SK8396A3 - Antagonists and agonists of human interleukin-10 - Google Patents

Abstract

Agonists and antagonists of human IL-10 are provided by this invention which are based upon modification of the termini of mature human IL-10. Also provided are compositions and methods for supplying or inhibiting the biological activity of human IL-10. Such compositions may be useful in the treatment of diseases characterized by inappropriate Th responses. Nucleic acids encoding the agonists and antagonists, recombinant vectors and transformed host cells comprising such nucleic acids, and methods for making the agonists and antagonists using the transformed host cells are also provided.

Description

Human interleukin-10 antagonists and agonists

Technical field

The present invention relates to human interieukin-10 antagonists and agonists, and to compositions and methods for their preparation and use. These agonists and antagonists are formed by substitution or deletion of the carboxy- or amino-terminus of amino acids in adult human interieukin-10.

BACKGROUND OF THE INVENTION

Interieukin-10 (IL-10) is a cytokine capable of mediating a number of actions or effects. ID10 has been isolated from both mouse and human cells and is involved in controlling the immune response of various classes or sets of CD4 ⁺ T helper (Th) cells. These Th cells can be divided into different pools that differ in their cytokine production profiles. Two of these pools are called Th1 and Th2 cells.

Th1 T cell clones consist of interieukin-2 (ILr2) and gamma interferon (IFN-γ), whereas Th2 T cell clones secrete IL-10, interieukin-4 (IL-4) and interleukin-5 (DL-5), generally after activation by antigens and lectins. Both classes of Th cell clones produce cytokines such as tumor necrosis factor-α (TNF-α), interleukin-3 (IL-3) and granulelocyte-macrophage colony stimulation factor (GM-CSF). The third category of Th cells (ThO) simultaneously comprises ID2, IFN-γ, IL-4, IL-5, TNF-α, IL-3 and GM-CSF.

Different types of Th1 and Th2 cytokine production by cells partly reflect their roles in response to various pathogens. For example, Th1 cells are involved in successful cellular responses to a variety of intercellular pathogens. They are also involved in delayed-type hypersensitivity reactions. Th2 cells are associated with humoral responses that are characterized by antibody production. In most situations, the immune system develops a Th response that is most effective in eliminating a particular antigen or pathogen, but not always.

For example: leishmaniasis is characterized by a defective Thl response. This defect can be demonstrated using in vitro assays, such as the assay described by Clerici et al. [J. Clin. Irrvest. 84: 1983 ($ 1 89)]. Using one such in vitro assay, it has been shown that a Th1 response defect can be attributed to endogenous levels of JL-10, since Th1 function can be corrected by the addition of neutralizing antibodies against IL-10.

Since leishmaniasis and other diseases are characterized by a defective Th1 response attributable to the inappropriate action of endogenous IL-10, there is a need for IL-10 agonists and antagonists to treat such diseases.

SUMMARY OF THE INVENTION

The present invention addresses this need by providing means and methods for providing or inhibiting the biological activity of human IL-10.

More specifically, the present invention provides human H 1 10 antagonists that include adult human IL-10 modified by replacing the lysine residue at position 157 with an acidic amino acid residue or by deleting one or more amino acid residues in a region containing about 12 carboxyterminal residues.

The amino acid sequences of such three embodiments are defined by the following sequences I, Π, and ΙΠ:

Ser Gly Gin Gly Thr Gin Ser Glu Asn Ser Cys Thr His Phe Pro

5 10 15

Gly Asn Leu For Asn Met

25 30

Val Lys Thr Phe Phe Gin Met

Lys Glu Ser Phu Leu Glu Asp Phe

5560

Leu Ser Glu Met Gin Phe Tyr Leu Glu Glu Val Met Pro Gin Ala 65 70 75 80 Glu same time gin Asp For Asp Lys Ala His Val Asn Ser Leu Gly Glu 85 90 95 same time Leu Lys Thr Leu Ar Leu Ar Leu Ar; g Arg Cy with His Arg Phe Leu 100 105 110 for Cys Glu same time Lys Ser Lys Ala Val Glu Gin Val Lys Asn, Ala Phe 115 120 125 same time Lys Leu gin Glu Lys Gly White Tyr Lys Ala Met Ser Glu Phe Asp 130 135 140

Phe Asn Tyr Glu Ala Tyr Met Thr Met Glu Arg Asn

145 150 155 160

Ser Gli Gin Gly Thr Gin Ser Glu Asn Ser Cys Thr His Phe Pro 15 1015

Gly Asn Leu For Asn Met

2530

Val Lys Thr Phe Phe Gin Met

Lys Glu Ser Phu Leu Glu Asp Phe

5560

Leu Ser Glu Met ile Gin Phe Tyr Leu Glu Glu Val Met Pro Gin Ala

70 7580

Glu Asn Gin Asp For Asp Lys Ala His Val Asn Ser Leu Gly Glu

9095

Asn Leu Lys Thr Arg Leu Arg Arg Leu Arg Arg Arg Cys His Arg Phe Leu

100 105110

For Cys Glu Asn Lys Ser Lys Ala Glu Gin Val Lys Asn Ala Phe

115 120125

Asn Lys Leu Gin Glu Lys Gly White Tyr Lys Ala Met Ser Glu Phe Asp

130 135140 White Phe White Asn Tyr White Glu Ala Tyr Met Thr Met Lys

145 150 155

Ser Gli Gin Gly Thr Gin Ser Glu Asn Ser Cys Thr His Phe Pro 15 1015

Gly Asn Leu For Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg 20 2530

Val Lys Thr Phe Phe Gin Met

Lys Glu Ser Leu Leu Glu Asp Phe Lys Glu Tyu Leu Gly Cys Gin Ala 50 5560

Leu Ser Glu Met Il Gin Phe Tyr Leu Glu Glu Val Met Pro Gin Ala 65 70 7580

Glu Asn Gin Asp For Asp Lys Ala His Val Asn Ser Leu Gly Glu

9095

Asn Leu Lys Thr Arg Leu Arg Arg Leu Arg Arg Arg Cys His Arg Phe Leu

100 105110

For Cys Glu Asn Lys Ser Lys Ala Glu Gin Val Lys Asn Ala Phe

115 120125

Asn Lys Leu Gin Lys Gly White Tyr Lys Ala Met Ser Glu Phe Asp 130 135140 White Phe White Asn Tyr White Glu Ala Tyr Met Thr Met

145150

155 (ΠΙ)

The invention further provides a nucleic acid encoding a human IL-10 antagonist, comprising adult human IL-10 modified by replacing the lysine residue at position 157 with an acidic amino acid residue or by deleting one or more amino acid residues in a region containing about 12 carboxyterminal residues. Recombinant vectors comprising such a nucleic acid and host cells comprising such a recombinant vector are also provided by the invention.

The invention further provides a method of producing a human IL-10 antagonist comprising adult human IL-10 modified by replacing a lysine residue at position 157 with an acidic amino acid residue or by deleting one or more amino acid residues in a region containing about 12 carboxyterminal residues. said host cells under conditions where the nucleic acid encoding the antagonist is expressed.

The present invention still further provides a method for inhibiting the biological activity of IL-10, comprising contacting cells carrying IL-10 receptors with an effective amount of a human IL-10 antagonist, including adult human IL-10 modified by replacement of the lysine residue at position 157 an acidic amino acid residue or a deletion of one or more amino acid residues in a region containing about 12 carboxyterminal residues.

The present invention further provides a human IL-10 agonist that includes adult human IL-10 modified by deletion of one to eleven amino-terminal amino acid residues.

Nucleic acids encoding these agonists, recombinant vectors, and transformed host cells comprising such nucleic acids, methods for preparing antagonists, and pharmaceutical compositions comprising one or more IL-10 agonists or antagonists and pharmaceutically acceptable carriers are also provided by the invention.

All references cited herein are hereby incorporated by reference in their entirety.

The antagonists of the invention are useful for the treatment of diseases such as leishmaniasis, which are characterized by a defective Th1 response attributable to endogenous IL-10. They may also be useful in the treatment of diseases associated with IL-10-mediated immunosuppression or IL-10 overproduction, such as B-cell lymphomas. In addition, antagonists are useful for studies illuminating the mechanism of action of IL-10 and for rational drug design, since they show strong receptor binding, separate from effector function. After immobilization on a solid support, the antagonists can be used for affinity purification of soluble forms of the IL10 receptor in which the transmembrane region is deleted.

The IL-10 viral protein (BCRFI, or vIL-10) of Epstein Bar Virus (EBV) also has the biological activity of IL-10 and is believed to bind to IL-10 receptors. It is believed that expression of vIL-10 activity of EBV gives the virus certain survival benefits in terms of its ability to be infected, replicated and maintained in the host organism. The ability of vIL-10 to negatively regulate IFN-γ production by both cells and NK cells, together with its enhancing effect on B cell viability, suggests that vIL-10 may suppress antiviral immunity while promoting the potential of EBV to transform human B cells.

The IL-10 antagonists of the invention may therefore be useful for effectively promoting viral immunity to EBV or other viruses. For more on the potential use of IL-10 antagonists, see e.g. in Howard et al. J. Clin. Immunol. 12: 239 (1992).

Three representative preparations of IL-10 mutant antagonists of the invention are set forth in the Examples below. In one instance, the new residue at position 157 of the adult human IL-10 sequence is replaced by a gyutamic acid residue (Sequence I). In other cases, three (sequence II) or four (sequence III) amino acid residues are deleted from the carboxyl terminus of human IL7. These antagonists are referred to below as K157E, CA3, and CA3 antagonists, respectively. CA4.

As used herein, the term "maternal human IL-10" is defined as a protein without a leader sequence (a) having an amino acid sequence substantially identical to sequence IV defined herein below

Ser Gli Gin Gly Thr Gin Ser Glu Asn Ser Cys Thr His Phe Pro 15 1015

Gly Asn Leu For Asn Met

2530

Val Lys Thr Phe Phe Gin Met

4045

Lys Glu Ser Phu Leu Glu Asp Phe

5560

Leu Ser Glu Met I Gin Phe Tyr Leu Glu Glu Val Met Pro Gin Ala

70 7580

Glu Asn Gin Asp For Asp Lys Ala His Val Asn Ser Leu Gly Glu

9095

Asn Leu Lys Thr Arg Leu Arg Arg Leu Arg Arg Arg Cys His Arg Phe Leu

100 105110

For Cys Glu Asn Lys Ser Lys Ala Glu Gin Val Lys Asn Ala Phe

115 120125

Asn Lys Leu Gin Glu Lys Gly White Tyr Lys Ala Met Ser Glu Phe Asp

130 135140 white Phe white Asn Tyr white Glu Ala Tyr Met Thr Met Lys white Arg Asn

145 150 155 160 (IV), and (b) has the biological activity common to native IL-1β. This includes natural allelic variants and other variants having one or more conservative and (b) has biological activity that is common to native ILr10. This includes natural allelic variants and other variants having one or more conservative amino acid substitutions [Grantham, Science 185,862 (1974)] that do not substantially impair biological activity. Such conservative substitutions include groups of synonymous amino acids, e.g. as described in U.S. Pat. No. 5,017,691 to Lee et al.

It will be understood that, although the following embodiments are currently preferred, other modifications of the carboxyl terminus of human IL-10 may be made to generate other antagonists. For example, it would be possible to produce an effective antagonist by replacing the lysine residue at position 157 with an aspartic acid residue instead of a glutamic acid residue. As used herein, the term "acidic amino acid residue" is thus defined to include both an aspartic acid residue and a glutamic acid residue.

More or less extensive deletions may also be performed. One or more amino acid residues may be deleted, including about 12 carboxyterminal residues. Preferably, about 8 terminal residues are deleted, more preferably 3 or 4 terminal residues.

Surprisingly, it has been found that up to 11 amino acid residues can be deleted from the amino terminus of adult human IL-10. These truncated variants, which may have different pharmacokinetic properties compared to ILr10 alone, have the biological activity of adult human IL-10 as measured by the MC / 9 mast cell stimulation assay. Therefore, they are useful in the treatment of all indications susceptible to treatment with IL-10 alone. They are also useful for some of the purposes described above for antagonists of the invention, e.g. for affinity purification.

Because they have the biological activity of human IL-10 but have a shortened amino acid sequence, these variants are also referred to herein as "human Π ^ ΙΟ agonists".

It is generally accepted that the cysteine residue at position 12 is essential for biological activity. Indeed: by deleting the first 12 residues, including this one

These aminothermal deletions may be combined with the above carboxyterminal modifications to form antagonists having the characteristics described below, but possibly other pharmacokinetic properties. These antagonists are also part of the invention.

Nucleic acids encoding IL-10 agonists and antagonists are also part of this invention. Those of skill in the art will recognize that due to the degeneracy of the genetic code, there are many different nucleic acids that can encode each agonist and antagonist. In particular, the codons used can be selected based on convenient construction and optimal expression in prokaryotic and eukaryotic systems.

Preferably, nucleic acids encoding agonists and antagonists are prepared using a polymerase chain reaction (PCR) [Saiki et al., Science 239: 487 (1988)], as exemplified in Daugherty et al. [Nucleic Acid Res. 19: 2471 (1991)], to modify the cDNK encoding human IL-10. Such cDNK is well known in the art and can be prepared using standard methods described e.g. in International Patent Application Publication No. WO 91/00349. Clones containing sequences encoding human IL-10 are also deposited at the American Type Culture Collection (ATCC), Rockville, Maryland, under numbers 68191 and 68192.

Alternatively, the DNA may be modified using well-known directed mutagenesis techniques. See e.g. Gillman et al., Gene 8: 81 (1979), Roberts et al., Nature 328: 731 (1987) or Innis (ed.), 1990, PCR Protocols: A Guide to Methods and Applications, Academic Press, New York, NY.

The nucleic acids of the invention can also be synthesized chemically using e.g. the solid phase phosphoamide method of Matteucci et al. [J. Am. Chem. Soc. 103: 3185 (1981)], the methods of Yoo et al. [J. biol. Chem. 764: 17078 (1989)] or other well known methods.

Recombinant vectors containing the preceding nullic acids are also part of this invention as well as host cells transformed with these vectors and methods for preparing agonists and antagonists.

Insertion of the DNA encoding one of the agonists and antagonists into one of a number of known expression vectors is readily accomplished, but the ends of the DNA and the vector contain compatible restriction sites. If this is not possible, it may be necessary to modify the ends of these DNAs and / or the vector by back-digesting the overlapping regions of the single stranded DNA created by restriction endonuclease digestion to create a blunt end or to achieve the same result by filling the single stranded ends with the appropriate DNA polymerase.

Alternatively, any desired site can be generated by ligating nucleotide sequences (linkers) to these termini. These linkers may contain specific oligonucleotide sequences that define the desired restriction site. The cleaved vector and DNA fragments can, if necessary, be modified by homopolymeric extension or PCR.

The antagonists of the invention are characterized by human IL-10 receptor binding affinities that are similar to IL-10 alone but are essentially devoid of biological activity. Preferably they will have less than 10% biological activity of human IL-10 in a standard assay, more preferably less than 1%.

Antagonists typically produce at least about 25% inhibition of IL-10 biological activity in cells that carry IL-10 receptors. Preferably, the degree of inhibition will be at least about 50%, more preferably at least 75%. The actual degree of inhibition may vary according to the particular biological activity measured.

Agonists and antagonists can be chemically synthesized by a suitable method, such as solely solid phase synthesis, partially solid phase methods, fragment condensation, or classical solution synthesis. Chemically synthesized polypeptides are preferably prepared by solid phase peptide synthesis, e.g. as described by Merrifield [J. Am. Chem. Soc. 85: 2149 (1963), Science 232: 341 (1986)] and Atherton et al., (Solid Phase Peptide Synthesis: A Practical Approach, 1989, IRL Press, Oxford).

Any agonist and antagonist prepared by any method can be purified using HPLC, gel filtration, ion exchange and partition chromatography, countercurrent separation or other well known methods.

The pharmaceutical compositions may be prepared by admixing one or more IL-10 agonists or antagonists, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

Useful pharmaceutical carriers can be any compatible non-toxic agent which is suitable for administering the compositions of the invention to a patient. The carrier may comprise water, alcohol, fats, waxes, and inert solids. A pharmaceutically acceptable adjuvant (buffering agent, dispersing agent) may also be incorporated into the pharmaceutical composition. In general, compositions useful for parenteral administration of such drugs are well known, e.g. Remington's Pharmaceutical Science, 18th Edition (Mack Publishing Company, Easton, P.A., 1990). Single-dose packaging is often preferred, e.g. in sterile form.

The administration of the agonists and antagonists is preferably parenteral, by intraperitoneal, intravenous, subcutaneous or mtramuscular injection or infusion, or any other acceptable systemic route. Alternatively, the antagonist may be administered by an implantable or injectable drug delivery system. Urquhart et al., Ann. Rev. Pharmacol. Toxicol. 24: 199 (1984), Lewis, ed., Controlled Release of Pesticides and Pharmaceuticals, 1981, Plenum Press, New York, New York, U.S. Pat. U.S. Patent Nos. 3,773,991 and 3,270,960]. Oral administration may also be accomplished by administering well known formulations that protect the antagonist from gastrointestinal proteases. See also Langer, Science 249: 1527 (1990).

Agonists and antagonists may also be administered by standard gene therapy techniques, including, e.g. direct injection of nucleic acid into tissues, use of recombinant viral vectors or liposomes, and implantation of transfected cells. See e.g. Rosenberg, J. Clin. Oncol. 10: 1992.

Agonists and antagonists may be administered alone or in combination with one or more other agents commonly used to treat conditions characterized by a defective Th response. For example, drugs such as interleukin-12 (IL-12) or gamma interferon (IFN-γ) may be co-administered with an antagonist. Insulin, cyclosporin, prednisone or azathioprine may be administered together with an agonist, e.g. when used as a replacement for IL-10 for the treatment or prevention of insulin-dependent diabetes mellitus (see co-pending U.S. application Ser. No. 07/955)

523 of October 1, 1992).

The co-administration of one or more agents may be concurrent (together with) or sequential (before or after) with respect to administration of the agonist or antagonist. All agents administered must be present in the patient at sufficient levels to be pharmaceutically effective. Typically, when the second agent is administered for about half-life of the first agent, the administration of the two agents is considered to be common.

Determination of the appropriate agonist or antagonist dosage for a particular situation is within the skill in the art. Generally, treatment is initiated at lower doses than optimal. Thereafter, the dosage is increased with small increments until the optimum effect under the conditions is reached. If desired, it may be appropriate to divide the total daily dose and administer it in portions.

An effective amount will be a dose that results in a demonstrable improvement in one or more clinical parameters or in a statistically significant improvement in response in one or more known Th functions; some of them, such as IL-2 production, are described above. This response can be measured in vitro using blood cells taken from a patient, e.g. as described in Clerici et al., supra. Such an in vitro assay may be performed prior to initiation of treatment to provide a baseline reference with which an improved response can be compared.

The actual amount and number of doses of agonists and antagonists and their pharmaceutically acceptable salts to a particular patient will be regulated at the discretion of the attending physician, taking into account factors such as the age, condition and size of the patient and the degree of symptom (s) being treated.

DETAILED DESCRIPTION OF THE INVENTION

The present invention can be explained by the following examples. Unless otherwise specified, the percentages given below for solids in solids, liquids in liquids, respectively. solids in liquids are based on the weight / weight, volume / volume, resp. weight / volume.

Reagents and general methods

Restriction endonucleases were obtained from Boehringer Mannheim (Indianapolis, IN), while the DNA ligation kit was purchased from Takara Biochem., Inc. (Berkley, CA). Taq polymerase and Pfu polymerase were obtained from Stratagene (La Jolla, CA). Recombinant human IL-10 (hIL-10) was prepared in a standard manner in Chinese hamster ovary (CHC) cells, essentially as described in Tsujimoto et al. [J. Biochem. 106: 23 (1989)]. Tissue culture medium, fetal bovine serum and glutamine were obtained from Gibco-BRL (Gaithesburg, MD). Oligonucleotide primers were synthesized by standard methods using an Applied Biosystems 380A, 380B or 394 DNA synthesizer (Foster City, CA).

Standard recombinant DNA methods were performed essentially as described by Sambrook et al. in Molecular Cloning: A Laboratory Manual, 2nd Edition, 1989, Cold Spring Harbor Laboratory Press, Plainview, New York.

transfection

Transient expression was performed as follows. COS cells (ATCC CRL 1651) were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 6 mM glutamine and penicillin / streptomycin. Transfection was performed by electroporation using Bio-Rad GENE PULSER® (Richmond, CA).

Cells were detached from the culture dishes by trypsin-EDTA treatment and suspended in fresh culture medium. About 5 x 10 ⁶ cells in a volume of 250 μΐ were mixed with 5 μg plasmid DNA and then electroporated at a voltage set at 0.2 V and a capacity set at 960 mFD.

After electroporation, cells were transferred to 10 cm culture dishes and cultured in 5% CO ₂ at 37 ° C for 6 hours in 10 ml DMEM-containing serum. When cells were attached to the dishes, the medium was removed by aspiration and replaced with serum-free medium. After 72 hours, the conditioned medium was collected for analysis.

Preparation of antagonists

Reconstruction of wild-type human IL-10 cDNK and expression vectors

To facilitate expression and manipulation, the coding region of the hIL-10 cDNK was generated by PCR using a hIL-10 vector based on pCDSRa [Viera et al., Proc. Natl. Acad. Sci. USA 88 '. 1172 (1991); the sequence deposited with GenBank under accession number M57627] as a template, although other known sources of this cDNK could be used.

Kozak's consistent vertebrate translation initiator [Kozak, Nucleic Acid Res. 20: 8125 (1987)] was introduced into the 5 'primer named B1789CC (sequence V):

GTCGACTGCA GCCGCCACCA TGCACAGCTC AGCACTGCTC TGTTGCCTGG TCCTCCTGAC 60 (V)

PslI site respectively. The EcoRI site was added to the 5 'primer of B1789CC, respectively. The 3 'primer named A1715CC (sequence VI):

ACGTCGAATT CTCAGTTTCG TATCTTCATT GTCATGTAGG C 41 (VI).

Using the above primers, hIL-10 cDNK was subjected to PCR in a 50 μΐ reaction mixture, with a 50 μΐ paraffin oil overlay, in a 0.5 ml Eppendorf tube. The reaction mixture typically contained 26.5 μΐ H ₂ O, 5 μΐ DNA polymerase buffer [final concentration in reaction: 10 mM Tris-HCl, pH 8.8, 50 mM KCl, 1.5 mM MgCl ₂ , 0.001% gelatin], 200 μΜ dlNTP, 60 ng template DNA, after 10 pmol of 5 'B1789CC and 3' A1715CC and 0.5 μΐ Taq polymerase (2 units) each.

The reaction was carried out in a PHC-1 thermocycler (Techne, Princeton, NJ) with 30 cycles of 95 ° C, 2 minutes for denaturation; 42 ° C, 2 minutes for duplex formation [annealing], and 70 ° C, 1 minute for synthesis. At the end of the 30th cycle, the reaction mixture was incubated for an additional 9 minutes at 72 ° C for extension.

The PCR mixture was subjected to electrophoresis in a 1.2% agarose Tris-acetate gel containing 0.5 µg / ml ethidium bromide. DNA fragments having the expected sizes were excised from the gel and purified with GENECLEAN ^R kit (La Jolla, CA). After gel extraction, the DNA product was digested with Pst I and Eco RI, isolated by gel electrophoresis and GENECLEAN ^R processing, and cloned as a Pst I / Eco ^{R I} restriction fragment into the pDSRG expression vector (ATCC 68233) and subsequently transferred to the pSC expression vector. , Gaithesburg, MD).

Vectors containing hIL-10 cDNKs were amplified in E. coli strain DH5α (Gibco-BRL) and the DNA sequence was verified by DNA sequencing. The hIL-10 expression vector, based on pSC.Sport, was used to transfect COS cells as well as to construct vectors with mutant hIL-10.

The resynthesized hIL-10 cDNK retained a unique BgBI site and a unique Äs / EII site, both present in wild-type cDNK. These two internal restriction sites, whose relative positions are schematically shown below, were then used to generate mutant hIL-10 cDNKs by cassette replacement.

Pst ---- BGM - ÄvZEII ------------------------- EcoRI

Carboxyterminal modification

To generate the C-terminal mutant ML-10 antagonist, mutant cDNK fragments corresponding to the wild-type λRI / FcoRI region of the hIL-10 wild-type cDNK were synthesized and used to replace the corresponding region in pSV. Sports the hIL-10 cDNK described above.

Mutant K157E, CA3, and CA4 antagonists of human hIL-10 cDNK were prepared by PCR using oligonucleotide primers complementary to the resynthesized hIL-10 cDNK sequence described above, with the proposed mutations pre-introduced into the 3'-end primers.

A 5 'primer named B3351CC, having an amino acid sequence defined by the VH sequence, was used to generate three mutant antagonists:

GGACTTTAAG GGTTACCTGG GTTGCCAAGC CTTGTCTGAG ATGATCCAGT TTTATCTAGA 60-GGAGGTGATG CCCCAAGCTG AGAAC 85 (VH)

The sequence of this 5 'primer was complementary to the internal sequence of the human hIL-10 cDNK, including the unique wild-type hIL-10 cDNK restriction site. The 3 'primers used to generate the antagonists had sequences complementary to the 3' terminal sequence of the cILK encoding hIL-10. These primers, followed by a sequence number defining their sequence, were as follows:

mutant: primer: Sequence number K157E C3481CC VIII CA3 C3482CC IX CA4 B3350CC X

AGCTGAATTC AGTTTCGTAT CTCCATTGTC ATGTAGGCTT CTATGTAGT 49 (in)

AGCTGAATTC ACTTCATTGT CATGTAGGCT TCTATGTAGT 40

AGCTGAATTC ACATTGTCAT GTAGGCTTCT ATGTAGT 37 (X).

Using the aforementioned primers, human IL-10 cDNK was subjected to PCR in a 50 μ zmesi reaction mixture, with a 50 μΐ paraffin oil overlay, in a 0.5 ml Eppendorf tube. The reaction mixture typically contained 26.5 μΐ H ₂ O, 5 μΐ DNA polymerase buffer [final concentration in reaction: 20 mM Tris-HCl, pH 8.2, 10 mM KCl, 2 mM MgCl ₂ , 6 mM (NH 4) ₂ SO , 0.1% Triton X-100 and 10 µg / ml bovine serum albumin (BSA) without nuclease], 200 μΜ dNTP, 40 ng template DNA, 10 pmol of 5 'primer B3351CC and one of the 3' primers and 0.5 μΐ pfu polymerase (2.5 units).

The reaction was carried out in a PHC-1 thermocycler (Techne, Princeton, NJ) with 22 cycles of 94 ° C, 2 minutes for denaturation; 50 ° C, 2 minutes for duplex formation; and 72 ° C, 1 minute for synthesis. At the end of cycle 22, the reaction mixture was incubated further

7.5 minutes at 72 ° C for extension.

The PCR mixture was processed by phenol-CHCl extraction, ethanol precipitation and then sequentially digested with Fe / EII and EcoRI. The restriction digestion products were electrophoresed in a 1% agarose / Tris-acetate acetate gel containing 0.5 µg / ml ethidium bromide. DNA fragments having the expected sizes were excised from the gel and obtained by phenol-CHCl ₃ extraction and ethanol precipitation.

After gel acquisition, BslEWEcoR1 fragments of hIL-10 mutants were used to replace the corresponding region of the hIL-10 wild-type DNA in the pSV.Sport vector. The hIL-10 mutant cDNKs in the pSV.Sport-based vector were propagated in E. coli strain DH5α and verified by DNA sequencing. The same expression vectors were used to transfect COS cells as described above.

Aminoterminal modification

To generate N-terminal variants of human IL-10, modified cDNK fragments corresponding to PsfUBg10 were synthesized by PCR. hIL-10 wild-type cDNA region, using a primer pair without the DNA template. The resulting fragments were used to replace the corresponding region of the hIL-10 wild-type DNA in the pSV.Sport vector. Thus, variants were generated in which 7 (variant ΝΔ7), 10 (variant ΝΔ10), 11 (variant ΝΔ11) and 12 (variant ΝΔ12) amino acids were deleted from the N-terminus of wild-type hIL-10. The primer pairs used to generate each variant with consecutive sequence numbers defining their sequences were as follows:

mutant: ΝΔ7 Primer (end): C3352CC (5 ') C3355CC (3 ') Sequence number XI XII ΝΔ10 C3353CC (5 ') XIII C3354CC (3 ') XIV

ΝΔ11 C3483CC (5 ') C3485CC (3 ') XV XVI ΝΔ12 C3484CC (5 ') XVII C3486CC (2) XVIII

GACGACGGTG GCTGCAGCCG CCACCATGCA CAGCTCAGCA CTGCTCTGTT GCCTGGTCCT 60

CCTGACTGGG GTGAGGGCCT CTGAGAACAG C 91 (XI)

GGCATCTCGG AGATCTCGAA GCATGTTAGG CAGGTTGCCT GGGAAGTGGG TGCAGCTGTT 60

CTCAGAGGCC TCTCGAGGAC 90 (ΧΠ)

GACGACGGTG GCTGCAGCCG CCACCATGCA CAGCTCAGCA CTGCTCTGTT GCCTGGTCCT 60

CCTGACTGGG GTGAGGGCCA GC 82 (XIII)

GGCATCTCGG AGATCTCGAA GCATGTTAGG CAGGTTGCCT GGGAAGTGGG TGCAGCTGTT 60

CCTCACCCCA GTCAGGAGGA C 81 XIV

GACGACGGTG GCTGCAGCCG CCACCATGCA CAGCTCAGCA CTGCTCTGTT GCCTGGTCCT 60

CCTGACTGGG GTGAGGGCCT GC 82

GGCATCTCGG AGATCTCGAA GCATGTTAGG CAGGTTGCCT GGGAAGTGGG TGCAGCTGTT 60

CACCCCAGTC AGGAGGAC 78

GACGACGGTG GCTGCAGCCG CCACCATGCA CAGCTCAGCA CTGCTCTGTT GCCTGGTCCT 60

CCTGACTGGG GTGAGGGCCA C81 (XVII)

GGCATCTCGG AGATCTCGAA GCATGTTAGG CAGGTTGCCT GGGAAGTGGG TGCAGCTGTT 60 CCCAGTCAGG AGGAC 75 (XVIII).

PCR was performed using 10 pmoles of each primer of said primer pairs as described above for the synthesis of C-terminal mutant antagonists. The PCR mixture was worked up by phenol-CHCl extraction, ethanol precipitation and then sequentially digested with Bgltt. and Psíl. The restriction digestion products were subjected to agarose gel electrophoresis as described above, and DNA fragments having the expected sizes were excised from the gel and obtained by phenol-CHCl extraction and ethanol precipitation.

After gel extraction, the PstI / BgM restriction fragments of hIL-10 variants were used to replace the corresponding region of the wild-type hIL-10 DNA in the pSV.Sport vector after digestion of this region by PstI / BgIII by digestion and ligation of the replacement fragment. The hIL10 mutant cDNKs in the pSV.Sport-based vector were propagated, verified and used as described above.

The resulting variants ΝΔ7, ΝΔ10, ΝΔ11 resp. ΝΔ12 had the amino acid sequences defined by residues 8-160, 11-160, 12-160, respectively. 13-160 of sequence IV, supra.

Antagonists of human IL-10 having N-terminal modifications

Antagonists having modifications at both the amino and carboxy termini can be readily prepared by combining the foregoing methods. For example, a ΝΔ7 / Κ157Ε antagonist can be generated by preparing a pSV.Sport vector containing a cDNK encoding a K157E antagonist by performing PCR using the 5 'primer B3351CC and the 3' primer C3481CC as described above. After preparation and isolation of the ΝΔ7 variant using the 5 'primer B3352CC and the 3' primer C3355CC as described above, a PstI / BglII restriction fragment of this variant is used to replace the corresponding K157E region of the mutant DNA in the pSV.Sport vector, after cleavage. of this region by PstU BglII by digestion and ligation of the replacement fragment.

Metabolic labeling

COS cells were transfected as described above with the pSV.Sport expression vector carrying cDNK inserts encoding human IL-10; K157E, CA3 or CA4 antagonists; or agonist variants of ΝΔ7, ΝΔ10, ΝΔ11 or ΝΔ12. Cells were then incubated in 10 cm culture dishes in 5% CO 3 at 37 ° C for 48-72 hours in serum containing culture medium. After this incubation, the culture plates were washed twice with phosphate salt buffer (PBS) and incubated in 5% CO ₂ at 37 ° C for 30 minutes with 8 ml / dish of DMEM without methionine and supplemented with dialyzed FBS and glutamine. The medium was removed by aspiration and replaced with 500 μΐ of methionine-free medium containing 250-300 pCi of ³⁵ S-methionine (DuPontNEN, Boston, MA; specific activity 43.3 mCi / ml).

The cells were incubated in 5% CO ₂ at 37 ° C for 5 hours, then 10 μΐ of a 1.5 mg / ml stock of L-methionine was added to the dishes and incubated for 30 minutes. The labeled conditioned medium was collected and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis [SDS PAGÉ; Laemmli, Nature 227 (680) (1970)] in 10 to 20% gels under non-reducing conditions, and the gels were dried and autoradiographed using standard methods and Kodak XAR film.

Autoradiography showed various labeled bands for human IL-10; K157E, CA3 and CA4 antagonists; and agon7 and ΝΔ10 agonists, all of whom migrated with apparent molecular weights of about 16 to 18 kilodaltons. When identical

Under the conditions of cell transfection and culture, all three antagonists, carboxytennin mutants of human IL-10, were expressed at a slightly reduced level of about 2 to 4 times less than IL-10. The ot7 amino-terminal agonist variant expression was comparable to IL-10, while the ΝΔ10 variant was expressed at a level about four-fold lower than IL-10. The expression of variants ΝΔ11 and ΝΔ12 was too low to be detected by this method.

ELISA analyzes

To further quantify levels of mutant human IL-10 antagonists in conditioned COS cell media, immunosorbent enzyme analysis (ELISA) assays were performed essentially as described by Abrams et al. [Immunol. Rev. 127: 5 (1992)]. Two monoclonal antibodies, specific for different epitopes of human IL-10, designated 9D7 and 12G8, were prepared by standard methods and used as capture and / or capture antibodies. detection reagent. Serially diluted conditioned media was tested in these assays using purified human IL-10 as a standard. The limit of detection in this assay was about 1 ng / ml and, in culture media after 72 hours of incubation, the levels of IL-10 were typically measured in the range of 100-300 ng / ml.

Thus, relative levels of IL-10 and antagonists have been found to correlate well with results obtained by metabolic labeling, suggesting that epitopes recognized by the monoclonal antibodies used are not in the mutated regions. In typical assays, the god for human IL-10, K157E, CA3, CA4, ΝΔ7, ΝΔ10, ΝΔ11, respectively. ΝΔ12 expression levels of 133, 80, 63, 48, 139, 28, 23, respectively. 6.5 ng / ml.

Biological tests

Human IL-10 and representative IL-10 mutant antagonists were assayed for activity using mouse mast cells and human peripheral mononuclear cells (PBMC).

The mast cell stimulation assay was performed essentially as described by O'Garrom et al. [Others. Immunol. 2, 821 (1990)] and Thompson-Snipes et al. [J. Exp. Med. 173, 507 (1991)]. Briefly: per 5 x 10 ³ MC / 9 cells (ATCC CRL 8306) per well in 100 μΐ assay medium [RPMI-1640 containing 10% fetal bovine serum (FBS), 50 μΜ β-mercaptoethanol, 2 mM glutamine and penicillin (streptomycin) in a 96-well microtiter plate, was treated for 48 hours with varying amounts of IL-10 or one of the IL-10 antagonists. Then, 25 microliters of 5 mg / ml MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] (Sigma, St. Louis, MO) was added to each well and the plate was incubated for 3 hours. up to 5 hours. Cells were then lysed using 10% SDS with 10 mM HCl and absorbance at 570 nm was measured.

Human IL-10 and variants ΝΔ7, ΝΔ10 and ΝΔ11 were active in this assay, but no activity was observed for variant ΝΔ12. None of the carboxytermally mutant antagonists were active even when tested at a concentration of up to 375 ng / ml (about 100 times the amount of IL-10 that exhibits high activity).

To measure how IL-10 antagonists inhibit lipopolysaccharide-induced cytokine synthesis (LPS), human peripheral mononuclear cells (PBMCs) from healthy donors were obtained and isolated by FICOLLe ^R gradient centrifugation [Boyum, Scand. J. Clin. Lab. Invest. Suppl: 77 (1966)]. Aliquots of PBMC were transferred to wells (10 ⁵ cells per well in 200 μΐ RPMI-1640 medium containing 5% FBS, penicillin / streptomycin, non-essential amino acids, sodium pyruvate and 2 mM glutamine) of 96 well microtiter plates.

Human IL-10 was added to some wells at a constant 100 µM concentration with or without a 100-fold molar excess (10 nM) of the IL-10 antagonist (as measured by ELISA). This was immediately followed by addition

LPS (Sigma) into each well to a final concentration of 80ng / ml. Positive and negative IL-10 controls were incubated in parallel with medium conditioned by COS cells transfected with a vector expressing IL-10 and IL-10, respectively. plasmid pSV.Sport. The latter control conditioned medium was used to dilute all samples. All assays were performed in duplicate and verified in subsequent assays using other cell batches.

Plates were incubated in a humidified 5% CO 2 atmosphere at 37 ° C for 24 hours, then supernatant liquids were collected and stored at -20 ° C for later analysis. Levels of IL-6, IL-α, and TNFα in the collected samples were measured by ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.

All antagonists were found to reverse the inhibitory activity of IL10 on cytokine synthesis in this assay, as shown in Table 1.

Table 1

Percentage of residual IL-10 activity *

sample IL-6 IL- TNF-? buffer 100 100 100 antibody 0 0 0 K157E 21 27 51 CA3 12 13 39 CA4 19 27 61

* The inhibitory effect of human IL-10 on the synthesis of labeled cytokines was measured in the presence of control buffer, a saturating amount of neutralizing anti-IL-10 monoclonal antibody, and a 100-fold molar excess of three IL-10 antagonists.

Similar assays performed with varying amounts of IL-10 mutant antagonists in the absence of IL-10 showed that no antagonist had inhibitory activity on cytokine synthesis. No inhibitory activity could be detected with any of the antagonists at concentrations up to 100 pM, including.

A mixed lymphocyte response (MLR) assay was performed to test the effect of IL-10 antagonists on T cell activity. Human PBMCs were isolated as described above. Stimulator PBMCs were prepared by treating these cells with 50 mg / ml mitomycin C (Sigma, St. Louis, MO) for 20 minutes at 37 ° C.

x 10 ⁵ corresponding PBMCs and stimulator cells were mixed in each well of a 96-well microtiter plate together with varying amounts of human IL-10 or one of K157E, CA3 or CA4 antagonists in a total amount of 200 µΐ (in triplicate). Cells were incubated with 5% CO ₂ at 37 ° C for 6 days, then the cultures received a pulse of 1 µCi of tritiated thymidine ([ ³ H] -TdR; 15.6 Ci / mmol, NEN, Boston, MA) per well for 16 hours. . Lysates were collected on a filter using a 96 well apparatus (Skatron, Inc., Sterline, VA) and measured in a β-counter (Pharmacia LKB Nuclear Inc., Gaithesburg, MD).

It has been found that antagonists do not have the ability to inhibit MLR at a concentration of 1 ng / ml. In contrast, human IL-10 exhibited 82% inhibition of MLR at this concentration.

Receptor Binding Assays

Pure human IL-10 (net of about 99%) was radioiodinated by the method ^r Enzymobead (Bio-Rad, Richmond, CA) according to manufacturer's instructions. Approximately 4 x 10 ⁵ transfected COS cells expressing human IL-10 receptor cDNK were pelleted by centrifugation at 200 xg for 10 minutes, washed in binding buffer (PBS, 10% fetal bovine serum, 0.1% NaN ₃ ), and resuspended in 200 xg. μΐ binding buffer containing [ ¹²⁵ I] -human IL-10 (specific radioactivity 225 pCi / pg) at 150 pM concentration, with serially diluted conditioned medium from COS cells expressing cDNA encoding human IL-10 or one of the mutant antagonists of the invention.

After incubation at 4 ° C for 2 hours, the cells were centrifuged at 200 xg for 10 minutes, each cell pellet was resuspended in 100 µL of IL-10-free binding buffer, overlaid with 200 ml of 10% glycerol in binding buffer in extended centrifuge tubes, centrifuged. at 200 xg for 10 minutes at 4 ° C and the tubes were rapidly frozen in liquid nitrogen. Cell pellets were then cut into measuring tubes and measured on a CLINGAMMA ^R 1272 counter (Pharmacia LKB). Non-specific binding was determined by binding in the presence of a 500 to 1000 fold molar excess of unlabeled human IL-

10th

The results are shown in Table 2, where it can be seen that all IL-10 antagonists were nearly as effective in competing for receptor binding as IL-10 alone.

Table 2 Inhibition of radiolabeled IL-10 binding sample IC ₅₀ o (pM) Human IL-10 100 K157E 136 ± 65 CA3 172 ± 28 CA4 120 ± 9

♦ The data presented, which are averages of 2 independent assays, are the concentrations of unlabeled human IL-10 or said IL-10 antagonist, which produced 50% inhibition of the binding of radiolabeled IL-10 to cellular receptors.

Many modifications and variations of this patent may be made without departing from the scope and scope thereof, as will be apparent to those skilled in the art. The specific embodiments described herein are provided by way of example only, and the invention is limited only by the terms of the appended claims.

Claims (12)

A human IL-10 antagonist comprising adult human Ib-10 modified by replacing a lysine residue at position 157 with an acidic amino acid residue or by deleting three or four amino acid residues in the carboxyterminal region. The antagonist of claim 1 having the amino acid sequence defined by sequence I, Π or m Ser Pro Gly Gin Thr Gin Ser Glu Asn Ser Cys Thr lEfis Phe Pro 15 1015 Gly Asn Leu For Asn Met 20 2530 Val Lys Thr Phe Phe Gin Met 35 4045 Lys Glu Ser Phu Leu Glu Asp Phe 50 5560 Le Glu Glu Met He Gin Phe Tyr Le Glu Glu Val Met Pro Gin Ala 65 70 7580 Glu same time gin Asp for Asp He Lys His His Val Asn Ser Gly Glu 85 90 95 same time Leu Lys Thr Leu Arg Leu Arg with Arg Phe Leu 100 105 110 for Cys Glu same time Lys Ser Lys Ala Glu Gin Val Lys Asn Ala, Phe 115 120 125 same time Lys Leu gin Glu Lys Gly He Tyr Ser Glu Phe Asp 130 135140 He Phe Ile Asn Tyr ile Glu Ala Tyr Met Thr Met Glu He Arg Asn 145 150 155160 Ser Glu Gin Gly Thr Gin Ser Glu Asn Ser Cys Thr His Phe Pro 1 5 1015 Gly Asn Leu For Asn Met 20 2530 Val Lys Thr Phe Phe Gin Lys Asp Gin Leu Asp Asn Leu Leu Leu (I) 35 4045 Lys Glu Ser Phu Leu Glu Asp Phe 50 55 60 Leu Ser Glu Met He Gin Phe Tyr Leu Glu Glu Val Met for gin Ala 65 70 75 80 Glu same time gin Asp For Asp He Lys Ala His wall Asn Ser Leu Gly Glu 85 90 95 Asn Leu Lys Thr Arg Leu Arg Arg Leu Arg Arg Cys Hís Arg Phe Leu 100 105110 For Cys Glu Asn Lys Ser Lys Ala Glu Gin Val Lys Asn Ala Phe 115 120125 Asn Lys Leu Gin Lys Gly He tyr Lys Ala Met Ser Glu Phe Asp 130 135140 Ile Phe He Asn Lyr De Glu Ala 'tyr Met Thr Met Lys 145 150 155 Ser Gli Gin Gly Thr Gin Ser Glu Asn Ser Cys Thr His Phe Pro 15 1015 Gly Asn Leu For Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg 20 2530 Val Lys Thr Phe Phe Gin Met 35 40 45 Lys Glu Ser Leu Leu Glu 'Tyi  Leu Gly Cys Gin Ala 50 55 60 Leu Ser Glu Met De Gin Phe Tyr Leu Glu Glu wall Met Pro Gin Ala 65 70 75 80 Glu same time gin Asp For Asp He Lys Ala His wall same time Ser Leu Gly Glu 85 90 95 Asn Leu Lys Thr Arg Leu Arg Arg Leu Arg Arg Arg Cys His Arg Phe Leu 100 105 110 for Cys Glu same time Lys Ser Lys Ala Val Glu Gin Val Lys Asn Ala, Phe 115 120 125 same time Lys Leu gin Glu Lys Ile Tyr Lys Ala Met Ser Glu Phe Asp 130 135 140 He Phe He Asn Tyr De Glu and Tyr Met Thr Met 145 150 155 (M) 3. A nucleic acid encoding a human IL-10 antagonist comprising an adult human 11 ^ 10 modified by replacing the lysine residue at position 157 with an acidic amino acid residue or by deleting three or four amino acid residues in the carboxyterminal region. A nucleic acid according to claim 3 encoding a human IL-10 antagonist having the amino acid sequence defined by sequence I, Π, or ΙΠ, as set forth in claim 2. A recombinant vector comprising the nucleic acid of claim 3, which is capable of directing expression of the nucleic acid. A host cell comprising the recombinant vector of claim 5. 3.1 A method of preparing a human IL-10 antagonist comprising adult human IL10 modified by replacing the lyses of a new residue at position 157 with an acidic amino acid residue or deleting one or more amino acid residues in a region containing 12 carboxyterminal residues, comprising culturing the host cells of claim 8 under conditions where the nucleic acid is expressed. 8. The method of claim 7, wherein the nucleic acid encodes a human IL-10 antagonist in which 1 to 11 amino acid residues have been deleted from the amino terminus. The method of claim 7, wherein the nucleic acid encodes a human IL-10 antagonist having the amino acid sequence defined by sequence I, Π, or Πζ as set forth in claim 3. 10. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a human IL-10 antagonist comprising adult human IL-10 modified by replacing the lysine residue at position 157 with an acidic amino acid residue or deleting three or four amino acid residues in the carboxyterminal region. Pharmaceutical composition according to claims 10 and 3, characterized in that the antagonist has an amino acid sequence defined by sequence I, Π or UL. Use of a human IL-10 antagonist, which comprises adult human IL-10 modified by replacing the lysine residue at position 157 with an acidic amino acid residue or deleting three or four amino acid residues in the carboxytemrinal region, for the manufacture of a medicament for inhibiting the biological activity of IL-10.