutes. After incubation, the mixture was stirred against 2 1 of PBS overnight at 4'C and stored at 4 ° C.

Example 1.2

Preparation of TWF-biotin (bio-BIF)

This chapter shows some examples of biotin-TNF conjugate preparation protocols.

Example 1.2.1 (preparation of bio-UiF code fB)

60 µl of a TNF solution (0.5 mg / ml), 6 µl 1 M Na-carboriate, pH 6.8, 6 µl of AH-BNHS, 3 mg / ml was pipetted into an Eppendorf tube. ml in DMSO (TNF / Biotin molar ratio: 1/66). The mixture was incubated at room temperature (23/24 "C) for 3 hours and mixed with 7.5 µl of a 1 M lysine solution. After further incubation for 1 h at room temperature, 240 µl of RPMI solution, 10% FCS, 2 mM glutamine, 100 IU / ml penicillin, 100 pg / ml streptomycin, amphotericin B 250 ng / ml The mixture was then dialyzed against 21 of 0.9% NaCl overnight at 4 * C. (3 changes) and preserved -20 * C.

Example 1.2.2 (preparation of bio-TNF code # C, code fD, code # E, code fF, codeffG)

The bio-TNF product cod. #C is prepared as described in Example 1.1.1 except for the use of a conjugation buffer at pH 7.8.

By way of comparison, bio-TNF solutions are also prepared (code # D, code # E, code # F, code # G) using other incubation buffers, various TNF / biotin molar ratios (see table 1 ).

Example

Determination of the biological activity of biotinylated THF on RMA Thy 1.1 C 1.2 cells

The purpose of this experiment is to determine the specific biological activity of different bio-TNF preparations obtained under different conjugation conditions (see example 1.1), by means of a cytotoxicity test on RMA Thy 1.1 C1.2 cells.

Procedure:

To each well of a 96-well flat bottom plate (Costar 3595) were added:

a) 60,000 RMA Thy 1.1 Cl.2 cells (Mycoplasma free) in 50 μΐ of RFMI, FCS 5%, glutamine 2 mM, penicillin 100 IU / ml, streptomycin 100 yg / ml, amphotericin B 250 ng / ml 2-mercaptoethanol 50 nM, G418500 pg / ml (KFMI-Full);

b) 50 μΐ of standard or sample solution in RPMI-Complete at the desired concentration;

c) 10 μΐ of Actinomycin-D 330 ng / ml in RPMI-Conpleto.

The plate was then incubated for 24 h at 37 "C, 5% CO2. In addition, 10 μΐ of thìazolyl blue (MTT) solution, 5 mg / ml in PBS, was added to each well. After further incubation for 4 h at 37" C, 5% CO2, 100 μΐ of lysing solution (N, N-dimethylformamide 33% (v / v), sodium dodecyl sulfate 20% (w / v), in water, at pH 4.7 with acid was added to each well acetic acid). The solutions in the various wells were mixed with the aid of a multichannel pipette and incubated for 24 h at 37 "C. The absorbency at 570 nm and 650 nm (reference) of each well was then read by means of a multi-channel plates.

The cytotoxic activity was calculated by interpolating the absorbency values on a calibration curve obtained with non-biotinylated TNF.

Results

Table 1

Biological activity of bio-TNF, measured by cytotoxicity test on EMA cells

a) Measured using a calibration curve constructed using non-biotinylated TNF {code # A)

Comparison of the binding capacities and the binding and dissociation kinetics of bio-TNF and TNF from cells pre-treated with antibodies and neutravidine

The aim of the experiment is to demonstrate that the construction of artificial receptors on tumor callules by means of the pretargeting system with biotinylated antibodies and neutravidine significantly increases the total amount of bio-TNF that can be bound.

to cells with respect to the maximum amount binding to natural receptors.

Furthermore, the aim of this experiment is to evaluate the association and dissociation times of bio-TNF from natural and artificial receptors (neutravidine) from cells pre-treated with antibodies and neutravidine.

Procedure

The experiment was conducted by treating the MRA thy 1.1 C1.2 dog cells described in examples 1.5 and 1.6. Total bound TNF was detected using an indirect method based on the use of rabbit anti-TNF polyclonal antibodies and fluorescinated goat anti-rabbit IgG antibodies and analysis by FACS.

Kinetics of association

50,000 cells in 50 μΐ PBS / FCS 2% were seeded in wells of a round bottom plate, mixed with 1 μΐ of mAb bio-19E12.0.5 mg / mL in PBS / FCS 2% (final antibody concentration 10 pg / ml), and incubated for 10 min on ice.

Cells were washed twice by adding 200 μΐ / well of 2% PBS / FCS and centrifuging for 2 min at 1300 rpm. Cells were resuspended by vortexing and mixed COTI 50 μΐ / well of PBS / FCS 2% and with 1 μΐ neutravidine 2.5 mg / ml in PBS / FCS 2% (final concentration of neutravidine 50 mg / ml). After incubation for 10 min on ice, the cells were washed again twice with 2% PBS / FCS, as above.

50 μΐ of 2% PBS / FCS, 1 μl of TNF or bio-TNF at a concentration of 22.2 pg / ml (final concentration of TNF and bio-TNF 450 ng / ml) were then added to each well, and the specimens were incubated for 1 h on ice.

After a further washing with 2% PBS / FCS, 50 μΐ of anti-TNF rabbit polyclonal serum (Genzyme code # IP300) 1: 1000 in 2% PBS / FCS was added to each well and incubated for 10 min on ice. .

Cells were washed twice by addition of 2% PBS / FCS (200 μl / well) and centrifugation. Subsequently, 50 µl of fluorescein-conjugated goat anti-rabbit-FITC antiserum was added to each well in 2% PBS / FCS and incubated for 10 min on ice.

After a final washing with 2% PBS / FCS the samples were resuspended in 200 µl of 2% PBS / FCS and analyzed by FACS.

Dissociation kinetics

100,000 cells in 50 µl 2% PBS / FCS were seeded into 8 wells of a round bottom plate, mixed with 1 µl of bio-19E12 mAb, 0.5 mg / mL in PBS / FCS 2 * (final antibody concentration 10 mg / ml), and incubated for 10 min on ice.

Cells were washed twice by adding 200 μl / well of 2% PBS / FCS and centrifuging for 2 min at 1300 rpm. Cells were resuspended by vortexing and mixed with 50 μl / well of 2% PBS / FCS and 1 pi neutravidine 2.5 mg / ml in 2% PBS / FCS (final neutravidine concentration 50 pg / ml) . After incubation for 10 min on ice, the cells were washed again twice with 2% PBS / FCS, as above.

50 μΐ of 2% PBS / FCS, 1 μl of TNF or bio-TNF at a concentration of 22.2 pg / ml (final concentration of TNF and bio-TNF 450 ng / ml) were then added to each well, and the specimens were incubated for 1 h on ice.

The 8 samples were washed with 200 μΐ of 2% PBS / FCS by centrifugation (2 times). Cells were resuspended in 50 μΐ of RPMI, FCS 51, 2 mM glutamine, 100 IU / ml penicillin, 100 pg / ml streptomycin, 250 ng / ml amphotericin B, and at various times, fixed with 0.25% paraformaldehyde for 1 h to 4 ®C.

After an internal washing with 2% PBS / FCS, 50 μΐ of anti-TNF rabbit polyclonal serum (Genzyme code # IP300) 1: 1000 in 2% PBS / FCS was added to each well and incubated for 10 min on ice. .

Cells were washed again twice by addition of 2% PBS / FCS (200 μΐ / well) and centrifugation. Subsequently, 50 μΐ of fluorescein-conjugated goat anti-immunoglobulin rabbit antiserum (goat antirabbit-FITC) 1/1000 in 2% PBS / FCS was added to each well and incubated for 10 min on ice.

After a final wash with 2% PBS / FCS the samples were resuspended in 200 μΐ of 2% PBS / FCS and analyzed by FACS.

Results

Pre-targeting of cells with neutravidine results in an increase of at least 10-20 times in the binding of bio-TNF to cells, compared to the binding obtainable only with natural receptors (determined in the absence of neutravidine or using non-biotinylated TNF). The greatest increase was observed with bio-TNF code # B (biotinylated at pH 6.8) (data not shown).

Furthermore, as can be seen in figure 1 (lower panel) the residence time of the bio-TNF on RMA cells pretreated with antibodies and avidin is about 30 times higher (t ^ / 2 = 7 h) than the residence time of the TNF on the same cells (ti / 2 = 0.22 h).

In conclusion, the binding experiments show that by exploiting the pre-targeting strategy with antibodies and neutravidine according to the object of the invention it is possible to increase the maximum quantity of bio-TNF bound to tumor cells by about 10-20 times and that this quantity it is able to remain on the cell membrane for about 30 times longer.

1-5

Comparison between the cytotoxicity of TNF and bio-TNF on tunoral cells pretreated with biotinylated antibodies and neutravidine, in vitro, in the presence of actinomycin D

The purpose of this example is to demonstrate the ability of a biotinylated / avidin antibody system to deliver TNF-biotin on the cell surface in a biologically active form and to increase cytotoxicity in vitro, compared to that obtainable simply by exploiting natural receptors.

For this purpose, RMA Thy 1.1 CI.2 cells, the monoclonal anti-human Thy 1 antibody, biotinylated (mAb bio-19E12), neutravidine, and bio-TNF cod # B were used.

The experiment was carried out by sequential incubation and cell washing with a) bio-19E12 mAb, b) neutravidine, c) bio-TNF.

Since it is known that the binding of TNF to natural receptors induces the synthesis of protective factors, such as Mn superoxide dismutase, in order to inhibit the production of these factors and enhance the cytotoxic activity of TNF, the cytotoxicity test was performed in the presence of a transcription inhibitor (actinomycin D).

Procedure:

Each well of a 96-well round bottom plate (Costar 3595) was added 1,000,000 cells in 50 μΐ PBS / FCS 2% and 1 μl of a bio-19E12,0.5 mg / mL mAb solution . The suspension was incubated for 10 min on ice. Cells were washed twice by adding 200 μΐ / well of 2% PBS / FCS and centrifuging for 2 min at 1300 rpm. Cells were resuspended by vortexing and mixed with 50 μ / well of 2% PBS / FCS and 1 μΐ 0.5 mg / ml neutravidine in 2% PBS / FCS. After incubation for 10 min on ice , the cells were again washed twice with 2% PBS / FCS, cxxn and above.

50 μΐ of 2% PBS / FCS, 1 μΐ of TNF or bio-TNF at the desired concentration were then added to each well and incubated for 15 min on ice. After a further wash with 2% PBS / FCS, the cells of each well were resuspended in 1.5 ml of RFMI, 5% FCS, 2 mM glutamine, 100 IU / ml penicillin, 100 pg / ml streptomycin, 250 amphotericin B ng / mL, 2-mercaptoethanol 50 nM, G418500 pg / mL (full RFMI).

The cells were then seeded into 96-well flat-bottom plates (60,000 cells / 100 μl / well), mixed with 10 μΐ / well of an actinomycin D solution, 330 ng / ml in RPMI-complete, and incubated for 24 h at 37'C, 5% C02.

Cell viability was determined as described in Example 1.3.

Results

The results of the experiment conducted in the presence of actinomycin D are shown in fig. 2. As can be seen, while the dose of TNF or bio-TNF required to kill 50% of the cells (LD50) (which manifests itself with a 50% decrease in absorbance) is higher than 100,000 U / ml, in the absence of neutravidine, the LD50 of bio-TNF in the presence of neutravidine is about 2,000-3,000 U / ml. Furthermore, while the cytotoxicity of TNF is not influenced by the presence or absence of neutravidine, the cytotoxicity of bio-TNF is, as expected, strongly dependent on the presence of neutravidine. This indicates that: a) TNF and bio-TNF are weakly cytotoxic to BMA cells, in the absence of neutravidine, b) the presence of "artificial" neutravidine receptors on the membrane of RMA cells strongly increases the cytotoxicity of bio-TNF, c) bio-TNF bound to the membrane through a bridge of antibody-biotin-avidin is able to interact with its natural receptors and trigger cytotoxic effects.

In an experiment conducted in the absence of actinomycin D. both TNF and bio-TNF were found to be devoid of cytotoxic activity (results not shown).

Example 16

Comparison between tumorigenicity in vivo (in mice) of RMA thy 1.1 cl.2 cells pretreated with biotinylated antibodies, neutravidine and with TNF or TNF-biotin, in the absence of Actinomycin D

The aim of this experiment is to demonstrate a reduced tumorigenicity in vivo of cells pretreated with bio-TNF bound to the cell surface through the antibody-biotinaneutravidine system (artificial receptor), compared to cells pretreated with TNF or with bio-TNF bound exclusively to the receptors. natural.

The model used is based on the subcutaneous administration of RMA Thyl.l Cl.2 cells pre-treated in vitro. The experiment was conducted using 4 groups of female C57 BL6 mice (5 mice each) and measuring the diameter of the tumor mass over several days. Mice were treated with pretreated dog cells shown in Table 2.

It is important to remember that the doses of TNF and bio-TNF used in this study (50,000 U / ml and 10,000 U / ml, respectively) are such as to determine partial cytotoxic effects only in the presence of actinomycin D, as demonstrated in example 1.5 (figure 2). In the present experiment the cells were treated entirely in the absence of actinomycin D, that is, in conditions in which both TNF and bio-TNF are free of cytotoxicity.

Table 2

Experimental scheme of the treatment of RMA Thyl.l C1.2 cells inoculated in tapi

Bio-19E12Neutrav mAb mouse group. TNF bio-TNF (#) (N.) (10 pg / ml) (10 pg / ml) (50000 U / ml) (10000 U / ml) 1 5

2 5

3 5

4 5

Procedure:

180,000 cells in 50 μΐ PBS / 2% FCS were seeded into four wells of a round bottom plate, mixed with 1 μΐ of bio-19E12 mAb, 0.5 mg / mL in 2% PBS / FCS, and incubated for 10 min in ice.

Cells were washed twice by adding 200 μΐ / well of 2% PBS / FCS and centrifuging for 2 min at 1300 rpm. Cells were resuspended by vortexing and mixed with 50 μ / well of 2% PBS / FCS and 1 μΐ neutravidine 0.5 mg / mL in 2% PBS / FCS. After incubation for 10 min on ice, the cells were washed again twice with 2% PBS / FCS, as above.

50 μΐ of 2% PBS / FCS, 1 μΐ of TNF or bio-TNF at concentrations of 50,000 and 10,000 U / ml, respectively, were then added to each well and incubated for 15 min. in ice

After a further washing with 2% PBS / FCS, the cells from each well were resuspended in 200 μΐ of PBS, transferred to Eppendorf tubes, and mixed with 1.2 ml of PBS. Using insulin syringes, 30,000 cells / 200 μΐ / mouse were injected subcutaneously in the groin. The mean diameter (lateral and longitudinal) of the tumor mass was determined at various times by using a caliper.

The results obtained are shown in table 3. As can be seen, the treatment of cells with mAb bio-19E12 / neutravidine / bio-TNF (group 4) caused a reduction in the tumorigenicity of KMA Thyl.l Cl.2 cells, in mice , compared to mice treated in the same way but without bio-TNF (group 2). Unlike the pre-treatment of cells with bio-19E12 mAb / neutravidine / TNF (group 3) did not produce a significant reduction in tumorigenicity compared to the control group (group 2). In a further experiment conducted with 50,000 U / ml of bio-TNF, instead of 10,000, the volume of the tumor mass after 17 days was below the detection limit (15 mnP) (data not shown).

These results indicate that bio-TNF bound to the cell membrane by means of an antibody-biotin / neutravidine "bridge" is able to trigger effector functions such as to reduce the tumorigenicity of KMA thy 1.1 C1.2 cells in vivo.

Furthermore, it is interesting to observe that although the dose of bio-TNF employed in the treatment of RMA cells was such as to cause cell lysis, in the presence of actinomocin D (see example 1.5), and although the treatment of cells in this experiment was carried out in absence of actinomycin D, ie in conditions such as TNF is not able to exert direct cytotoxic effects, this treatment has in any case determined a> 5-fold reduction in the volume of the tumor mass after 17-19 days from inoculation. This suggests that the observed reduced tumorigenicity is due to indirect host effects on tumor cells (inflammatory, immune effects, etc.)

Table 3

Diameter and volume of the tumor masses in the four groups of mice treated according to the scheme shown in table 2 several days after inoculation

_ <* 1> n <»>

a) starvation of longitudinal and lateral diameters

Example 2

In this example an application of the invention based on the system is demonstrated:

Pathological target: Δ ■ B c-initiator of the release of torcine where:

Pathological target = murine lymphoma expressing the human Thy 1.1 antigen (KMA Thyl.l C1.2 cells);

A = human anti-Thy 1 monoclonal antibody, biotinylated (mAb bio-19812}. B = neutravidine

C-inducer of cytokine release = biotin-lipopolysaccharide conjugate (LPS)

and where

(:) represents a non-covalent interaction, while

(-) represents a covalent bond.

Example 2.1

Preparation of LPS-biotin

Materials

Lipopolysaccharide from Salmonella minesota (LPS): Sigma code L2137 lot. 101H4029

Biotinamidocaproylhydrazide: Sigma B-3770

Tris base: BDH code 103156X

Sodium-M-periodate: Sigma S-1878

Sodium-acetate: BDH code 10235

Sodium-azide: J.T.Baker code 9099

gollicervi

- "Stop solution": 0.1 M Tris-HCl pH 7.5 (in water).

- "Labeling solution": 100 mM Na-acetate, 0.02% Na-azide, pH 5.5.

Method

100 microliters LPS (0.8 mg / ml in labeling solution) were mixed with 50 microliters of Na-H-periodate (30 mM in labeling solution). The mixture was incubated at room temperature in the dark for 30 minutes. After incubation, the mixture was dialyzed against distilled water (4 candii of 1 h each).

50 microliters of biotin-hydrazide (0.5 mg / ml in labeling solution) were added to the sample and the mixture was incubated for 1 hour at room temperature.

50 ml of "stop solution" was added to the sample. The mixture was dialyzed against distilled water (4 changes of 1 b each) and stored at -20 ° C.

Example 2.2

control of the preparation of LPS-hiotin

Materials

Sodium clonyrus (NaCl), BDH code 10241.

(NaH2F04 * H20): BDH, code 102454R.

Sodium hydroxide (NaOH): BDH, code 10252.

BSA, albumin, bovine: Sigma A4503.

Tween 20 (polyoxyethylene (20) sorbitan monolaurate: BEH lot.ZA1645815.

Streptavidin: SPA (Antibiotic Products Company) SAS1-610.

STV-HHP: Sigma S 5512.

OPD tablets, 5 mg: Sigma P 6912.

H2S04: BDH 10276-5G.

H ^: Carlo Erba A902011404.

Solutions

- PBS: 0.15 M NaCl, 0.05 M Na-phosphate pH 7.3.

- PBS-3% BSA

- PBS-0.5% BSA-0.05% Tween

- PBS-0.05% Tween

- OPD: 2 tablets in 15 ml of distilled water and 20 ml of Η2θ2 ·

- ios H2SO4

Method

The LPS-biotin preparation was incubated in a PVC microplate (Becton Dickinson code 3912) with 10 micrograms / ml streptavidin in PBS, 100 microliters / well; 1 has 37'C. The plate was washed three times with PBS, then blocked with PBS-3% BSA, 200 μl / well for 2h at room temperature.

Serial dilutions (1: 2) of biotin-LPS in PBS-0.5% BSA-0.05% Tween were prepared. The plate was washed again three times with PBS. 100 microliters / well of sample were added, incubating 1 h at 37'C.

The plate was washed three times with PBS. To each well was added 100 microliters of Streptavidin-HKP 1: 2000 in PBS / BSA 0.5% / Tween 0.05%. After incubation for 1 h at 37 ° C, the plate was washed three times with 0.05% PBS-Tween. 100 microliters of OPD solution was added to each well.

The reaction was stopped with 100 microliters of 10% H2SO4.

Results

Optical densities measured

Biotin-LPS (ndcrogranmi / inl) Optical Density (492 in)

0.3 1.150

0.1 1.107

0.03 0.986

0.01 0.670

Example 2.3

Comparison between the in vivo tunorigenicity (in mice) of RMA t ± y 1.1 cl.2 cells pretreated with biotinylated antibodies, neutravidine and with LPS or LPS-biotin

The aim of this experiment is to demonstrate reduced in vivo tunorigenicity of cells pretreated with bio-LPS bound to the cell surface via the antiobody-biotinaneutravidine system (artificial receptor), compared to cells pretreated with LPS.

The model used is based on the subcutaneous administration of RMA Thyl.l Cl.2 cells pre-treated in vitro. The experiment was conducted using 3 groups of female C57 BL6 mice (5 mice each) and measuring the diameter of the tumor mass over several days. Mice were treated with pretreated cells as indicated in Table 4.

Table 4

Experimental scheme of the treatment of RMA Thy l.l C1.2 cells inoculated in mice.

Bio-19E12 Neutrav mAb mouse group. LPS bio-LPS (#) (n.) (10 pg / ml) (10 pg / ml) (2 yg / ml) (2 pg / ml)

The procedure of the experiment is identical to that reported in Example 1.6 except for the use of LPS or biotin-LPS and instead of TNF and biotin-TNF, respectively.

Results

The results obtained are shown in table 5. As can be seen, the treatment of cells with mAb bio-19E12 / neutravidine / bio-LPS (group 3) resulted in a reduction in the tumorigenicity of RMA Thyl.l C1.2 cells in mice , compared to mice treated in the same way but without bio-LPS (group 1) or with LPS (group 2).

These results indicate that the bio-LPS bound to the cell membrane by an antibody-biotin / neutravidine "bridge" is able to trigger effector functions that reduce the tumorigenicity of RMA thy 1.1 Cl.2 cells in vivo.

The two mice that survived the thirtieth day from inoculation (see Table 5) were inoculated with fresh, untreated collide RMA thy 1.1 in the other flank, in order to verify a possible immune response triggered by the previous treatment. As can be seen

in table 5 the two mice also survived the second inoculum. Since the inoculated cells had not been treated since the first injection, these results indicate that the first treatment induced an innunitary "memory".

Table 5

Survival rate in the three groups of mice treated according to the scheme shown in table 4 at several days after inoculation

Days elapsed since first inoculation

a) Second inoculation: the two surviving mice were re-administered fresh untreated RMA Thy 1.1 cells.

Relevant bibliography

Aggarwal B.B. and Pocsik E. Cytokines from clone to Clinic. Arch. Biochem.Biophys. 292, 335-359 (1992).

Beutler, B., and A. Cerami. (1989). The biology of caehectin / TNF - a primary mediator of the host responso.Ann.Rev. Immurici. 7: 625.

Carswell, E.A., L.J. Old, R.L. Kassel, S. Greene, N. Fiore, and B. Williamson. 1975.An endotoxin-induced serum factor that causes necrosis of tumors.Proc.Nati.Acad.Sci.U.S.A.72: 3666.

Corti A., Bagnasco L., Cassarti C. (1994) Identification of an epitcpe 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 in vivo levels.FEBS Lett. 285,199-212.

Hoogenboom, HR., Volkaert G., and Rausw C.M. (1991) Construction and expression of antibody-TNF fusion proteins. Molecular Immunology 28: 1027-1037.

Lienard, D., Ewalenko, P., Delmotte J.J, Renar, N., Lejeune, P. (1992) High-dose recombinant tumor necrosis factor in contoination with interferon gamma and Melphalan in isolation perfusion of the lini "for melanoma and sarcoma .J.Clin Oncol. 10: 52-60.

Loetscher H., Steueber D., Banner D., Mackay F., Lesslauer H. (1993) Human TNF alpha mutant with exclusive specificity for the 55 kDa or 75-kDa TNF receptors.J.Biol.Chem.268: 26350- 26357.

Rathien DA, Furphy L.J. and Aston R. (1992) Selective enhancement of the tunor necrosis activity of TNFalpha with monoclonal antihody 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. Scissi, M. Meroni, M. Mariani, M.Vignali, A.G.Siccardi and F. Fazio. (1992). Twostep tumor 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. Scasse1lati and F. Fazio. (1993). Biotinylated monoclonal antibodies: thèir potential for diagnosis and therapy of cancer. Curr.0p.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 DR., Yates SW (1992) Cancer chemotheraphy. Bxperiences with TNF administration in humans. In Turaor Necrosis Factor: The Molecules and their emerging roles in medicine, ed. B. Beutler Raven Press, Ltd, New York, ρρ 283-406

Claims (15)

1. Pharmaceutical compositions in the form of associated preparations for sequential use in therapy which include: a) a pathological anti-target compound conjugated to a ligand of an at least ternary ligand / anti-ligand / ligand system; b) an anti-ligand complementing the ligand of compound a); c) a cytokine conjugated to a ligand complementary to the anti-ligand b), with the condition that the ligand / anti-ligand / ligand interaction is characterized by an affinity at least one order of magnitude higher than the affinity between cytokine and its receptors natural. 2. Compositions according to claim 1, wherein the ligand or biotin and the anti-ligand are avidin, streptavidin or neutravidin. 3. Compositions according to claim 1 or 2, wherein the compound a) and healthy cytokine both conjugated to biotin and the constituent b) is avidin, streptavidin or neutravidin. 4. Compositions according to any one of the preceding claims, wherein the pathological anti-target compound is an antibody. 5. Compositions according to claim 4, wherein the antibody is monoclonal. 6. Compositions according to claim 4 or 5, wherein the antibody is an antibody directed towards tumor antigens. 7. Compositions according to any one of the preceding claims, wherein the cytokine is the tumor necrosis factor (TNF). 8. Compositions according to claim 7, wherein the tumor necrosis factor is biotinylated. 9. Compositions according to claim 8, wherein the tumor necrosis factor is biotinylated in the amino-terminal region. 10. Tumor necrosis factor conjugated to a ligand in the amino-terminal region. 11. Tumor necrosis factor according to claim 10, wherein the ligand is conjugated to the a-amino group of residue 1. Tumor necrosis factor according to claim 10 or 11, wherein the ligand is biotin. 13. Tumor necrosis factor according to claim 10 or 11, wherein the ligand is a biotinylated protein or protein fragment. 14. Process for the preparation of the tumor necrosis factor of claims 10-13 which comprises the reaction of the tumor necrosis factor with biotin-6-aminocaproyl-N-hydroxysuccinimide at pH between 5.5 and 7.5. 15. Biotinylated tumor necrosis factor obtainable by mixing subunits between biotinylated tumor necrosis factor and non biotinylated tumor necrosis factor in a ratio of 1: 3 for 24-72 hours at 4 <*> C.