WO1996004305A1

WO1996004305A1 - Hybrid molecules useful in the treatment of tumors, their preparation and use in pharmaceutical compositions

Info

Publication number: WO1996004305A1
Application number: PCT/EP1995/003060
Authority: WO
Inventors: Antonio Mele; Luigi Rotondaro; Mario Di Loreto; Laura D'alatri
Original assignee: Menarini Ricerche S.P.A.
Priority date: 1994-08-04
Filing date: 1995-08-01
Publication date: 1996-02-15
Also published as: IT1271688B; ITMI941694A0; ITMI941694A1; AU3255295A

Abstract

The present invention relates to hybrid molecules useful in the treatment of tumors, comprising the human G-CSF molecule linked to a monoclonal antibody or fragments thereof or to a ligand which recognizes a specific antigen; it is also described the use of such hybrid molecules to stimulate in vivo an immune response against tumor cells expressing the target antigen.

Description

HYBRID MOLECULES USEFUL IN THE TREATMENT OF TUMORS. THEIR PREPARATION

AND USE IN PHARMACEUTICAL COMPOSTUONS

Field of the invention

The present invention relates to hybrid molecules useful in the treatment of tumors and in particular to hybrid molecules comprising the human G-

CSF molecule linked to a monoclonal antibody or fragments thereof or to a ligand which recognizes a specific antigen.

State of the art

An increasing number of experimental evidences show that the release of low doses of some cytokines, directly at the site of tumor growth, leads:

1) to the activation of antineoplastic reactions, which depend on the host; and 2) to tumor suppression.

The local release of cytokines was obtained: 1) by injection of cytokines into the tumor site; or 2) by transfer of cytokines genes into tumor cells.

In the first case, in vivo studies in mice showed that peritumoral or perilymphatic injections of different cytokines as IL-lβ, IL-2,

gamma-interferon (gamma-IFN) and IL-7 can activate a strong antineoplastic immune response (Forni G. et al., 1993. J- Immunother., :4. 253-257).

A number of complete or partial clinical remissions after regional treatment of localized tumors with tumor necrosis factor-α (TNF-α) , IL-2, IFN-α, -β or -gamma, were also described as clinical experimentation. In the second case, it was observed that the transfer and the expression, in urine tumor cells, of genes encoding several cytokines as IL-2, IL-4, IL-7. gamma-IFN. TFN-α, granulocyte colony stimulating factor (G-CSF) bring to the rejection of the engineered tumor cells in syngenic animals (Colombo M.P. et al., 1992, Cancer Res.. 52, 48 3- 87). According to these studies, it was suggested to transfer cytokine genes into tumor cells of oncological patients in order to stimulate an antitumor immune response (Russel S.J., 1990, Immunol. Today, 11, 196- 200).

At present such a therapy, even though attractive, does not seem avalable for humans. In fact, the data published up to now were obtained using different kinds of tumors engineered to release different doses of cytokines and these data are not always consistent or seem to be in conflict. It is known that the same cytokine - which in an experimental model can be able to activate antitumor immune responses, when it is made available at the site of tumor growth by gene transfer or by peritumoral injections - can also be produced spontaneously by tumor cells without detectable antitumor effect.

Moreover, many cell lines depending on growth factors, when transfected with the proper cDNA, acquire the capacity of autocrine growth and are tumorigenic in vivo in experimental animals. These results show that cytokines can not be used indiscriminately in the anti-tumoral therapy (Blankenstein T. , et al., 1991. Curr. Opinion Immunol.. 3, 694-698). Alternatively to the above cited two methods of local release of cytokines, two patent applications (EP 0439095 A2 and W0 92/08495) propose to carry the cytokines of interest (including IL-2, TNF-α, TNF-fi and GM-CSF) to the target tissue by using the binding specificity of immunoconjugates obtained according to recombinant techniques and consisting of antibodies linked to cytokines. Such molecules, taking advantage of the binding specificity of antibodies and of the biological activity of cytokines. can produce, according to the authors of the above cited patent applications, a localized amplification of the immune response, enabling the in vivo selective destruction of tumor cells (or infected cells).

Recently, Gillies S.D. et al.(1993, Bioconjugate Chem. , 4, 230-235) described pharmacokinetic properties of immunoconjugates, obtained by recombinant techniques, which use IL-2, TNF-α. TNF-β or GM-CSF linked to an antibody specific for an antigen associated to tumors. The aforesaid molecular hybrids use cytokines showing a well-known antitumor activity, acting either directly on the tumor cells. TNF-α and TNF-β, or indirectly by means of mechanisms mediated by host, IL-2; the possible antitumor role of GM-CSF is suggested by the activity of this cytokine on the maturation and/or the functions of specialized cells presenting the antigen.

The idea of using immunoconjugates in order to specifically carry cytokines to the target cells and stimulate an immune response is based on an apparently usual aspect of reactions activated by cytokines which is known to the skilled men of the art (Forni G. et al., 1993. J- Immunother., 14, 253-257).

In fact, it has been observed that the characteristics of the reaction activated by cytokines are not affected by the way in which a useful local concentration of cytokine is obtained. Similar mechanisms of reactions are activated either if the cytokine is repeatedly injected at the tumor site or if it is secreted by cells engineered by the cytokine gene, as it was observed with IL-2, IFN-gamma and IL-4. As it is known, at present, the antitumor reaction activated by G-CSF does not seem to follow this general behaviour, but shows peculiar features. In fact, although the engineering of tumor cells with a vector for G-CSF can inhibit the tumor growth in vivo , it is known that such antitumor activity is not activated when G-CSF is injected peritumorally, also if at high doses (Colombo M.P. et al.,1991. J- Exp. Med.. 173, 889-897): this behaviour is in compliance with the clinical tests on G-CSF which, contrary to most of the cytokines with activity of immune stimulation, did not show antitumor activity of G-CSF in treated oncological patients. Moreover, in confirmation of the present small comprehension of the action of cytokines i n vivo , the above cited authors note that the transfer of the vector for G-CSF in a different cell line, a murine fibrosarcoma, does not suppress the tumorigenicity in vivo (Colombo M.P. and Parmiani G. , 1991. Immunol. Today, 12, 249-250). It will be understood that, at the time the invention was realized, it was not possible to suppose or foresee that hybrid molecules, based on the use of G-CSF, could be used to carry this cytokine to the tumor growth site, activating an antitumor response. Su-nmary of the invention The authors of the present invention have surprisingly found that the new hybrid molecules consisting of the G-CSF human molecule linked to a monoclonal antibody or fragments thereof or to a ligand which recognize a specific antigen are able to stimulate in vivo an immune response, against human tumor cells expressing the target antigen, which is stronger than the immune response obtainable with the mixture comprising the monoclonal antibody or ligand and G-CSF. The mixture comprising the antibody and G-CSF shows an activity higher than the mixture comprising the ligand and G-CSF according to the fact that the antibody per se has an antitumor activity which the ligand has not .

More particularly , the present invention relates to hybrid molecules consisting of the human G-CSF molecule linked to a murine monoclonal antibody or fragments thereof or to a ligand specific for a cell surface receptor. Preferably, said cell surface receptor is the human Epidermal Growth Factor Receptor (EGF-R) .

Moreover , the present invention relates to the process for the preparation of said hybrid molecules by chemical means or by means of recombinant DNA techniques , and to their use in pharmaceutical compositions for the treatment of tumors and to said pharmaceutical compositions comprising said hybrid molecules in combination with a pharmaceutically acceptable carrier and/or excipient . The word immunocytokines as herein used indicates hybrid molecules consisting of monoclonal antibodies or fragments thereof , able to bind the specific antigen, linked to G-CSF.

Brief description of the drawings

Figure 1 shows a SDS-PAGE electrophoresis , under reducing conditions ( PhastSystem , Pharmacia ; PhastGel 4-15$ Gradient Pharmacia LKB ) of fractions comprising immunocy tokine , obtained by gel filtration on a G3000 SW XL TSK-gel (Tosohaas) column. The proteins were visualized by silver/staining .

Figure 2 shows a SDS-PAGE electrophoresis performed according to Figure 1 , with the exception that non-reducing conditions were used . Detailed description of the invention Hybrid molecules according to the present invention are prepared by chemical means or according to recombinant techniques . Such hybrid molecules were prepared, by chemical means, as described by Thorpe P.E. et al., 1981. in Eur. J. Biochem., 116, 447-454. The chemical linkage between the antibody and recombinant human G-CSF (rhG-CSF) was obtained using N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), a heterobifunctional reagent. This reagent is used in order to introduce 2- pyridyl disulfide groups into both the antibody and rhG-CSF; then the two molecules were conjugated by means of the formation of a disulfide bridge.

Thanks to this method it is also possible to prepare immunocytokines of smaller molecular sizes wherein only antibody fragments able to bind the specific antigen [Fab'; F(ab)2_l are used. Such immunocytokines show advantageous uses deriving from: i) a favourable phaπnacokinetics; ii) a reduced immunogenicity; iii) an increased capacity to penetrate tissues and to reach solid tumor masses. In order to increase the yields and stabililty in vivo of the hybrid molecules it was also considered the possibility to prepare molecules according to the present invention by means of recombinant techniques. Many groups of scientists produced fusion proteins wherein the sequences encoding the whole antibody, or fragments thereof able to bind the antigen, were linked to genes encoding the proteins of interest. Some examples of fusion proteins containing the human G-CSF were described (Chadwick D.E. et al., 1993, Leuk. Lymphoma, 11, 249-262). The G-CSF used in the present invention is preferably the recombinant human G-CSF (rhG-CSF). Such rhG-CSF is produced by means of a CHO cell line engineered as described in the patent application PCT/EP94/03488. The monoclonal antibody used in the present invention is an antibody which recognizes a cell surface receptor. Preferable is the cell surface receptor EGF-R, and more in particular the antibody Mint5- Mint5 is a murine monoclonal antibody (MoAb) specific for the human Epidermal Growth Factor Receptor (EGF-R) and it is described in the patent application PCT/EP94/02969-

The hybridoma producing the Mint5 was filed at the DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen Gmbh) with the access number DSM ACC2150.

With reference to the antibody fragments used in the present invention, the Fab' and F(ab) of the antibody Mint5 are preferred. The ligands used in the hybrid molecule, according to a characteristic of the invention, are any fragments having the capacity to bind a cell surface receptor, preferably EGF-R.

Some specific embodiments according to the present invention are hereinafter described in the following examples. Example 1 Conjugation of the MoAb Mint5 to the G-CSF

The immunocytokine was obtained by chemical reaction between the MoAb Mint5 and the recombinant human G-CSF (rh-G-CSF), both derivatized with N-succinimidyl-3-(2-pyridyldithio)-proprionate (SPDP) as described by Thorpe P. et al., 1981, Eur. J. Biochem., 116, 447-454. Derivatization of the recombinant human G-CSF

21 μl of a 20 mM solution of SPDP in ethanol were added to a solution of 1.6 mg of rhG-CSF in 3 ml of 50 mM, pH 9-0, sodium borate buffer; NaCl 1-7% (weight/volume) ; n-butanol 0.5% (volume/volume) . After 30 minutes of incubation at room temperature, the sample was centrifuged to remove the particulate matter and applied on a column of Sephadex G-25 pre- equilibrated with 0.1 M, pH 7-5. sodium phosphate buffer; 0.1 M NaCl; 1 mM EDTA; the derivatized protein was eluted from the column using the same buffer.

The derivatization ratio was calculated on an aliquot of the eluted material, according to the value of optical density at 343 nm and 280 nm before and after reduction with final 2 mM of dithiothreitol (DDT) . Derivatization of MoAb Mint5

5 μl of a 20 mM solution of SPDP in ethanol were added to 2.44 mg of MoAb Mint5 in 2 ml of 20 mM, pH 7-8, sodium phosphate buffer; NaCl 150 mM. After 30 minutes of incubation at room temperature, the sample was centrifuged, applied on a column of Sephadex G-25 pre-equilibrated with 0.1 M, ph 4.5, sodium acetate buffer; 0.1 M NaCl; 1 mM EDTA; and eluted using the same buffer. The derivatization ratio was calculated on an aliquot of the eluted material, according to the value of optical density at 343 nm and 280 nm before and after reduction with final 25 mM of dithiothreitol (DDT). Conjugation

The sample of derivatized antibody, eluted from the column, was reduced using final 25 mM DDT. After 30 minutes of incubation, at room temperature, the sample was centrifuged and charged on a column of Sephadex G-25 equilibrated with 0.1 M, pH 7-5. sodium phospate buffer; 0.1 M NaCl; 1 mM EDTA. The elution was carried out with the same buffer and the antibody eluted from the column was directly added to the solution containing the derivatized rhG-CSF. The reaction mixture was incubated, at room temperature, for 24 hours and at the end of the reaction it was centrifuged and fractioned by gel filtration, using a column TSK-gel G3000 SW XL (Tosohaasj equilibrated in 50 mM, pH 6.6, sodium phosphate buffer; 150 mM NaCl. The same buffer was used to elute the material from the column. Example 2

Biochemical characterization of the immunocytokine

The fractions obtained by gel filtration from the column TSK-gel G3000 SW XL ( Tosohaas ) were analysed by SDS-PAGE and Western-blot ; the electrophoretic separations were performed under reducing and non- reducing conditions (Fig. l and 2 , respectively) on 4-15% polyacrilamide gel , in gradient (PhastSystem . Pharmacia , PhastGel Gradient 4- 15$ - Pharmacia LKB ) . The Western-blot analysis of the fractions was performed using as primary antibody a rabbit anti-human G-CSF polyclonal antibody (Genzyme) and as secondary antibody a Peroxidase-conjugated goat IgG fraction to rabbit IgG-whole molecule (Number of the product: 55676) obtained from Cappel . The analytical control thus performed enabled to identify both the different components purified from the reaction mixture , and the fractions comprising immunocytokine. In particular, it was obtained:

A ) a fraction ( the number 6 ) containing material at high molecular weight , immunoreactive with the rabbit anti -human G-CSF polyclonal antibody and migranting in SDS-PAGE as a diffuse band; B ) a fraction ( the number 7 ) containing 4 distinct bands , which are immunoreactive with the rabbit anti-human G-CSF polyclonal antibody and migranting with an apparent molecular weight consistent with the presence of the antibody Mint5 linked to 1 ; 2 ; 3 or 4 molecules of G-CSF. with a prevalence of the form antibody/G-CSF in a ratio of 1 : 2; C ) a fraction ( the number 8) containing three distinct bands : two bands immunoreactive with the human anti-G-CSF antibody and migranting with an apparent molecular weight consistent with the presence of the antibody Mint linked to 1 or 2 molecules of G-CSF, with a prevalence of the form antibody/G-CSF in a ratio of 1:1, and a third band visible only by staining the proteins with silver and migranting with an apparent molecular weight comparable to that of the Mint5 non-conjugated with G- CSF;

D) a fraction (the number 9) containing two distinct bands: a band of immunoreactive material with molecular size consistent with that expected for the form antibody/G-CSF in a ratio of 1:1 and a band, quantitatively predominant, put in evidence only by staining the proteins with silver and corresponding to the antibody Mint5 non-conjugated with the G-CSF.

Fractions 7, 8 and 9 were dosed according to their content of G-CSF using a ELISA test kit for human G-CSF (Biotrak, RPN 2150, Amersham) . The same fractions were dosed according to the content of proteins using the Bio- Rad Protein Assay (BI0RAD - Number of catalogue: 500-0006) and Mint5 as a standard.

The following Table shows the results of these assays:

G-CSF μg/ml protein μg/ml

Fraction 7 3.17 44.1

Fraction 8 2.51 69.3 Fraction 9 0.60 53-9

Example 3

Biological properties in vitro of the immunocytokine

The biological activity of rhG-CSF of the immunocytokine (fractions 7. 8 and 9 of Example 2) was measured in vitτo in a cell proliferation assay using a murine cell line depending on the growth factor, 32DC13 (Valtieri

M. et al., 1987, J. Immunol., 138. 3829-3835).

In the proliferation tests, r-Met-hG-CSF (Granulokine , Roche) was used - li ¬

as reference standard.

Using the quantitative data obtained with the ELISA test, it results that the rhG-CSF of the immunocytokine shows a biological activity which is, always considering the variability inherent to a bioassay, comparable to that of r-Met-hG-CSF (Granulokine^fl) .

The capacity of the immunocytokine (fraction 7 of the Example 2) to bind specifically the target antigen of the antibody Mint5. that is EGF-R, was verified with a radioimmunological assay. For such purpose, A-431 cells were fixed on 96-wells plates and then incubated with the Mint5 or the immunocytokine. The binding of the immunocytokine to A-431 cells was assayed, with and without rhG-CSF at a concentration of 2 μg/ml, using an anti-murine immunoglobuline secondary antibody marked with I ^J . The results obtained indicated that the immunocytokine maintains the capacity to specifically bind EGF-R. The A- 431 human cell line (ATCC CRL 1555). obtained from an epidermoid carcinoma, was provided by the Istituto Nazionale per lo Studio e la Cura dei Tumori, E Experimental Oncology Department. Milan, Italy; such line shows about 2.5 x 10 binding sites for anti-EGF-R antibodies. Example 4 Biological property in vivo of the immunocytokine

The capacity of the immunocytokine to be localized in the tumor was studied in vivo by subcutaneous injection of 2 x 10 Calu-3 cells in the right flank of athymic mice BALB/c-nu/nu. The human cell line Calu-3 (ATCC HTB 55). obtained from a pulmonary adenocarcinoma, was provided by the Istituto Nazionale per lo Studio e la Cura dei Tumori, E Experimental Oncology Department, Milan, Italy; such line shows about 6 x 10 4 binding sites for anti-EGF-R antibodies. Athymic mice BALB/c-nu/nu were obtained from ossan (Correzzana-Milan, Italy). Two weeks after the inoculum of the cells, the immunocytokine (fraction 7 of the example 2)) was marked with I * and peritumorally injected. After 24 hours, a study of scintigraphy was carried out acquiring data and images by means of a gamma chamber Star Cam 3000 (General Electric). Then, the animals were killed and the radioactivity present in the single organs was detected with a gamma counter Cobra (Packard Camberra) . The performed analysis showed an intense accumulation of radioactivity at the tumor level.

The antitumor activity of the immunocytokine was assayed i n v i vo as inhibition capacity of the tumor take. The assay was carried out dividing the animals into three groups. Group 1: mice injected with tumor cells and receiving, once a day and by peritumoral injection, a dose immunocytokine (fraction 7 of the Example 2) equal to 60 ng of G-CSF/animal in a volume of 150 μl in a saline- phosphate solution (PBS) (assay of the rhG-CSF contained in the immunocytokine was carried out by ELISA method). Group 2: Control animals injected with tumor cells and receiving, once a day and by peritumoral injection, a dose of rh-G-CSF equal to 60 ng of G- CSF/animal and a dose of Mint5 equal to 600 ng/animal in a volume of 150 μl of PBS.

Group 3: Control mice injected with tumor cells and receiving, once a day and by peritumoral injection, a volume of 150 μl of PBS. Experiments were performed by subcutaneous injection of 2 x 10 Calu-3 cells in the right flank of athymic mice BALB/c-nu/nu. The animals were killed 7 or 12 days after the inoculum of Calu-3 cells. Tumors were subjected to a histopathology test.

The performed analysis showed: i) higher inhibition of the tumor growth in the treated group (group 1) than in the control groups; ii) presence of reactive infiltrate consisting of mixed inflammatory cells immersed in a fibrous component particularly abundant in the tumor inoculum site of group 1. These results show that the immunocytokine is able to stimulate an antitumor immune response more intense than that obtained when the two molecules (Mint5 and G-CSF) are separately used.

The hybrid molecules according to the present invention are useful for the preparation of pharmaceutical compositions, in the antitumor therapy, in combination with a pharmaceutical acceptable carrier and/or excipient. Furthermore, the present invention relates to said pharmaceutical compositions comprising said hybrid molecules and a pharmaceutical acceptable carrier and/or excipient.

Claims

CLAIMS 1 . Hybrid molecules , useful in the antitumoral treatment , consisting of a G-CSF molecule linked to a monoclonal antibody or fragment thereof or to a ligand able to recognize a specific antigen . 2. Hybrid molecules according claim 1 , wherein said G-CSF is human G-CSF. 5 - Hybrid molecules according to claim 2 , wherein said G-CSF is recombinant human G-CSF (rh-G-CSF) . 4 ' . Hybrid molecules according to claim 1 , wherein the antibody or fragment thereof is an antibody specific for a cell surface receptor. 5 - Hybrid molecules according to claim 4 , wherein the cell surface receptor is EGF-R. 6. Hybrid molecules according to claims 4, wherein said antibody is the antibody produced by the DSM ACC2150 cell line. 7• Hybrid molecules according to claim 4, wherein the antibody fragments able to bind the specific antigen are the fragments Fab' or F(ab>2 of the antibody produced by the DSM ACC2150 cell line. 3. Hybrid molecules according to claim 1, wherein said ligand is any fragment able to bind a cell surface receptor. - Hybrid molecules according to claim 8, wherein said cell surface receptor is EGF-R. 10. Hybrid molecules according to claims 1-9, wherein said G-CSF is chemically linked to an antibody or fragments thereof or to a ligand '.hich recognizes a specific antigen. 11. Process for the preparation of a hybrid molecule according to claims 1-8 consisting of chemically linking the G-CSF molecule to an antibody or fragments thereof or to a ligand able to recognize a specific antigen. 12. Process for the preparation of a hybrid molecule according to claims 1-9. according to the technique of the recombinant DNA. 13- Use of the hybrid molecules according to claims 1-9 for the preparation of a pharmaceutical composition in combination with a pharmaceutically acceptable carrier and/or excipient. 14. Pharmaceutical composition, useful in the antitumor treatment, comprising a hybrid molecule according to claims 1-9 and a pharmaceutically acceptable carrier and/or excipient.