WO1998041229A1

WO1998041229A1 - USE OF IL-12p40 AS IMMUNOSTIMULANT

Info

Publication number: WO1998041229A1
Application number: PCT/EP1998/001473
Authority: WO
Inventors: Gottfried Alber; Frank Brombacher
Original assignee: F. Hoffmann-La Roche Ag
Priority date: 1997-03-19
Filing date: 1998-03-13
Publication date: 1998-09-24

Abstract

The present invention relates to the use of IL-12p40 for the preparation of medicaments for the treatment of diseases correlated with T cell mediated immunity.

Description

USE OF IL-12p40 AS IMMUNO STIMULANT .

The present invention provides the use of the interleukin-12 subunit p40 (IL-12p40) for the preparation of medicaments for the prophylaxis and treatment of diseases correlated with T cell mediated immunity.

Interleukin 12 (IL-12), formerly called natural killer cell stimulatory factor (Kobayashi M., et al., J. Exp. Med. 170:827-845, 1989) and cytotoxic lymphocyte maturation factor (Stern A.S., et al., Proc. Natl. Acad. Sci. USA 87:6808-6812, 1990), has potent anti-tumor and antimetastatic activity in several murine tumor models (Brunda M. J., et al., J. Exp. Med. 178:1223-1230, 1993; Nastala C. L., et al., J. Immunol. 153:1697-1706, 1994). Although the mechanism through which IL-12 exerts its anti-tumor effects is not completely understood, it has been shown that IL-12 induces a variety of biological effects on natural killer and T cells in vitro (Manetti R., et al, J. Exp. Med. 179:1273-1283, 1994; Wu C. Y., et al., J. Immunol. 151:1938-1949, 1993; Tripp C. S., et al., Proc. Natl. Acad. Sci. USA 90:3725- 3729, 1993; Seder R. A., et al., Proc. Natl. Acad. Sci. USA 90:10188-10192, 1993; Bloom E. T., et al, J. Immunol. 152:4242-4254, 1994; Cesano A., et al., J. Immunol. 151:2943-2957, 1993; Chan S. H., et al., J. Immunol. 148:92-98, 1992). Activation of cytotoxic T lymphocytes by IL-12 is considered crucial in its anti-tumor activity (Brunda M. J., et al., J. Exp. Med. 178:1223-1230, 1993). The IL-12 anti-tumor effect is partially maintained in severe combined immune deficient (SCID) and nude mice, both of which are T cell-deficient, and in CD8*-depleted euthymic mice (Brunda M. J., et al., J. Exp. Med. 178:1223-1230, 1993; O oole M., et al., J. Immunol. 150:294A, 1993). The results indicate that IL-12 has potent in vivo antitumor and antimetastatic effects against murine tumors and demonstrate as well the critical role of CD8⁺ T cells in mediating the antitumor effects against subcutaneous tumors.

IL-12 is a heterodimeric molecule with an approximate molecular weight of about 75 kD consisting of two disulfide-linked subunits: IL- 12p35, having an approximate molecular weight of about 35 kD, and IL- 12p40, having an approximate molecular weight of about 40 kD (Kobayashi M. et al., J. Exp. Med. 173:869, 1991; Stern, A. S. et al., Proc. Natl. Acad. Sci. USA 87:6808, 1990; Wolf, S. F. et al., J. Immunol. 146:3074, 1991; Gubler, U. et al, Proc Natl. Acad. Sci. USA 88:4143, 1991). The IL-12p40 subunit shares amino acid sequence homology with the interleukin-6 receptor (IL-6R) and therefore belongs to the cytokine receptor superfamily, whereas IL-12p35 has a distant but significant relationship to the IL-6/G-CSF cytokine family. IL-12p40 has been proposed to inhibit the biological activities of IL-12 (European Patent Application No. 625354). It has been speculated that the p35/p40 heterodimer could represent a cytokine (p35) and soluble cytokine receptor (p40) complex, with the cellular IL-12 receptor providing function analogous to the IL-6 signal transducing protein, gpl30 (Gearing, D. P. and D. Cosman, Cell 66:9, 1991; Merberg, D. M., et al., Immunology Today 13:77, 1992).

The present invention provides the use of the interleukin-12 subunit p40 for the preparation of medicaments for the prophylaxis and treatment of diseases correlated with T cell mediated immunity. It was observed that the agonistic activity of IL-12p40 in specific immunity described in a classical model of cellular immunity offers the possibility of prophylactic and therapeutic applications. In a preferred embodiment, the present invention provides the use of the interleukin-12 subunit p40 (IL-12p40 or p40) as immunostimulating agent. Accordingly, IL-12p40 may be used in vaccination as an adjuvant or in cancer therapy.

It is another object of the present invention to provide the use of IL-12p40 for the manufacture of medicaments for the treatment of infections, especially of chronic infections.

Chronic infections may be caused by viral agents. Consequently, IL- 12p40 is especially useful for the manufacture of medicaments for prophylaxis and treatment of viral infections. In preferred embodiments, the invention provides the use of IL-12p40 for the treatment of hepatitis, papilloma, human immunodeficiency or herpes virus infection. It is yet another object of the present invention to provide the use of interleukin-12 for the manufacture of medicaments for the treatment and prophylaxis of bacterial infections, for example tuberculosis, salmonellosis or listeriosis. Further, the invention comprises the use of interleukin-12 p40 for the manufacture of medicaments for the treatment and prophylaxis of parasitic infections, like malaria, leishmaniosis or schistosomiasis. It is yet another object of the present invention to provide the use of IL-12p40 for the treatment and prophylaxis of the diseases mentioned above and the use in cancer treatment.

The above medicaments may contain one or more additional compounds having agonistic activity useful for the prophylaxis and treatment of diseases correlated with T cell mediated immunity, like IFNα, IFNγ, 11-2, etc.

The invention also comprises the corresponding methods for treatment of the above mentioned diseases by administering a therapeutically effective amount of IL-12p40.

Brief description of the drawings

Figure la. Survival of mice infected with 500 cfu (colony forming units) of L. monocytogenes. C57BIJ6 mice("wt"), IL-12p35-/- ("P35-/-") and IL-12p40^_/- ("p40' ^_") mice were infected i.v. and survival of mice was monitored daily.

Figure lb. Survival of mice infected with 4,000 cfu of L. monocytogenes. C57BL/6 mice (" t"), IL-12P35"/- ("P35-/-") and IL-12p40^"/- ("p40''"") mice were infected i.v. and survival of mice was monitored daily for 56 days. Survival status did not change after day 25.

Figure 2. Organ load in mice infected with L. monocytogenes for two days (i.p.) and five days (i.v.). C57BL/6 mice ("wt"), IL-12P35^"'- ("p35-/-") and IL-12p40-/- ("P40-/-") mice were infected i.p. with 50,000 cfu of . monocytogenes for two days or i.v. with 1,000 cfu for five days. The number of viable bacteria in organ homogenates was determined by plating 10-fold serial dilutions on trypticase-soy broth agar plates. Plates were incubated at 37°C and the number of colony forming units (cfu) were counted after 24 hours. Analysis of spleen on day five represents the average of five pooled spleens.

Figure 3. Cytokine mRNA in CD4+ and CD8+ T cells from wild-type and

IL-12-deficient mice infected with L. monocytogenes for 5 days. C57BL/6, IL- 12p35-/- ("P35-/-") and IL-12P40"'- (^,,p40- -^M) mice were infected i.v. with 1,000 cfu of L. monocytogenes for five days. CD4⁺ and CD8⁺ T cells were purified from spleens of five infected mice. RNA was purified, reverse-transcribed and the cDNA used for PCR-mediated amplification of the designated cytokines. The relative specific mRNA levels for the designated cytokines in CD4⁺ and CD8⁺ T cells form wild-type C57BL/6 mice ("wt") were arbitrarily defined as 1 unit.

Figure 4. In vitro production of IFN-γ by spleen cells of mice infected with L. monocytogenes for 5 days. C57BIJ6 mice ("wt"), IL-12p45 -I- ("p35- /-") and IL-12P40-/- ("P40-/-") mice were infected i.v. with 1,000 cfu of L. monocytogenes for five days. Suspensions of spleen cells were incubated for 48 hours with medium or 2 x 10⁸ HKLM/ml. Supernatants were subsequently analyzed for IFN-γ. Cells from normal C57BL/6 mice ("wt") and from IL-12-deficient mice produced similar amounts of IFN-γ (170- 214 ng/ml) when stimulated with anti-CD3 (5 mg/ml).

Figure 5. Organ load in mice infected i.v. with L. monocytogenes for 14 days. C57BIJ6 mice ("wt"), IL-12p45 -'- ("p35-/-") and IL-12P40" - ("p40- /"") mice were infected i.v. with 1,000 cfu for 14 days. The number of viable bacteria in organ homogenates was determined by plating 10-fold serial dilutions on trypticase-soy broth agar plates. Plates were incubated at 37°C and the number of colony forming units were counted after 24 hours.

Figure 6a. Liver load of mice infected i.v. with 1,000 cfu of L. monocytogenes for 9 days with or without treatment with recombinant homo- dimeric p40. C57BL/6 mice ("wt") and IL-12P40-/- ("P40-'-") mice were infected i.v. with 1,000 cfu for 9 days. IL-12p40"^/" mice were given 25 μg of purified recombinant homodimeric IL-12p40 i.p. daily starting one day prior to infection until day 8 post infection. The number of viable bacteria in organ homogenates was determined by plating 10-fold serial dilutions on trypticase-soy broth agar plates. Plates were incubated at 37°C and the number of colony forming units were counted after 24 hours.

Figure 6b. Survival of mice infected i.v. with 1,700 cfu of L-monocyto- genes and reconstituted with recombinant monomeric or homodimeric p40. C57BL/6 mice ("wt") and IL-12p45-/- ("P35-'-") and IL-12P40^"'- ("p40-/- ") mice were infected i.v. with 1,700 cfu for 7 days. IL-12p40^~/_ mice were given 25 μg of purified recombinant monomeric or homodimeric IL-12p40 i.p. daily starting one day prior to infection until day 8 post infection. Survival of mice was monitored daily. Figure 7. Liver burden of immunized mice on day two and day 13 post challenge with 60,000 cfu of L. monocytogenes. C57BL/6 mice ("wt") ("wt"), IL-12P45-/- ("_P35-/-") and IL-12P40^"'- ("P40^"7"") mice were infected i.v. with 300 cfu of L. monocytogenes and 15 days later challenged with 60,000 cfu. At the time of challenge all mice had cleared the primary infection. Two days and 13 days post challenge the number of viable bacteria in liver homogenates was determined by plating 10-fold serial dilutions on trypticase-soy broth agar plates. Plates were incubated at 37°C and the number of colony forming units were counted after 24 hours.

Figure 8. Serum IL-12p40 levels in immunized mice rechallenged with L. monocytogenes. C57BL/6 mice ("wt") and IL-12p35 -'- ("P35-/-") and IL-12p40-/- ("P40-/-") mice were infected i.v. with 300 cfu of L. monocytogenes and 15 days later challenged with 60,000 cfu. Two days post challenge immunoreactive IL-12p40 was measured in serum of five mice per group.

The present invention is directed to the use of the interleukin-12 subunit p40 for the manufacture of a medicament for the prophylaxis and treatment of diseases correlated with T cell mediated immunity. Especially, the invention comprises the use of IL-12p40 for the manufacture of medicaments useful as immunostimulants.

The term "immunostimulant" describes drugs capable of increasing the resistance of an organism against stress of variable origin. These types of drugs achieve their effects primarily by nonspecific mechanisms of actions. Immunostimulants generally stimulate, in an non-antigen dependent manner, the function and efficiency of the nonspecific immune system in order to counteract for example microbial infections or immunosuppressive states like cancer. In addition, immunostimulants can be used as adjuvants.

Accordingly, the present invention provides the use of IL-12p40 for the prophylaxis and treatment of diseases correlated with an immunosuppressive condition but with maintained presence of T cells.

The invention includes the use of the interleukin-12 subunit p40 for the preparation of medicaments for the treatment of all forms of infectious diseases, especially chronic infectious diseases. Examples of infections which can be treated by IL-12p40 are viral, bacterial or parasitic infections. Viral infections may be caused for example by hepatitis, papilloma, human immunodeficiency or herpes virus. Examples for bacterial infections are tuberculosis, salmonellosis or listeriosis. Examples for parasitic infections are malaria, leishmaniosis or schistosomiasis.

The invention further provides the use of IL-12p40 as adjuvant. IL- 12p40 is useful for improving the immune response obtained with any particular antigen in a vaccine. Although some antigens are administered in vaccines without an adjuvant, there are many antigens that lack sufficient immunogenicity to stimulate a useful immune response in the absence of an effective adjuvant. IL-12p40 also improves the immune response obtained form "self-sufficient" antigens, in that the immune response obtained may be increased or the amount of antigen administered may be reduced.

For example, IL-12p40 can be used as an adjuvant in immunisation against the infectious diseases mentioned above.

The invention also provides the use of IL-12p40 for the manufacture of a medicament for the prophylaxis and treatment of cancer by specifically enhancing T cell function to recognize tumor antigens and to lyse tumor cells.

The terms "p40", "IL-12p40" or "p40 subunit" include the natural and recombinant p40 subunit of interleukin-12 as well as derivatives thereof: The term comprises fragments as well as monomer and polymer forms of the p40 subunit and fusion proteins: i.e. p40 subunit derivatives comprising the amino acid sequence of natural IL-12p40 or partial sequences thereof together with amino acid sequences derived from other proteins. The protein according to the invention may optionally contain an initiator methionine.

The above terms also comprise non-naturally occurring IL-12p40 analogous subunits having amino acid sequences which are analogous to the amino acid sequence of IL-12p40 or its fragments. Such IL-12p40 analogue subunits are proteins in which one or more of the amino acids of the natural IL-12p40 or its fragments have been replaced or deleted without loss of the mentioned IL-12p40 activity. Such analogous may be produced by known methods of peptide chemistry or by known methods of recombinant DNA technology such as site directed mutagenesis. Furthermore the above terms also include "functional derivatives". This term refers to derivatives of the IL-12p40 monomers and polymers, which may be prepared from the functional groups occurring as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e. they do not destroy the activity of the protein and do not confer toxic properties on compositions containing it. These derivatives may include, for example, polyethylene glycol side-chains which may mask antigenic sites and extend the residence of the p40 protein as defined above in body fluids. Other derivatives include aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues formed with acyl moieties (e.g. alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl groups (for example that of seryl- or threonyl residues) formed with acyl moieties.

IL-12p40 dependent activity is characterized by reduction of microbial numers associated with reduced mortality as defined in the Examples.

In principle, all IL-12p40 proteins which are encoded by a nucleic acid which hybridize under moderately stringent conditions to a nucleic acid which encodes the protein of SEQ ID NO: 1 and which show the above described activity are suitable for the described use. "Moderately stringent conditions" are described by Sambrook et al. (Molecular Cloning: A Laboratory Manual; NY, Cold Spring Harbor Laboratory Press, 1989) and include the use of a washing solution and hybridization conditions known in the art. An example of moderately stringent conditions are conditions such as overnight incubation at 37°C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 μl/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C.

A preferred embodiment of the invention is the use of a human IL-12p40 protein. The most preferred p40 protein is that of SEQ ID NO:l.

The IL-12p40 proteins of this invention show an agonistic function in T cell mediated immunity. The agonistic function may be determined as described in the Examples. In accordance with the present invention, IL-12p40 proteins are obtained in pure form. The preparation of IL-12p40 is described in detail in European Patent Application Publication No. 0 433 827 and International Patent Application WO90/05147. Biologically active analogues and fragments of the p40 subunit may be prepared. These biologically active proteins may be produced biologically using standard methods of the recombinant DNA technology or may be chemically synthesized in an amino acid synthesizer or by manual synthesis using well-known liquid or solid phase peptide synthesis methods. In a similar way analogues, fragments and proteins comprising the amino acid sequence of IL-12p40 together with other amino acids can be produced. All of these proteins may then be tested for the corresponding biological activity.

This invention provides evidence that the agonist activity is a new biological activity of IL-12p40 (Examples 1-6). Despite the potency of IL-12 in inducing IFN-γ, the examples show that the considerable amount of IFN-γ produced by splenocytes of Listeria-infected IL-12-deficient mice is still sufficient for resistance against low and medium doses of Listeria. In this respect IL-12-deficient mice differ very much from mice unable for IFN-γ activation, like IFN-γ or IFNγR-deficient mice. The latter are highly susceptible and die during the first week after low dose infection. (Harty, J. T. and Bevan, M. J., Immunity 3:109-117, 1995). These mice show multiple macrophage defects (Harty, J. T. and Bevan, M. J., Immunity 3:109-117, 1995; Huang, S., et al., Science 259:1742-1745, 1993). In addition, IFNγ-R"^/_ macrophages are impaired in inhibiting bacteria escape form the phagosome to the cytoplasm, demonstrating a strong listericidal defect. A similar defect was also observed in NF-IL-6^_/" macrophages, indicating a common pathway of this important macrophage effector function. Clearly, the IL-12-independent IFN-γ levels during bacterial infection with Listeria monocytogenes appeared to be sufficient for the relative resistance towards low and medium inocula observed in IL-12- deficient mice. Early IFN-γ is produced by NK cells activated by Listeria- stimulated macrophage-derived IL-12 (Rogers H. W., et al., The Immunologist 3/4:152-156, 1995). The Examples indicate that either NK cells or other cells have the potential to produce some initial IFN-γ independent of IL- 12.

Only high inocula caused lethality in IL-12-deficient mice and most mice died within the first week of the infection. At these doses insufficient IFN-γ production and aberrant granulomatous lesions led to uncontrolled bacterial growth and subsequent mortality. The latter observation is in agreement with data from anti-IL-12-treated mice which also succumbed to infection (Tripp , C. S., et al., J. Immunol. 152:1883- 1887, 1994), and shows the important protective role of bioactive IL-12 in bacterial infections. The role of IL-12 for protection against bacteria is limited to innate immunity. This is a period of an infection where the commitment to a type 1 or type 2 T cell response takes place. Consequently, IL-12 has a function in innate mechanisms by activating optimal IFN-γ production and initiation of T cell differentiation, since in both mutant mice a reduced Thl polarization and lower IFN-γ and IL-10 levels in CD8+ T cells from mutant mice than from wild-type mice were found.

Since IL-12p35^_/'" mice is able to clear Listeria comparable to wild type mice these data further suggest that T cell dependent effector mechanisms against Listeria are not regulated by IL-12. During that period of infection specific immunity depends on IL-12p40 as shown in the inability of IL-12p40"^/" mice to clear Listeria during primary or secondary responses.

In resistance against Listeria the function of IL-12p40 is agonistic during IL-12-independent specific immunity. The antagonistic potency of mainly homodimeric IL-12p40 against IL-12 can be excluded in this situation. This of course may be different in IL-12-induced proinflammatory reactions since IL-12p40 can block the binding of IL-12 to the IL-12 receptor (Gillessen S. et al., Europ. J. Immunol. 25:200-206, 1995; Ling, P. et al., J. Immunol. 1545:116-127, 1995).

Treatment with recombinant monomeric and homodimeric IL-12p40 is able to partially reconstitute resistance (Fig. 6). This makes it unlikely that the ligand IL-12p40 has to associate with an unrelated molecule (different from p35 to become functional. A currently proposed model suggests that the βl subunit is the docking subunit for p40. The β2 subunit appears to act as signal transducing subunit for p75. Therefore, the antagonistic activity by homodimeric IL-12p40 described in the prior art is believed to result from blocking the binding of IL-12p40 to the βl subunit of the IL-12 receptor. The agonistic activities by IL-12p40 may be mediated by an unknown receptor molecule. Pharmaceutically acceptable formulations of IL-12p40 in connection with this invention can be made using formulation methods known to those of ordinary skill in the art. These formulations can be administered by standard routes. In general, the formulations may be administered parenterally (e.g., intravenous, subcutaneous or intramuscular) with topical, transdermal, oral, or rectal routes also being contemplated. In addition, the formulations may be incorporated into biodegradable polymers allowing for sustained release of IL-12p40, the polymers being implanted in the vicinity of where drug delivery is desired, for example, at the site of a tumor. The biodegradable polymers and their use are described, for example, in detail in Brem et al. (1991) J. Neurosurg. 74, 441-446. The dosage of IL-12p40 will depend on the condition being treated, the particular compound, and other clinical factors such as weight and condition of the human or animal and the route of administration of IL-12p40. It is to be understood that the present invention has application for both human and veterinary use. For parenteral administration to humans, a dosage of between approximately 10⁵ ng IL-12p40 to 1 ng/kg body weight, preferably between approximately 3000 ng to 30 ng/kg 1 to 3 times a week is generally sufficient. It will however be appreciated that the upper and lower limit given above can be exceeded when this is found to be indicated.

The formulations include those suitable for parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intratracheal, and epidural) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association IL-12p40 and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the IL-12p40 with liquid carriers. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, seated ampoules and vials, and may be stored in a freeze-dried (lyophilized) conditions requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the administered ingredient.

For the preparation of tablets, coated tablets, dragees or hard gelatine capsules the compounds of the present invention may be admixed with pharmaceutically inert, inorganic or organic excipients. Examples of suitable excipients for tablets, dragees or hard gelatine capsules include lactose, maize starch or derivatives thereof, talk or stearic acid or salts thereof.

Suitable exicpients for use with soft gelatine capsules include for example vegetable oils, waxes, fats, semi-solid or liquid polyols etc. For the preparation of solutions and syrups, excipients which may be used include for example water, polyols, saccharose, invert sugar and glucose. For injectable solutions, excipients which may be used include for example water, alcohols, polyols, glycerine, and vegetable oils. For suppositories, and local or percutaneous application, excipients which may be used include for example natural or hardened oils, waxes, fats and semi-solid or liquid polyols. The pharmaceutical compositions may also contain preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts for the variation of osmotic pressure, buffers, coating agents or antioxidants. They may also contain other therapeutically valuable agents.

With respect to the use of IL-12p40 as adjuvant it will be apparent to one of ordinary skill in the art that the precise amounts of IL-12p40 and antigen needed to produce a given effect will vary with the particular compounds and antigens, and with the size, age and condition of the subject to be treated. The amounds needed can easily be determined using methods known to those of ordinary skill in the art. The adjuvants and vaccines of the invention are generally administered by injection, particularly intramuscular injection, preferably into a large muscle. In general, an initial vaccination is administered using the desired antigen and an appropriate formulation. The vaccination is "boosted" several weeks later (usually 2-6 weeks, for example, 4-6 weeks) using the adjuvans of the invention. Generally, 1-2 ml of a vaccine is administered to a human subject in the practice of the invention. This invention is further illustrated by the following Examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims.

EXAMPLES

Materials and Methods

a) Mice

IL-12p35^~'^~ and IL-12 p40^~'" mice have been generated and characterized by Mattner et al. (Europ. J. Immunol., 26:1553-1559, 1996) and Magram et al. (Immunity, 4:471-481, 1996). IL-12p35A and IL-12 p 0-/" mice were backcrossed five times to C57BL/6. Mice were bred in BRL (Fϋllingsdorf/Basel), Switzerland in SPF condition and used for experiments at an age of 6 to 10 weeks. Mice were kept in filter-cap cages during the experiments.

b) Bacteria and infection of mice

Virulent Listeria monocytogenes were grown in tryptose-soy broth (Difco, Detroit, Mich.). Aliquots of log-phase growing cultures were stored at -70°C until use. For each experiment, a vial was thawed, washed once in saline, and diluted in endotoxin free PBS before injection. Mice were injected intravenously (i.v.) into the tail vein or intraperitoneally (i.p.) with 200 ml PBS with or without bacteria. The number of viable bacteria in the inoculum and in organ homogenates was determined b y plating 10-fold serial dilutions on trypticase blood agar plates. Plates were incubated at 37°C and the numbers of colony forming units (cfu) were counted after 24 hours.

Heat killed L. monocytogenes (HKLM) were prepared by incubating bacteria at 60°C for 60 min. Killed bacteria (2 x 10⁹/ml) were stored in PBS at -70°C. c) Histology

Mice were killed by cervical dislocation, their organs removed whole, cut in pieces, and fixed in 10% formalin solution. Tissues were dehydrated in ethanol and embedded in paraffin. Five mm sections were cut and stained with hematoxylin and eosin, naphthol-AS-D-chloroacetatesterase (NACE) for special visualization of neutrophils. Silver stain was used to visualize Listeria (Dieterle, Arch. Neurol. Psych. 73-80, 1924). All studies were done with a Zeiss microscope with image analysis software (SIS, Munster, Germany) for the computerized morphometry.

d) Purification of CD4+ and CD8+ T lymphocytes

For purification of the CD4⁺ subset, spleens were collected, teased to a single cell suspension, erythrocytes lysed, and incubated with mAbs 3.155 against CD8 and Ml/70.15.11.5. HL against Mac-1. For purification of the CD8+ cell subsets splenocytes were incubated with anti-CD4 and anti-Mac- 1. Cells were separated in a magnetic field using anti-Ig-coated ferrous beads (Milan Analytica AG, La Roche, Switzerland). The purity of the CD4⁺ and of the CD8+ population exceeded 90% as assessed by FACS analysis (Becton Dickinson & Co.).

e) Reconstitution with p40

Mice were given recombinant murine monomeric or homodimeric IL-

12p40 i.p. at 25 μg daily during the infection starting one day prior to infection.

f) Stimulation of cells and cytokine production

Spleens were collected and erythrocyte-lysed single cell suspensions were stimulated at 5 x 10⁶ cells per ml with HKLM (2 x 10⁸ cell equivalents/ml) as indicated in a final volume of 1 ml at 37°C and in an atmosphere of 7.5% CO2. Cells were cultured in IMDM supplemented with 5% heat-inactivated FCS, L-glutamine, 2-ME and HEPES. Culture super- natants were collected after 24-72 h of stimulation and stored at -20°C until use. g) Determination of cytokine levels in blood and supernatant

IFN-γ was measured by ELISA using rat IgGl mAbs AN18 and XMG1,2. IL-4 was measured by ELISA using rat IgGl mAb 11B11 and rat mAb BVD6-24G2, obtained from PharMingen (Lugano, Switzerland).

h) RT-PCR

Total RNA was isolated from cells by the single-step guanidinium thiocyanate procedure. Complementary DNA was synthesized for 1.5 hours at 37°C in a 10 ml reaction volume containing: 16 U/ml moloney murine leukemia virus (MuMLV) reverse transcriptase (Gibco BRL, Paisley, Scotland), 48 pg/ml random hexamers (Biolabs, Beverley, USA), 0.4 mM of each dNTP, 0.7 U/ml RNase-inhibitor (Promega, Heidelberg, Germany), 25 mM Tris (pH 8.3), 37.5 mM KC1, 1.5 mM MgCl₂, 5 mM dithiotreitol.

PCR was performed in 50 ml containing: 0.25 mM of each dNTP, 0.25 mM 5' and 3' primers, 10 mM Tris (pH 9.0), 50 mM KC1, 0.1% Gelatine, 1.5 mM MgCl₂, 0.1% (v/v) Triton X-100) and 0.2 U Taq polymerase (Stehelin AG, Basle, Switzerland) for 35 cycles (20 sec 94°C, 20 sec at 60°C and 30 sec at 72°C). Thereafter, 20 ml of the reaction product was analyzed on a 1.5% agarose gel in Tris borate-EDTA buffer containing 0.2 mg/ml ethidium bromide.

i) Quantification of cytokine mRNA by competitive RT-PCR

Competitive PCR was performed essentially as described by co- amplifying constant amounts of cDNA in the presence of four-fold dilutions of internal competitor multiple plasmids, pNil (Kopf et al., J. Exp. Med. 184:1127 and pMUS (Shire, Europ. Cyto. Netw. 4:161-162, 1993). Five ml of competitor fragment was added to the PCR reaction at fourfold dilutions in 9 dilution steps (1 to 9) form a stock concentration of 3.73 ng/ml (1 x 10⁶ molecules/ml) In all experiments, control PCR without cDNA or without cDNA and competitor fragment was performed to exclude false positives. To avoid amplification of genomic DNA, primers from different exons were used. Relative quantification of cDNA was done by calculating how much of the competitor fragment was required to achieve equal amounts of product described by others (Bouaboula, J. Biol. Chem. 267:21830-21838, 1992). In order to compare different samples, cDNAs were first standardized to levels of β2-microglobulin transcripts. The calculated amounts of cytokine cDNA were then normalized with the β2-microglobulin levels.

Example 1

Increased mortality of IL-12p35^_ - and IL-12p40^_/- mice by Listeria

Mice were infected with a sublethal Listeria dose of 500 cfu (low dose) or

4,000 cfu (high dose) and mortality was followed during the course of infection. Wild-type and both mutant mouse strains survived low dose infection as monitored daily for 17 days (Fig. la). Increased susceptibility of mutant mice was seen during high dose infection where 90% of the IL-12p35"'^~ and 70% of the IL-12p40"'" mice succumbed to infection during the first two weeks post infection, whereas wild-type C57BIJ6 mice (LD50 of C57BL/6 = 20,000 cfu) survived sublethal infection with low (10%) mortality (Fig. lb). These results suggest that IL-12 deficient mice are able to successfully resist low dose Listeria, but show an increased susceptibility to higher doses.

Example 2

Normal bacterial burden but increased granulomatous lesions in IL- 12p35"/^~ and IL-12p40^~/^~ mice during innate immunity

The bacterial burden was determined in IL-12p35^"'^" and IL-12p40^~'^~ mice and C57BL/6 control mice at different days during L. monocytogenes infection (Fig. 2). On day 2 after infection with 50,000 cfu of Listeria (i.p.) and on day 5 after infection with 1,000 cfu of Listeria (i.p.) the bacteria load recovered form liver and spleen were similar in IL-12-deficient and wild-type

Table 1: Granulomatous lesions in liver of mice infected with L. monocytogenes for 5 days

Mice no. of granu- Size of granuloma loma/field (μm² x 10³)

wt 8.5 82.3 ± 48.0

p35-/- 16.2 304.4 ± 116.4

p40-/- 12.2 285.5 ± 102.5

C57BL/6 mice ("wt"), IL-12p35-/- ("p35-/-) and IL-12p40-'- ("p40") mice were infected i.v. with 1,000 cfu of L. monocytogenes for five days. Liver lobes of infected mice were fixed in formalin solution and processed for histology. Sections were stained with hematoxylin and eosin, naphthol-AS-D-choroacetateesterase for special visualization of neutrophils. Samples were analyzed with a Zeiss microscope with image analysis software (SIS, Munster, Germany) for the computerized morphometry.

mice with slightly increased burdens in mutant liver at day 2. Despite the comparable bacterial load at day 5, granulomatous lesions in the infected organs of mutant mice were more abundant in number and size, indicating aberrant granulomatous structure in mutant mice (Table 1). The cellular composition of the granulomas (macrophages, lymphocytes and neutrophils) was similar in wild-type and both mutant mice but the necrotic center was 3-4-fold bigger in mutant mice than in wild-type mice.

Example 3

Th2 polarization in IL-12p35-/- and IL-12p40-/- mice

The potential of IL-12p35^~/^~ and IL-12p40^~/" mice to mount a polarized Thl/Th2 and a Tcl/Tc2 cytokine response 5 days after infection with L. monocytogenes was determined. Splenic CD4+ and CD8⁺ T cells (from experiment shown in Fig. 2, day 5) were purified by magnetic beads (purity >90%), and their specific transcripts quantitatively determined by competitive RT-PCR. CD4+ cells form IL-12P35"/- and IL-12p40-/- mice expressed four-fold lower IFN-γ mRNA and two-fold higher IL-4 mRNA levels, when compared to expression levels of wild-type CD4+ cells (Fig. 3). IL-2 and IL-10 specific transcript levels were indistinguishable form wild-type CD4⁺ specific cytokine levels. CD8⁺ cells from IL-12p35^{- ,~} and IL-12P40"/" mice expressed twofold and fourfold reduced IFN-γ mRNA levels, and fourfold and six-fold reduced IL-10 mRNA, respectively, when compared to wild-type CD8⁺ cells. IL-4 expression was not detected in CD8⁺ cells. These results indicate that the absence of endogenous IL-12p75 favours a Th2 polarization which is associated with reduced IFN-γ production by CD8⁺ cells. This polarization did not affect resistance to Listeria at the onset of the specific response, since mutant mice showed a bacterial burden comparable to wild- type mice (see Fig. 2, day 5). The reduced IFN-γ and IL-10 transcript levels in mutant mice derived CD8⁺ T cells indicate regulation by IL-12p75.

Interestingly, transcript levels for interferon-inducing factor (Okamura et al., Nature, 378:88-91, 1995) or now called IL-18 were enhanced in T cells from IL-12p35^"'^~ mice but not from IL-12p40^~'^~ mice as revealed by semi- quantitative PCR. Reduced but still considerable levels of IFN-γ protein were detectable in supernatants from ex vivo restimulated splenocytes derived from mutant mice (Fig. 4).

Example 4

Specific immunity during primary responses is normal in IL-12p35^~'^~ mice but impaired in IL-12p40"'~ mice

To determine if absence of IL-12 plays a role in specific immunity to

Listeria infection, mutant and control mice were either infected with 200 cfu (low dose) or 1,000 cfu (medium dose), avoiding mortality. At day 14 post infection, bacterial load in liver and spleen was measured from individual mice. Low dose infection led to sterile elimination of Listeria from liver and spleen (<10 cfu/organ) in wild-type and both mutant mice groups. However, medium dose infection was followed by sterile elimination of Listeria from liver and spleen in control and IL-12p35^~'^~ mice (<10 cfu/organ), whereas IL- 12p40^~'^~ mice were unable to clear Listeria with up to 10⁸ cfu in the liver and 10³ cfu in the spleen of these mice (Fig. 5). Sterile elimination of Listeria in IL-12p35^~/" mice after low and medium dose infection strongly suggest that IL-12 is indispensable for T cell specific immunity. The impaired sterile immunity of IL-12p40"/" mice, seen after medium dose infection suggests a positive role of IL-12p40 for T cell specific elimination, since IL-12p40 is produced by wild-type and IL-12p35^~'^~ mice during secondary responses (see below and Fig. 8). Example 5

Reconstitution with IL-12p40 reduces bacterial burden and prolongs survival of IL-12p40^~'^~ mice during primary responses

In order to determine if the impaired clearance of Listeria infected IL- 12p40^"'" mice is due to the lack of IL-12p40 production, we treated IL-12p40^~'^~ mice with homodimeric IL-12p40 (25 mg/mouse/day) during Listeria infection with 1,000 cfu and determined the bacterial burden in lever at day 9 post infection, in comparison to untreated infected mutant or normal mice (Fig. 6a). As expected, high bacterial load (10⁷ cfu) was found in the liver of IL-12p40"/^~ mice, whereas lower organ burden (100 cfu) was recovered from control mice, since the latter cleared the invader. IL-12p40^_/" mice supplemented with IL-12p40 showed 1000-fold lower organ burden compared to untreated infected IL-12p40"^/_ mice, demonstrating reconstitution of T cell- dependent clearance due to IL-12p40 treatment. Splenic CD4⁺ and CD8+ T cell derived from IL-12p40^~/" mice and restimulated with anti-CD3 produced 2-4-fold lower levels of IFN-γ than T cells derived from wild-type mice. Interestingly, similar levels of IFN-γ were found in T cell cultures from treated or untreated IL-12p40"'^~ mice indicating that treatment with recombinant IL-12p40 does not regulate IFN-γ production.

In another experiment ILrl2p40^~'^~ mice had been infected with a lethal dose of Listeria and treated with either recombinant murine monomeric or dimeric p40. As shown in Fig. 6b, both treatment with monomeric and homodimeric IL-12p40 was able to prolong survival of IL-12p40^~'^~ mice. Taken together reconstituting IL-12p40^~'^~ mice with recombinant murine IL- 12p40 is able to reduce bacterial load and to prolong the survival of Listeria- infected mutant mice indicating an agonistic role of IL-12p40 in resistance to Listeria.

Example 6

IL-12p40 is necessary for secondary responses

Secondary responses against L. monocytogenes are dominated by memory T cells. To further address the role of IL-12 and of IL-12p40 for specific T cell responses, secondary responses against L. monocytogenes were studied in immunized IL-12p35^~'" and IL-12p40^~'^~ mice. For immunization mice were infected with a low dose of L. monocytogenes (300 cfu) and 15 days later rechallenged with a normally lethal dose for wild-type mice. At the time point of rechallenge all mice had cleared the primary infection. Mice were analyzed 2 and 13 days after rechallenge (Fig. 7). At day 2, liver from control mice and IL-12p35^~/^~ mice had comparable bacterial load, whereas in the liver of IL-12p40^~'" mice a 25-fold increase was observed. In serum of those mice IL-12p40 was detectable in infected wild-type and IL- 12p35^"'^" mice but not in infected IL-12p40^~'^~ mice indicating endogenous production of IL-12p40 during secondary responses against Listeria (Fig. 8). Striking differences in bacterial load were observed at day 13 post infection. Control mice and IL-12p35^"''" mice had already cleared the infection, whereas IL-12p40^~'^~ mice were defective in clearing Listeria carrying up to 10⁷ cfu of Listeria in liver. Normal bacterial burden early after rechallenge and normal sterile elimination in IL-12p35"'^~ mice clearly demonstrate that IL-12 plays no role in specific immunity to Listeria. Increased bacterial burden, early after rechallenge and lack of elimination in IL-12p40^~'^~ mice indicates that IL-12p40 plays an important role in secondary immunity to Listeria.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: F. HOFFMANN-LA ROCHE AG

(B) STREET: Grenzacherstrasse 124 (C) CITY: Basle

(D) STATE: BS

(E) COUNTRY: Switzerland

(F) POSTAL CODE (ZIP) : CH-4002

(G) TELEPHONE: 061 - 688 51 08 (H) TELEFAX: 061 - 688 13 95

(I) TELEX: 962292/965542 hlr ch

(ii) TITLE OF INVENTION: USE OF IL-12p40 AS IMMUNOSTIMULANT (iii) NUMBER OF SEQUENCES: 1

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: Apple Macintosh (C) OPERATING SYSTEM: System 7.1 (Mac)

(D) SOFTWARE: Word 5.0

(2) INFORMATION FOR SEQ ID NO : 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 306 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 : lie Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr 1 5 10 15

Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu 20 25 30

Glu Asp Gly lie Thr Trp Thr Leu Asp Gin Ser Ser Glu Val Leu Gly 35 40 45

Ser Gly Lys Thr Leu Thr lie Gin Val Lys Glu Phe Gly Asp Ala Gly 50 55 60

Gin Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu 65 70 75 80

Leu Leu His Lys Lys Glu Asp Gly lie Trp Ser Thr Asp lie Leu Lys 85 90 95 Asp Gin Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys 100 105 110

Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr lie Ser Thr 115 120 125

Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gin 130 135 140

Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly 145 150 155 160

Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gin Glu Asp Ser Ala 165 170 175

Cys Pro Ala Ala Glu Glu Ser Leu Pro lie Glu Val Met Val Asp Ala 180 185 190

Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe lie Arg 195 200 205

Asp lie lie Lys Pro Asp Pro Pro Lys Asn Leu Gin Leu Lys Pro Leu 210 215 220

Lys Asn Ser Arg Gin Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp 225 230 235 240

Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gin Val Gin 245 250 255

Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr 260 265 270

Ser Ala Thr Val lie Cys Arg Lys Asn Ala Ser lie Ser Val Arg Ala 275 280 285

Gin Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro 290 295 300

Cys Ser 305

Claims

1. The use of IL-12p40 for the manufacture of a medicament for the prophylaxis and treatment of diseases correlated with T cell mediated immunity.

2. A use according to claim 1 wherein IL-12p40 is used as immunostimulating. agent.

3. A use according to claim 1, wherein IL-12p40 is used as adjuvant.

4. The use according to any one of claims 1 - 3 for the prophylaxis and treatment of infections.

5. A use according to any one of claims 1 -4 for the prophylaxis and treatment of chronic infections.

6. A use according to any one of claims 1 - 5 for the prophylaxis and treatment of viral infections.

7. A use according to any one of claims 1 - 6 for the prophylaxis and treatment of a hepatitis, papilloma, human immunodeficiency or a herpes virus infection.

8. A use according to any one of claims 1 - 5 wherein the infection is a bacterial infection.

9. A use according to claim 8 wherein the infection is tuberculosis, salmonellosis or listeriosis.

10. A use according to any one of claims 1 - 5 wherein the infection is a parasitic infection.

11. A use according to claim 10 wherein the infection is malaria, leishmaniosis or schistosomiasis.

12. A use according to any one of claims 1 - 5 in cancer treatment.

13. The use of IL-12p40 in the treatment of a disease as defined in any one of claims 1 to 12.

14. IL-12p40 for the treatment of diseases as mentioned in any one of claims 1 to 12.

15. IL-12p40 in combination with one or more additional immunostimulating agents for the treatment of diseases as mentioned in any of claims 1 to 12.

16. Method for treatment of diseases as mentioned in any of claims 1- 12 comprising administering a therapeutically effective amount of IL-

12p40.

17. The invention as hereinbefore defined.