WO1998041229A1 - Utilisation du il-12p40 comme immunostimulant - Google Patents

Utilisation du il-12p40 comme immunostimulant Download PDF

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WO1998041229A1
WO1998041229A1 PCT/EP1998/001473 EP9801473W WO9841229A1 WO 1998041229 A1 WO1998041229 A1 WO 1998041229A1 EP 9801473 W EP9801473 W EP 9801473W WO 9841229 A1 WO9841229 A1 WO 9841229A1
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mice
infection
treatment
use according
cfu
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PCT/EP1998/001473
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Gottfried Alber
Frank Brombacher
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F. Hoffmann-La Roche Ag
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Publication of WO1998041229A1 publication Critical patent/WO1998041229A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/208—IL-12
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • 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.
  • IL-12p40 interleukin-12 subunit p40
  • Interleukin 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).
  • 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.
  • IL-12 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).
  • SCID severe combined immune deficient
  • nude mice both of which are T cell-deficient, and in CD8*-depleted euthymic mice (Brunda M.
  • 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
  • IL- 12p35 having an approximate molecular weight of about 35 kD
  • IL- 12p40 having an approximate molecular weight of about 40 kD
  • 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.
  • 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.
  • IL- 12p40 is especially useful for the manufacture of medicaments for prophylaxis and treatment of viral infections.
  • 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.
  • 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 IFN ⁇ , IFN ⁇ , 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.
  • FIG. 1 Survival of mice infected with 500 cfu (colony forming units) of L. monocytogenes.
  • FIG. 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.
  • FIG. 1 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.
  • 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.
  • FIG. 4 In vitro production of IFN- ⁇ 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 10 8 HKLM/ml. Supernatants were subsequently analyzed for IFN- ⁇ .
  • Cells from normal C57BL/6 mice (“wt") and from IL-12-deficient mice produced similar amounts of IFN- ⁇ (170- 214 ng/ml) when stimulated with anti-CD3 (5 mg/ml).
  • FIG. 1 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.
  • FIG. 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 ⁇ g 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.
  • FIG. 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 ⁇ g 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.
  • 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.
  • the invention comprises the use of IL-12p40 for the manufacture of medicaments useful as immunostimulants.
  • 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.
  • 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.
  • 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.
  • bacterial infections are tuberculosis, salmonellosis or listeriosis.
  • 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.
  • 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.
  • p40 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.
  • 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.
  • 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.
  • acyl moieties e.g. alkanoyl or carbocyclic aroyl groups
  • O-acyl derivatives of free hydroxyl groups for example that of seryl- or threonyl residues
  • IL-12p40 dependent activity is characterized by reduction of microbial numers associated with reduced mortality as defined in the Examples.
  • 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.
  • 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 ⁇ l/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C.
  • 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
  • 20 ⁇ l/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.
  • 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.
  • IL-12p40 This invention provides evidence that the agonist activity is a new biological activity of IL-12p40 (Examples 1-6).
  • the examples show that the considerable amount of IFN- ⁇ produced by splenocytes of Listeria-infected IL-12-deficient mice is still sufficient for resistance against low and medium doses of Listeria.
  • IL-12-deficient mice differ very much from mice unable for IFN- ⁇ activation, like IFN- ⁇ or IFN ⁇ R-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).
  • 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).
  • IFN ⁇ -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.
  • IFN-independent IFN- ⁇ 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- ⁇ 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- ⁇ independent of IL- 12.
  • IL-12 has a function in innate mechanisms by activating optimal IFN- ⁇ production and initiation of T cell differentiation, since in both mutant mice a reduced Thl polarization and lower IFN- ⁇ and IL-10 levels in CD8+ T cells from mutant mice than from wild-type mice were found.
  • mice 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.
  • IL-12p40 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).
  • IL-12p40 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 ⁇ l subunit is the docking subunit for p40. The ⁇ 2 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 ⁇ l subunit of the IL-12 receptor.
  • the agonistic activities by IL-12p40 may be mediated by an unknown receptor molecule.
  • 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.
  • a dosage of between approximately 10 5 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).
  • 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.
  • the compounds of the present invention may be admixed with pharmaceutically inert, inorganic or organic excipients.
  • 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.
  • excipients which may be used include for example water, polyols, saccharose, invert sugar and glucose.
  • excipients which may be used include for example water, alcohols, polyols, glycerine, and vegetable oils.
  • excipients which may be used include for example natural or hardened oils, waxes, fats and semi-solid or liquid polyols.
  • 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.
  • 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.
  • 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 (F ⁇ llingsdorf/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.
  • 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.
  • cfu colony forming units
  • HKLM Heat killed L. monocytogenes
  • 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.
  • NACE naphthol-AS-D-chloroacetatesterase
  • 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.
  • mAbs 3.155 against CD8 and Ml/70.15.11.5 HL against Mac-1.
  • 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.).
  • mice were given recombinant murine monomeric or homodimeric IL-
  • Spleens were collected and erythrocyte-lysed single cell suspensions were stimulated at 5 x 10 6 cells per ml with HKLM (2 x 10 8 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.
  • IFN- ⁇ 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).
  • 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 MgCl 2 , 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 MgCl 2 , 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.
  • cDNAs were first standardized to levels of ⁇ 2-microglobulin transcripts. The calculated amounts of cytokine cDNA were then normalized with the ⁇ 2-microglobulin levels.
  • mice were infected with a sublethal Listeria dose of 500 cfu (low dose) or
  • 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 ( ⁇ m 2 x 10 3 )
  • mice 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.
  • SIS Zeiss microscope with image analysis software
  • 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 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.
  • IL-12p35 ⁇ / ⁇ and IL-12p40 ⁇ /" mice 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- ⁇ 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- ⁇ 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.
  • mutant mice showed a bacterial burden comparable to wild- type mice (see Fig. 2, day 5).
  • the reduced IFN- ⁇ and IL-10 transcript levels in mutant mice derived CD8 + T cells indicate regulation by IL-12p75.
  • 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- ⁇ protein were detectable in supernatants from ex vivo restimulated splenocytes derived from mutant mice (Fig. 4).
  • 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- ⁇ than T cells derived from wild-type mice.
  • similar levels of IFN- ⁇ were found in T cell cultures from treated or untreated IL-12p40"' ⁇ mice indicating that treatment with recombinant IL-12p40 does not regulate IFN- ⁇ production.
  • IL-12p40 is necessary for secondary responses
  • mice and IL-12p35 " ''" mice had already cleared the infection, whereas IL-12p40 ⁇ ' ⁇ mice were defective in clearing Listeria carrying up to 10 7 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.

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Abstract

L'invention concerne l'utilisation du IL-12p40 pour la préparation de médicaments permettant de traiter des maladies associées à l'immunité assurée par les lymphocytes T.
PCT/EP1998/001473 1997-03-19 1998-03-13 Utilisation du il-12p40 comme immunostimulant WO1998041229A1 (fr)

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AU70349/98A AU7034998A (en) 1997-03-19 1998-03-13 Use of il-12p40 as immunostimulant
PCT/EP1998/001473 WO1998041229A1 (fr) 1997-03-19 1998-03-13 Utilisation du il-12p40 comme immunostimulant

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001047546A2 (fr) * 1999-12-28 2001-07-05 Brown University Research Foundation Methodes pour l'immunotherapie antitumorale utilisant des cytokines et produits correspondants

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990005147A1 (fr) * 1988-11-10 1990-05-17 Genetics Institute, Inc. Facteur stimulateur de cellules cytotoxiques naturelles
EP0433827A2 (fr) * 1989-12-22 1991-06-26 F. Hoffmann-La Roche Ag Facteur de maturation de lymphocytes cytotoxiques et anticorps monoclonaux dirigés contre celui-ci
WO1992005256A1 (fr) * 1990-09-18 1992-04-02 Genetics Institute, Inc. Facteur stimulateur de cellules tueuses naturelles
US5571515A (en) * 1994-04-18 1996-11-05 The Wistar Institute Of Anatomy & Biology Compositions and methods for use of IL-12 as an adjuvant
WO1997000321A1 (fr) * 1995-06-14 1997-01-03 Commonwealth Scientific And Industrial Research Organisation Modulateurs de la reponse immunitaire et leur utilisation
WO1997046263A1 (fr) * 1996-06-05 1997-12-11 Powderject Vaccines, Inc. Therapie genique antitumorale par interleukine 12

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990005147A1 (fr) * 1988-11-10 1990-05-17 Genetics Institute, Inc. Facteur stimulateur de cellules cytotoxiques naturelles
EP0433827A2 (fr) * 1989-12-22 1991-06-26 F. Hoffmann-La Roche Ag Facteur de maturation de lymphocytes cytotoxiques et anticorps monoclonaux dirigés contre celui-ci
WO1992005256A1 (fr) * 1990-09-18 1992-04-02 Genetics Institute, Inc. Facteur stimulateur de cellules tueuses naturelles
US5571515A (en) * 1994-04-18 1996-11-05 The Wistar Institute Of Anatomy & Biology Compositions and methods for use of IL-12 as an adjuvant
WO1997000321A1 (fr) * 1995-06-14 1997-01-03 Commonwealth Scientific And Industrial Research Organisation Modulateurs de la reponse immunitaire et leur utilisation
WO1997046263A1 (fr) * 1996-06-05 1997-12-11 Powderject Vaccines, Inc. Therapie genique antitumorale par interleukine 12

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001047546A2 (fr) * 1999-12-28 2001-07-05 Brown University Research Foundation Methodes pour l'immunotherapie antitumorale utilisant des cytokines et produits correspondants
WO2001047546A3 (fr) * 1999-12-28 2001-12-06 Univ Brown Res Found Methodes pour l'immunotherapie antitumorale utilisant des cytokines et produits correspondants

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