CN1127529A - Agonists and antagonists of human interleukin-10 - Google Patents

Abstract

The present invention provides agonists and antagonists of human IL-10 which are based on modification of the terminus of mature human IL-10. Compositions and methods for imparting or inhibiting the biological activity of human IL-10 are also provided. These compositions are useful in the treatment of diseases characterized by inappropriate Th responses. Nucleic acids encoding these agonists and antagonists, recombinant vectors and transformed host cells containing these nucleic acids, and methods for producing agonists and antagonists using transformed host cells are also provided.

Description

Agonists and antagonists of human interleukin-10

Background of the invention

The present invention relates to agonists and antagonists of human interleukin-10, compositions thereof and methods of making and using them. These agonists and antagonists are obtained by introducing amino acid substitutions or deletions at the carboxy-terminus and/or amino-terminus of mature human interleukin-10.

Interleukin 10 (IL-10) is a cytokine capable of mediating a number of actions or effects. IL-10 has been isolated from both mouse and human cells. IL-10 is involved in controlling the immune response of different classes or subsets of CD4 ⁺ T helper (Th) cells. These Th cells can be divided into different subgroups distinguished by their cytokine production patterns. Two of these subgroups are called Th1 and Th2 cells.

Th1 cell clones produce interleukin-2 (IL-2) and gamma-interferon (IFN-γ), while Th2 cell clones usually secrete IL-10, leukocytes after activation by antigen or mitogenic lectins Interleukin-4 (IL-4) and Interleukin-5 (IL-5). Both classes of Th cell clones also produce cells such as tumor necrosis factor-α (TNF-α), interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) white. The third type of Th cells (Th0) simultaneously produce IL-2, IFN-γ, IL-4, IL-5, TNF-α, IL-3 and GM-CSF.

The different cytokine production patterns of Th1 and Th2 cells reflect in part their roles in the response to various pathogens. For example, Th1 cells are involved in successful cell-mediated responses to various groups of intracellular pathogens. These cells are also involved in delayed-type hypersensitivity. Th2 cells are associated with the humoral response, which is characterized by antibody production. In most cases, the Th response generated by the immune system is most effective in clearing a particular antigen or pathogen, but not always.

Leishmaniasis, for example, is characterized by a defective Th1 response. This deficiency can be demonstrated using in vitro assays, such as that described by Clerici et al. (J. Clin. Invest. 84:1892, 1989). The association of defective Th1 responses with endogenous IL-10 levels was confirmed by using such an in vitro assay, in which Th1 function was restored by the addition of neutralizing anti-IL-10 antibodies.

Since leishmaniasis and other diseases are characterized by defective Th responses associated with inappropriate action of endogenous IL-10, agonists and antagonists of IL-10 are needed to treat these diseases.

Summary of the invention

The present invention fulfills the above needs by providing compositions and methods which provide or inhibit the biological activity of human IL-10.

More specifically, the present invention provides antagonists of human IL-10 comprising a lysine residue at position 157 replaced by an acidic amino acid residue, or a region containing about 12 carboxy-terminal residues. Mature human IL-10 modified by deletion of one or more amino acid residues.

The amino acid sequences of the three above-mentioned embodiments are defined by sequence numbers 1, 2 and 3 in the sequence table.

The present invention further provides a nucleic acid encoding a human IL-10 antagonist comprising a lysine residue at position 157 replaced by an acidic amino acid residue, or a deletion in a region containing about 12 carboxy-terminal residues. mature human IL-10 modified by one or more amino acid residues. The present invention also provides a recombinant vector containing the nucleic acid and a host cell containing the recombinant vector.

The present invention also provides a method for preparing an antagonist of human IL-10 comprising a lysine residue at position 157 replaced by an acidic amino acid residue, or a deletion in a region containing about 12 carboxy-terminal residues. A mature human IL-10 modified by one or more amino acid residues, the method comprising: culturing a host cell as described above under conditions for expressing a nucleic acid encoding the antagonist.

The present invention further provides a method of inhibiting the biological activity of IL-10, the method comprising: contacting a cell bearing an IL-10 receptor with an effective amount of a human IL-10 antagonist comprising a lysine due to 157 Mature human IL-10 modified with acid residues replaced by acidic amino acid residues or by deletion of one or more amino acid residues in a region containing about 12 carboxy-terminal residues.

The present invention further provides an agonist of human IL-10 comprising mature human IL-10 modified by deletion of 1-11 amino-terminal amino acid residues.

The present invention also provides a nucleic acid encoding the agonist, a recombinant vector containing the nucleic acid and a transformed host cell, a method for preparing the antagonist, and an IL-10 agonist or antagonist containing one or more IL-10 agonists or antagonists and a A pharmaceutical composition of a pharmaceutically acceptable carrier.

Detailed description of the invention

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

Antagonists of the invention are useful in the treatment of diseases such as leishmaniasis characterized by defective Th1 responses associated with endogenous IL-10. These antagonists are also useful in the treatment of diseases associated with IL-10-mediated immunosuppression or IL-10 overproduction, such as B-cell lymphomas. Furthermore, these antagonists are useful in studies aimed at elucidating the mechanism of action of IL-10 and in the rational design of drugs because they display strong receptor binding dissociated from effector functions. When immobilized on a solid support, these antagonists can be used for affinity purification of readily soluble forms of the IL-10 receptor that have lost the transmembrane domain.

The Epstein-Barr virus (EBV) viral IL-10 protein (BCRFI or vIL-10) also has the biological activity of IL-10, and it is speculated that it can bind to the IL-10 receptor. It is speculated that the vIL-10 activity expressed by EBV confers a viability advantage on the virus with respect to the ability to infect, replicate and/or maintain in the host. The ability of vIL-10 to negatively regulate IFN-γ production by T cells and NK cells, as well as its B cell viability-enhancing effect, suggest that vIL-10 can suppress antiviral immunity while enhancing the potential of EBV-transformed human B cells.

Therefore, the IL-10 antagonists of the present invention can also be used to effectively enhance antiviral immunity against EBV (and possibly other viruses). For a more detailed description of the potential use of IL-10 antagonists see Howard et al. (J. Clin. Immunol. 12:239, 1992).

The examples that follow disclose three representative embodiments of mutant IL-10 antagonists of the invention. In one embodiment, the lysine residue at position 157 of the mature human IL-10 sequence is replaced by a glutamic acid residue (sequence number 1). In another embodiment, three (sequence number 2) or four (sequence number 3) amino acid residues are deleted from the carboxy-terminus of human IL-10. These antagonists are referred to below as K157E, CΔ3 and CΔ4 antagonists, respectively.

The term "mature human IL-10" as used herein is defined as a protein lacking the leader sequence, which protein (a) has an amino acid sequence substantially identical to that defined by sequence number 4; (b) has an amino acid sequence identical to that of native IL-10 same biological activity. The term includes natural allelic and other variants having one or more conservative amino acid substitutions (Grantham, Science 185:862, 1974) that do not substantially affect biological activity. These conservative substitutions involve several sets of synonymous amino acids, as described, for example, in US Patent 5,017,691 to Lee et al.

It should be understood that while the above embodiments are presently preferred, other modifications to the carboxy terminus of human IL-10 may be made to generate other antagonists. For example, the lysine residue at position 157 can be replaced by an aspartic acid residue in place of a glutamic acid residue, resulting in a potent antagonist. Therefore, the term "acidic amino acid residue" as used herein is defined to include both aspartic acid residues and glutamic acid residues.

The degree of deletion can also be greater or lesser. One or more amino acid residues including about 12 carboxy-terminal residues may be deleted. Preferably about 8 terminal residues are deleted, more preferably 3 or 4 terminal residues are deleted.

It has now been surprisingly found that up to 11 amino acid residues can also be deleted from the amino terminus of mature human IL-10. These truncated variants, which may have different pharmacokinetic properties than IL-10 itself, possess the biological activity of mature human IL-10 as determined in the MC/9 mast cell stimulation assay described below. Thus, these variants can be used to treat any indication that is sensitive to treatment with IL-10 itself, as well as for some of the purposes described above for the antagonists of the invention, such as affinity purification.

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

However, the cysteine residue at position 12 is believed to be essential for biological activity. In fact, deletion of the first 12 residues including the cysteine residue produces a biologically inactive variant. Therefore, amino-terminal deletions were limited to deletions of one or more of the first 11 residues.

Such amino-terminal deletions can be combined with the carboxy-terminal modifications described above to generate antagonists with the characteristics described below but possibly different pharmacokinetic properties. These antagonists are also part of the invention.

Nucleic acids encoding IL-10 agonists and antagonists are also part of the invention. Of course, those skilled in the art will be well aware that, due to the degeneracy of the genetic code, there are many different nucleic acids that encode each agonist and antagonist. The specific codons used can be chosen for ease of construction and optimal expression in prokaryotic or eukaryotic systems.

Preferably, cDNA encoding human IL-10 is modified as described by Daugherty et al. (Nucleic Acids Res. 19:2471, 1991) and polymerase chain reaction (PCR) is used (Saiki et al., Science 239:487, 1988 ) to prepare nucleic acids encoding agonists and antagonists. Such cDNA is well known in the art and can be prepared using standard methods, for example as described in International Patent Application Publication WO 91/00349. Clones containing sequences encoding human IL-10 have also been deposited with the American Type Culture Collection (ATCC, Rockville, Maryland) under accession numbers 68191 and 68192.

Alternatively, DNA can be modified using well-known site-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 acid of the present invention can also be chemically synthesized, for example, using the method of Matteucci et al.'s phosphoramidite (phosphoramidite) solid phase carrier (J.Am.Chem.Soc.103:3185, 1981), the method of Yoo et al. (J.Boil Chem.764:17078, 1989) or other known methods.

Recombinant vectors containing the above nucleic acids, host cells transformed with the vectors, and methods for producing agonists and antagonists are also part of the present invention.

DNA encoding one of an agonist and an antagonist can be readily inserted into one of the many known expression vectors if both the DNA and the ends of the vector contain compatible restriction sites. If this is not possible, the ends of the DNA and/or vector must be modified, i.e., by re-digesting the single-stranded DNA overhang produced by restriction endonuclease cleavage, creating blunt ends, or by using an appropriate DNA polymerase The same effect is achieved by filling in the ends of single strands.

In addition, any desired site can be created by ligating nucleotide sequences (linkers) to the termini. These adapters may contain specific oligonucleotide sequences defining desired restriction sites. If necessary, the cleaved vector and DNA fragments can also be modified by adding homopolymer tails or PCR.

Antagonists of the invention are characterized by having a binding affinity for the human IL-10 receptor similar to that of human IL-10 itself, but are substantially inactive biologically. Preferably, these antagonists have less than about 10% of the biological activity of human IL-10, more preferably less than about 1%, as measured by standard assays.

These antagonists generally achieve at least about 25% inhibition of the biological activity of IL-10 in cells bearing the IL-10 receptor. The degree of inhibition is preferably at least about 50%, more preferably at least about 75%. The actual degree of inhibition will vary with the particular biological activity being measured.

Agonists and antagonists can also be chemically synthesized by suitable methods, for example by exclusion solid phase synthesis, partial solid phase, fragment condensation or classical solution synthesis. Chemically synthesized polypeptides are preferably prepared by solid-phase peptide synthesis, such as 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).

Agonists and antagonists, however prepared, can be purified, for example, by HPLC, gel filtration, ion exchange and partition chromatography, countercurrent distribution, and/or other known methods.

Pharmaceutical compositions can be prepared by mixing one or more IL-10 agonists or antagonists, or pharmaceutically acceptable salts thereof, with a physiologically acceptable carrier.

An acceptable pharmaceutical carrier can be any compatible nontoxic substance suitable for administering the compositions of the present invention to a patient. Carriers may contain sterile water, alcohols, fats, waxes and inert solids. Pharmaceutically acceptable adjuvants (buffers, dispersants) can also be incorporated into the pharmaceutical compositions. In general, compositions for parenteral administration of such drugs are well known (e.g., Remington's Pharmaceutical Science, 18th Ed., Mack Publishing Company, Easton, PA, 1990). Unit-dose packaging, eg, in sterile form, is often preferred.

Agonists and antagonists are preferably administered parenterally, ie, by intraperitoneal, intravenous, subcutaneous or intramuscular injection or infusion, or any other acceptable systemic method. In addition, antagonists may also be administered using implantable or injectable drug release systems (see, e.g., 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; US Patents 3,773,919 and 3,270,960). Oral administration may also be performed with known formulations that protect the antagonist from gastrointestinal proteases. See also Langer, Science 249:1527, 1990.

Agonists and antagonists can also be administered using standard gene therapy techniques, including direct injection of nucleic acids into tissues, use of recombinant viral vectors or liposomes, and implantation into transfected cells (see, e.g., Rosenberg, J. Clin. Oncol. .10:180, 1992).

Agonists and antagonists may be administered alone or in combination with one or more other agents commonly used in the treatment of disorders characterized by deficient Th responses. For example, drugs such as interleukin-12 (IL-12) or gamma-interferon (IFN-gamma) can be co-administered with the antagonist. If an agonist is used instead of IL-10 to treat or prevent insulin-dependent diabetes, it may be co-administered with insulin, cyclosporine, prednisone, or azathioprine (see co-pending U.S. application 07/955,523, 1992 Submitted October 1).

Such co-administration of one or more other agents may be performed concurrently (together) or sequentially (either before or after) with the administration of the agonist or antagonist. All agents administered should be present in the patient at levels sufficient to produce a therapeutic effect. In general, two agents are considered to be co-administered if the second agent is administered approximately within the half-life of the first agent.

Determination of the appropriate dosage of agonist or antagonist for a particular condition is within the ordinary skill of the art. In general, treatment is started with smaller doses that are less than optimal. The dose is then gradually increased until the optimum effect in the particular case is achieved. For convenience, the total daily dosage may be divided and administered during the day if desired.

An effective amount will be that which significantly improves one or more clinical parameters, and/or results in a statistically significant improvement in response to one or more known Th functions, some of which, such as IL-2 production, are already in described earlier. This response can be measured in vitro using blood cells collected from a patient, for example as described by Clerici et al. (supra). This in vitro assay can be performed prior to initiation of therapy, thereby providing a reference baseline. Improved responses can be compared to it.

The actual dosage and frequency of administration of agonists and antagonists, and pharmaceutically acceptable salts thereof, for a particular patient will be adjusted according to the judgment of the attending physician, taking into consideration factors such as the patient's age, physical condition and weight, and the factors such as the severity of symptoms.

Example

The invention is illustrated by the following examples. Unless otherwise indicated, percentages given below in solid-solid mixtures, liquid-liquid mixtures and solid-liquid mixtures are expressed in weight/weight, volume/volume and weight/volume, respectively.

Reagents and General Methods

Restriction endonucleases were obtained from Boerhringer Mannheim (Indianapolis, IN), and DNA ligation kits were purchased from Takara Biochem., Inc. (Berkeley, CA). Taq polymerase and Pfu polymerase were obtained from Stratagene (La Jolla, CA). Recombinant human IL-10 (hIL-10) was produced in Chinese hamster ovary (CHO) cells essentially as described by Tsujimoto et al. (J. Biochem. 106:23, 1989) by standard methods. Tissue culture medium, fetal calf serum and glutamine were purchased from Gibco-BRL (Gaithersburg, MD). Oligonucleotide primers were synthesized by standard methods using an Applied Biosystems Model 380A, 380B or 394 DNA Synthesizer (Foster City, CA).

Standard recombinant DNA methods were performed essentially as described by Sambrook et al. (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 the BioRad GENEPULSER( ^R) (Richmond, CA).

Cells were detached from the dish by trypsin-EDTA treatment and suspended in fresh medium. Approximately 5 x ¹⁰⁶ cells in a volume of 250 μl were mixed with 5 μg of plasmid DNA before electroporation. The voltage and capacitance were set at 0.2 volts and 960mFD, respectively.

After electroporation, cells were transferred to 10 cm dishes and incubated in 10 ml of serum-containing DMEM at 37 °C in 5% _CO for 6 h. After the cells have attached to the dish, the medium is aspirated and replaced with serum-free medium. After 72 hours, conditioned medium was collected for analysis.

Preparation of antagonists

Reconstruction of wild-type human IL-10 cDNA and expression vector

For the convenience of expression and manipulation, the hIL-10 vector based on pCDSRα (Vieira et al., Proc. The coding region of hIL-10 cDNA was generated by PCR method. But other known cDNA sources may have been used as well.

A Kozak vertebrate common translation initiation region (Kozak, Nucleic Acids Res. 20:8125, 1987) was introduced into the 5' primer (sequence number 5) designated B1789CC. In the 5' primer B1789CC and the 3' primer named A1715CC (sequence No. 6), a PstI site and an EcoRI site were introduced respectively.

Using the above primers, PCR was performed on hIL-10 cDNA in a 0.5 ml Eppendorf tube with a reaction mixture volume of 50 μl and a paraffin oil upper layer of 50 μl. The reaction mixture generally contains 26.5 μl H ₂ O, 5 μl Taq (Thermus aquaticus) DNA polymerase buffer (the final concentration in the reaction solution is: 10 mM Tris-HCl, pH 8.8, 50 mM KCl, 1.5 mM MgCl ₂ , 0.001 % (W/V) gelatin), 200 μM dNTPs, 60 ng template DNA, 10 picomoles each of 5' primer B1789CC and 3' primer A1715CC, 0.5 μl Taq polymerase (2 units).

The reaction was performed in a PHC-1 thermal cycler (Techne, Princeton, NJ) 30 times as follows: denaturation at 95°C for 2 minutes; annealing at 42°C for 2 minutes; synthesis at 70°C for 1 minute. After the 30th cycle, the reaction mixture was incubated at 72° C. for 9 minutes to carry out the elongation reaction.

The PCR mixture was electrophoresed on a 1.2% agarose Tris-acetate gel containing 0.5 μg/ml ethidium bromide. A DNA fragment of the expected size was excised from the gel and purified using the GENECLEAN( ^R) kit (La Jolla, CA). After recovery from the gel, the product DNA was digested with PstI and EcoRI, separated by gel electrophoresis and ^GENECLEAN® treatment, and cloned into the expression vector pDSRG (ATCC68233) as a PstI/EcoRI restriction fragment, and then transferred to the expression vector pSV. Sport (Gibco-BRL, Gaithersburg, MD).

The vector containing hIL-10 cDNA was propagated in Escherichia coli DH5α strain (Gibco-BRL), and the DNA sequence was confirmed by DNA sequencing. The hIL-10 expression vector based on pSV.Sport was used for COS transfection and the construction of mutant hIL-10 vector.

The resynthesized hIL-10 cDNA retained the unique BglII site and the unique BstEII site, both of which were present in the wild-type cDNA. These two internal restriction sites were later used to generate mutant hIL-10 cDNA by cassette replacement, and the relative positions of these two sites are shown schematically below:

PstI--------BgIII-----------BstEII-----------EcoRI

Carboxy-terminal modification

In order to produce hIL-10 C-terminal mutant antagonists, mutant cDNA fragments corresponding to the BstEII/EcoRI region of wild-type hIL-10 cDNA were synthesized by PCR, and these fragments were used to replace the corresponding regions in the above pSV.Sport hIL-10DNA.

Utilizing oligonucleotide primers complementary to the above resynthesized hIL-10 cDNA sequence, and pre-introducing specified mutations in the 3' end primers, the K157E, CΔ3 and CΔ4 mutation antagonists of human IL-10 were produced by PCR.

Three mutant antagonists were generated using a 5' primer designated B3351CC having the amino acid sequence defined by sequence number 7. The sequence of this 5' primer is complementary to the internal sequence of the human IL-10 cDNA containing the unique BstEII restriction site of the wild-type hIL-10 cDNA. The sequence of the 3' primer used to prepare the antagonist was complementary to the sequence at the 3' end of the cDNA encoding hIL-10. These primers and the sequence numbers defining their sequence are as follows:

Mutant Primer Sequence No.

K157E C3481CC 8

CΔ3 C3482CC 9

CΔ4 B3350CC 10

Using the above primers, PCR was performed on hIL-10 cDNA in a 0.5 ml Eppendorf tube with a reaction mixture volume of 50 μl and a paraffin oil upper layer of 50 μl. The reaction mixture generally contains 26.5 μl H ₂ O, 5 μl Pfu DNA polymerase buffer (the final concentration in the reaction solution is: 20 mM Tris-HCl, pH 8.2, 10 mM KCl, 2 mMMgCl ₂ , 6 mM (NH ₄ ) ₂ SO ₄ , 0.1% Triton x-100, 10 μg/ml nuclease-free bovine serum albumin (BSA)), 200 μM dNTPs, 40 ng template DNA, 10 pmol each of 5' primer B3351CC and one 3' primer, 0.5 μl pfu polymerized Enzyme (2.5 units).

The reaction was performed in a PHC-1 thermal cycler (Techne, Phnceton, NJ) for 22 cycles as follows: denaturation at 94°C for 2 minutes; annealing at 50°C for 2 minutes; synthesis at 72°C for 2 minutes. After the 22nd cycle, the reaction mixture was incubated at 72° C. for 7.5 minutes to carry out the elongation reaction.

The PCR mixture was processed by phenol- _CHCl3 extraction and ethanol precipitation, followed by digestion with BstEII and EcoRI sequentially. The restriction digests were electrophoresed on a 1% agarose/Tris-acetate gel containing 0.5 μg/ml ethidium bromide. DNA fragments of the expected size were excised from the gel and recovered by phenol- _CHCl3 extraction and ethanol precipitation.

After recovery from the gel, the corresponding region of the wild-type hIL-10 DNA in the pSV.Sport vector was replaced with the BstEII/EcoRI restriction fragment of the hIL-10 mutant. The hIL-10 mutant cDNA based on pSV.Sport was propagated in Escherichia coli DH5α strain and confirmed by DNA sequencing. COS cells were transfected with the same expression vector as described above.

N-terminal modification

To generate N-terminal variants of human IL-10, a modified cDNA fragment corresponding to the PstI/BglII region of wild-type hIL-10 cDNA was synthesized by PCR using a primer pair but without a DNA template. The corresponding region of the wild-type hIL-10 DNA in the pSV.Sport vector was replaced with the resulting fragment. This resulted in variants in which 7 (variant NΔ7), 10 (variant NΔ10), 11 (variant NΔ11) or 12 (variant NΔ12) were deleted from the N-terminus of wild-type hIL-10 residues. The primer pairs used to prepare each variant and the sequence numbers defining their sequence are as follows:

Variant Primer (end) Sequence number

NΔ7 C3352CC(5’) 11

C3355CC(3’) 12

NΔ10 C3353CC(5’) 13

C3354CC(3’) 14

NΔ11 C3483CC(5’) 15

C3485CC(3’) 16

NΔ12 C3484CC(5’) 17

C3486CC(3’) 18

PCR was performed with 10 picomoles of each primer of the indicated primer pairs as described previously for the synthesis of antagonists of C-terminal mutations. The PCR mixture was processed by phenol- _CHCl3 extraction and ethanol precipitation, followed by digestion with BglII and PstI sequentially. Restriction digests were electrophoresed on an agarose gel as described above, and DNA fragments of the expected size were excised from the gel and recovered by phenol- _CHCl3 extraction and ethanol precipitation.

After recovery from the gel, the corresponding region of the wild-type hIL-10 DNA in the pSV.Sport vector was excised by PstI/BglII digestion and ligated into the PstI/BglII restriction fragment of the hIL-10 variant, thereby replacing the wild-type with these fragments Corresponding region in hIL-10 DNA. The pSV.Sport-based hIL-10 mutant cDNA was propagated, confirmed and used as described above.

The resulting NΔ7, NΔ10, NΔ11 and NΔ12 variants have amino acid sequences defined by residues 8-160, 11-160, 12-160 and 13-160 of Sequence No. 4, respectively.

Human IL-10 antagonists with N-terminal modification

Combining the above methods facilitates the preparation of antagonists with both amino- and carboxy-terminal modifications. For example, an NΔ7/K157E antagonist can be prepared by PCR using 5' primer B3351CC and 3' primer C3481CC as described above to generate pSV.Sport containing a cDNA encoding a K157E antagonist. After preparing and isolating the NΔ7 variant fragment using 5' primer C3352CC and 3' primer C3355CC as described above, the corresponding region of the K157E mutant DNA in the pSV.Sport vector was replaced with the PstI/BglII restriction fragment of this variant by PstI This region was excised by /BglI digestion and a replacement fragment was ligated in.

metabolic markers

COS cells were transfected as described above with the expression vector pSV.Sport carrying a cDNA insert encoding human IL-10; the antagonist K157E, CΔ3 or CΔ4; or the agonist variants NΔ7, NΔ10, NΔ11 or NΔ12. Cells were then cultured in serum-containing medium for 48-72 hours in 10 cm dishes. After this culture, the culture dish was washed twice with phosphate buffered saline (PBS), and then incubated at 37°C _in 5% CO for 30 minutes. This time, 8ml/petri dish was added with dialyzed FBS and glutamic acid. Amide-free DMEM medium. The medium in each dish was aspirated and replaced with 500 μl of methionine-free medium containing 250-300 μCi ³⁵ S-methionine (DuPont NEN, Boston, MA; specific activity 43.3 mCi/ml).

Cells were incubated at 37 °C _in 5% CO for 5 h, then 10 μl of 1.5 mg/ml L-methionine stock solution was added to the dish and chased for 30 min. Labeled conditioned medium was collected and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE; Laemmli, Nature 227:680, 1970) in a 10-20% gel under non-reducing conditions, gel dried Autoradiography was then performed using standard methods and Kodak XAR film.

Autoradiography showed distinctly labeled bands for human IL-10; antagonists K157E, CΔ3 and CΔ4 and agonists NΔ7 and NΔ10, all migrating at an apparent molecular weight of approximately 16-18 kilodaltons. Under the same transfection and cell culture conditions, the expression levels of the three antagonists of the carboxy-terminal mutation of human IL-10 were all slightly lower, about 2-4 times lower than that of IL-10. Expression of the N-terminal agonist variant NΔ7 was comparable to that of IL-10, whereas the expression level of the NΔ10 variant was approximately 4-fold lower than that of IL-10. The expression of variants NΔ11 and NΔ12 was too low to be detected by this method.

ELISA analysis

For further quantitative analysis of mutant antagonist and human IL-10 levels in COS cell conditioned medium, enzyme-linked immunosorbent assay (ELISA) was performed essentially as described by Abrams et al. (Immunol. Rev. 127:5, 1992). Two monoclonal antibodies specific for different epitopes on human IL-10 were prepared by standard methods and designated 9D7 and 12G8, respectively, and were used as capture and detection reagents, respectively. Serial dilutions of conditioned media were tested in this assay and purified recombinant human IL-10 was used as a standard. The limit of detection of this assay is approximately 1 ng/ml, and after 72 hours of culture, IL-10 levels were typically measured in the range of 100-300 ng/ml in the culture medium.

The relative levels of IL-10 and antagonist were found to correlate well with the results obtained from metabolic labeling using the above method, indicating that the epitope recognized by the monoclonal antibody used was not in the mutated region. In a typical assay, the measured expression levels of human IL-10, K157E, CΔ3, CΔ4, NΔ7, NΔ10, NΔ11, and NΔ12 were 133, 80, 63, 48, 139, 28, 23, and 6.5 ng, respectively /ml.

biometrics

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

Mast cell stimulation assays were performed essentially as described by O'Garra et al. (Int. Immunol. 2:821, 1990) and Thompson-Snipes et al. (J. Exp. Med. 173:507, 1991). Briefly, 5×10 ³ MC/9 cells (ATCCCRL8306) were added to each well of a 96-well microtiter plate in 100 μl assay medium (RPMI-1640 containing 10% fetal bovine serum (FBS), 50 μM β- mercaptoethanol, 2 mM glutamine, and penicillin/streptomycin), were treated with varying amounts of human IL-10 or an IL-10 antagonist for 48 hours. Then 25 μl of 5 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma, St.Louis, MO) was added to each well, The titer plate was incubated for 3-5 hours. Cells were then lysed with 10% SDS containing 10 mM HCl, and absorbance was measured at 570 nm.

Human IL-10 and variants NΔ7, NΔ10 and NΔ11 were active in this assay, but variant NΔ12 was not observed to be active. None of the carboxy-terminal mutant antagonists were active, even when tested at concentrations as high as 375 ng/ml (approximately 100 times the amount of IL-10 that produced strong activity).

To determine the inhibitory effect of IL-10 antagonists on lipopolysaccharide (LPS)-induced cytokine synthesis, human peripheral blood mononuclear cells (PBMC) were obtained from healthy donors and separated by FICOLL® gradient centrifugation (Boyum, Scandinavia). . J. Clin. Invest. Supp 1:77, 1966). Aliquots of PBMC were transferred to individual wells of a 96-well microtiter plate ( ¹⁰⁵ cells/well in 200 μl RPMI-1640 medium containing 5% FBS, penicillin/streptomycin, non-essential amino acids, sodium pyruvate and 2mM glutamine).

Human IL-10 was added at a fixed concentration of 100 pM in some wells with or without a 100-fold molar excess (10 nM) of IL-10 antagonist (as determined by ELISA). LPS (Sigma) was then immediately added to each well to a final concentration of 80 ng/ml. Positive and negative IL-10 controls were incubated in parallel using the culture medium of COS cells transfected with IL-10 expressing vector or plasmid pSV.Sport, respectively. The conditioned medium of the latter control was used as a diluent for all samples. All assays were performed in duplicate and confirmed with subsequent assays using different batches of cells.

The titer plate was incubated at 37°C for 24 hours in a humidified atmosphere of 5% CO ₂ , then the supernatant was collected and stored at -20°C for later analysis. The levels of IL-6, IL-1α and TNFα in the collected samples were determined using ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.

All antagonists were found to reverse the inhibitory activity of IL-10 on cytokine synthesis in this assay, as shown in Table 1.

Table 1

% Residual IL-10 Activity ^*

Sample IL-6 IL-1α TNF-α

Buffer 100 100 100

Antibody 0 0 0 0

K157E 21 27 51

CΔ3 12 13 39

CΔ4 19 27 61

*The inhibitory effect of human IL-10 on the synthesis of the indicated cytokines was determined in the presence of control buffer, saturating amounts of neutralizing anti-IL-10 monoclonal antibodies, and 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 none of the antagonists had cytokine synthesis inhibitory activity. No inhibitory activity was detected for any antagonist at concentrations up to and including 100 pM.

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

In each well of a 96-well microtiter plate, mix about 1× ¹⁰⁵ response PBMCs and stimulator cells, and add different amounts of human IL-10 or one of K157E, CΔ3 or CΔ4 antagonists at the same time. The volume was 200 μl (triplicate experiments). Cells were incubated at 37°C with 5% CO ₂ for 6 days, and then 1 μCi of tritiated thymidine ([ ³ H]-TdR, 15.6 Ci/mmol, NEN, Boston, MA, ), which lasted 16 hours. The lysates were collected onto filters with a 96-well cell harvester (Skatron, Inc., Sterline, VA) and counted with a β-counter (Pharmacia LKB Nuclear Inc., Gaithersburg, MD).

These antagonists were found to be unable to inhibit MLR at a concentration of 1 ng/ml. In contrast, human IL-10 achieved 82% inhibition of MLR at this concentration.

receptor binding assay

Purified human IL-10 (approximately 99% pure) was radioiodinated using the ENZYMOBEAD ^(R) method (BioRad, Richmond, CA) following the manufacturer's instructions. Approximately 4 x ¹⁰ transfected COS cells expressing human IL-10 receptor cDNA were pelleted by centrifugation at 200 x g for 10 min, washed with binding buffer (PBS, 10% fetal bovine serum, 0.1% NaN ₃ ), and resuspended in 200 μl binding buffer containing [ ¹²⁵ I]human IL-10 (225 μCi/μg specific radiation) at a concentration of 150 pM, and at the same time, serially diluted conditioned medium of COS cells expressing human IL-10 encoding - 10 or the cDNA of 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 at the same temperature. The supernatant was then removed, and each cell pellet was resuspended in binding buffer without labeled IL-10, plated in 200 μl of binding buffer containing 10% glycerol in an elongated centrifuge tube, and incubated at 4°C. Centrifuge at 200 x g for 10 min and snap freeze in liquid nitrogen. Cell pellets were then cut into counting tubes and counted in a ^CLINIGAMMA® 1272 counter (PharmaciaLKB). Binding was performed in the presence of a 500- to 1000-fold molar excess of unlabeled human IL-10 to determine non-specific binding.

The results are shown in Table 2. As can be seen from Table 2, all IL-10 antagonists were nearly as effective as IL-10 itself in competing for receptor binding.

Table 2

Inhibition of radiolabeled IL-10 binding ^*

Sample _IC50 (pM)

Human IL-10 100

K157E 136±65

CΔ3 172±28

CΔ4 120±9

*The data shown are the average of 2 independent determinations and give the concentration of unlabeled human IL-10 or the indicated IL-10 antagonists that achieves a 50% inhibition of the binding of radiolabeled human IL-10 to the cellular receptor.

It will be apparent to those skilled in the art that many modifications and variations can be made in the present invention without departing from the spirit and scope of the invention. The specific embodiments described herein are given by way of example only, and the invention is to be limited only by the appended claims.

sequence table

(1) General information

(i) Applicant: Schering Corporation

(ii) Title of invention: agonists and antagonists of human interleukin-10

(iii) Sequence number: 18

(iv) Mailing address:

(A) Recipient: Schering-Plough Corporation

(B) Street: One Giralda Farms

(C) City: Madison

(D) State: New Jersey

(E) Country: USA

(F) Zip code: 07940

(v) in computer readable form:

(A) Media type: floppy disk

(B) Computer: Apple Macintosh

(C) Operating system: Macintosh 7.1

(D) Software: Microsoft Word5.1a

(vi) This application data

(A) Application Number: (B) Filing Date (C) Classification: (vii) Prior Application Data: US Patent Application 08/098,943(viii) Attorney Information: (A) Name: Lunn, Paul, G. (2 ) No. 1 sequence information: (i) Sequence feature: (A) Length: 160 amino acids (B) Type: amino acid (D) Topology: linear (ii) Molecular type: peptide (xi) Sequence description: No. 1 sequence: Ser Pro GLN GLN GLE GLN Sern Sern Sern Thr His PHE PRO1 5 10 15Gly Asn Leu Pro ASP Leu ARG ARG Ala Phe Serg

20 25 25 30Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu

35 40 45Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

50 55 60leu Serite Ile Gln PHE TYR Leu Glu Glu Val Met Pro Gln Ala65 75 80GLU Asn Gln ASP ILE LYS ALA His Val Leu GLY GLU

85 90 95 Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu

100 105 110Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe

115 120 125Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp

130                 135                 140Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Glu Ile Arg Asn145                 150                 155                 160(2)2号顺序信息：(i)顺序特征：(A)长度：157个氨基酸(B)类型：氨基酸( D) Topology: Linear (II) molecular type: peptide (xi) order description: 2 No. 2: Ser Pro GLY GLN GLN GLN Sern Sern Sern Thr HIS PHE PRO1 5 10 15Gly Asn Leu ARG Asn Met Leu ARG ASP Leu Arg Asp Ala Phe Ser Arg

20 25 25 30Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu

35 40 45Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

50 55 60leu Serite Ile Gln PHE TYR Leu Glu Glu Val Met Pro Gln Ala65 75 80GLU Asn Gln ASP ILE LYS ALA His Val Leu GLY GLU

85 90 95Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu

100 105 110Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe

115 120 125Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp

135 135 140ILE ILE Asn Tyr Ile Glu Ala Tyr Met THR MET LYS145 150 155 (2) Sequence Information: (i) Sequence Features: (A) length: 156 amino acids (b) Type: amino acid (d) topology structure : Linear (II) molecular type: peptide (xi) order description: 3rd order: Ser Pro GLY GLN GLN GLN Sern Sern Ser Cys Thr His PHE PRO1 5 10 15GLY Asn Met Leu ARG ARG ARG ARG Asp Ala Phe Ser Arg

20 25 25 30Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu

35 40 45Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

50 55 60leu Serite Ile Gln PHE TYR Leu Glu Glu Val Met Pro Gln Ala65 75 80GLU Asn Gln ASP ILE LYS ALA His Val Leu GLY GLU

85 90 95Asn Leu Lys Thr Leu Arq Leu Arg Leu Arg Arg Cys His Arg Phe Leu

100 105 110Pro Cys Glu Asn Lys Set Lys Ala VaL Glu Gln Val Lys Asn Ala Phe

115 120 125Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp

130 135 140ILE ILE Asn Tyr Ile Glu Ala Tyr Met THR MET145 150 155 (2) Sequence information: (i) Sequence features: (A) length: 160 amino acids (b) Type: amino acid (d) topology structure: Linear (II) molecular type: peptide (xi) order description: 4 order: Ser Pro GLY GLN GLY THR GLN Sern Sern Ser Cys Thr His PHE PRO1 5 10 15Gly Asn Met Leu ARG Asp Ala PHE Ser Arg

20 25 25 30Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu

35 40 45Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

50 55 60leu Serite Ile Gln PHE TYR Leu Glu Glu Val Met Pro Gln Ala65 70 80GLU Asn Gln Asp Ile Lys Ala His Val Leu GLY GLU

85 90 95 Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu

100 105 110Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe

115 120 125Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp

130 135 140ILE ILE Asn Tyr Ile Glu Ala Tyr Met THR MET LYS ILE ARG Asn145 150 155 160 (2) Sequence Information: (i) Sequence Features: (A) length: 60 base pair (b) type: Nucleic acid (C) chain number: single strand (D) topology: linear (xi) sequence description: No. 5 sequence: GTCGACTGCA GCCGCCACCA TGCACAGCTC AGCACTGCTC TGTTGCCCTGG TGTTGCCTGG TCCTCCTGAC 60 (2) No. 6 sequence information: (i) sequence feature: (A ) length: 41 base pairs (B) type: nucleic acid (C) chain number: single strand (D) topology: linear (xi) sequence description: 6 sequence: ACGTCGAATT CTCAGTTTCG TATCTTCATT GTCATGTAGG C 41(2)7 Sequence Information: (i) Sequence Features: (A) Length: 85 base pairs (B) Type: Nucleic Acid (C) Chain Number: Single Strand (D) Topology: Linear (xi) Sequence Description: No. 7 Sequence: GGACTTTAAG GGTTACCTGG GTTGCCAAGC CTTGTCTGAG ATGATCCAGTTTTATCTAGA 60GGAGGTGATG CCCCAAGCTG AGAAC 85(2) No. 8 sequence information: (i) sequence characteristics: (A) length: 49 base pairs (B) type: nucleic acid (C) strand number: single strand (D ) Topology: Linear (xi) Sequence Description: Sequence No. 8: AGCTGAATTC AGTTTCGTAT CTCCATTGTC ATGTAGGCTT CTATGTAGT 49 (2) Sequence No. 9 Information: (i) Sequence Features: (A) Length: 40 base pairs (B) Type: Nucleic acid (C) chain number: single strand (D) topology: linear (xi) sequence description: No. 9 sequence: AGCTGAATTC ACTTCATTGT CATGTAGGCT TCTATGTAGT 40 (2) No. 10 sequence information: (i) sequence characteristics: (A) length: 37 base pairs (B) type: nucleic acid (C) chain number: single strand (D) topology: linear (xi) sequence description: No. 10 sequence: AGCTGAATTC ACATTGTCAT GTAGGCTTCT ATGTAGT 37 (2) No. 11 sequence information: ( i) Sequence characteristics: (A) Length: 91 base pairs (B) Type: Nucleic acid (C) Chain number: Single strand (D) Topology: Linear (xi) Sequence description: No. 11 Sequence: GACGACGGTG GCTGCAGCCG CCACCATGCA CAGCTCAGCA CTGCTCTGTT GCCTGGTCCT 60CCTGACTGGG GTGAGGGCCT CTGAGAACAG C 91(2) No. 12 sequence information: (i) sequence characteristics: (A) length: 90 base pairs (B) type: nucleic acid (C) number of strands: single strand (D) topology : Linear (xi) Sequence Description: Sequence No. 12: GGCATCTCGG AGATCTCGAA GCATGTTAGG CAGGTTGCCT GGGAAGTGGG TGCAGCTGTT 60CTCAGAGGCC CTCACCCCAG TCAGGAGGAC 90(2) Sequence No. 13 Information: (i) Sequence Features: (A) Length: 82 base pairs (B) Type : Nucleic acid (C) Chain number: Single strand (D) Topology: Linear (xi) Sequence description: No. 13 sequence: GACGACGGTG GCTGCAGCCG CCACCATGCA CAGCTCAGCA CTGCTCTGTT GCCTGGTCCT 60CCTGACTGGG GTGAGGGCCA GC 82(2) No. 14 sequence information: (i) sequence characteristics : (A) Length: 81 base pairs (B) Type: Nucleic acid (C) Number of strands: Single strand (D) Topology: Linear (xi) Sequence description: No. 14 Sequence: GGCATCTCGG AGATCTCGAA GCATGTTAGG CAGGTTGCCT GGGAAGTGGG TGCAGCTGGC 60CCTCACCCCA GTCAGGAGGA C 81(2) No. 15 sequence information: (i) sequence characteristics: (A) length: 82 base pairs (B) type: nucleic acid (C) number of strands: single strand (D) topology: linear (xi) Sequence description: No. 15 sequence: GACGACGGTG GCTGCAGCCG CCACCATGCA CAGCTCAGCA CTGCTCTGTT GCCTGGTCCT 60CCTGACTGGG GTGAGGGCCT GC 82(2) No. 16 sequence information: (i) sequence characteristics: (A) length: 78 base pairs (B) type: nucleic acid (C) Number of Chains: Single Chain (D) Topology: Linear (xi) Sequence Description: Sequence No. 16 GGCATCTCGG AGATCTCGAA GCATGTTAGG CAGGTTGCCT GGGA AGTGGG TGCAGGCCCT 60CACCCCAGTC AGGAGGAC 78 (2) Sequence Information No. 17: (i) Sequence Features: (A) Length: 81 base pairs (B) type: nucleic acid (C) chain number: single strand (D) topology: linear (xi) sequence description: No. 17 sequence: GACGACGGTG GCTGCAGCCG CCACCATGCA CAGCTCAGCA CTGCTCTGTT GCCTGGTCCT 60CCTGACTGGG GTGAGGGCCA C 81(2)18 Number sequence information: (i) sequence characteristics: (A) length: 75 base pairs (B) type: nucleic acid (C) chain number: single strand (D) topology: linear (xi) sequence description: 18 sequence : GGCATCTCGG AGATCTCGAA GCATGTTAGG CAGGTTGCCT GGGAAGTGGG TGGCCCTCAC 60CCCAGTCAGG AGGAC 75

Claims (28)

1. An antagonist of human IL-10, which antagonist comprises substitution of the lysine residue at position 157 by an acidic amino acid residue, or due to deletion of one or more in a region containing about 12 carboxy-terminal residues Amino acid residues are modified mature human IL-10. 2. The antagonist of claim 1, wherein 1-11 amino acid residues have been deleted from the amino terminus. 3. The antagonist of claim 1 having an amino acid sequence defined by sequence number 1, 2 or 3. 4. A nucleic acid encoding an antagonist of human IL-10, said antagonist comprising substitution of a lysine residue at position 157 by an acidic amino acid residue, or a deletion in a region containing about 12 carboxy-terminal residues mature human IL-10 modified by one or more amino acid residues. 5. The nucleic acid of claim 4, which encodes an antagonist from which 1-11 amino acid residues have been deleted from the amino terminus. 6. The nucleic acid of claim 4, which encodes a human IL-10 antagonist having an amino acid sequence defined by sequence number 1, 2 or 3. 7. A recombinant vector comprising the nucleic acid of claim 4, capable of directing the expression of said nucleic acid. 8. A host cell containing the recombinant vector of claim 7. 9. A method for producing an antagonist of human IL-10, said antagonist comprising substitution of a lysine residue at position 157 with an acidic amino acid residue, or a deletion in a region containing about 12 carboxy-terminal residues One or more amino acid residues are modified mature human IL-10, the method comprising: cultivating the host cell of claim 8 under the condition of expressing the nucleic acid. 10. The method of claim 9, wherein said nucleic acid encodes an antagonist having 1-11 amino acid residues deleted from its amino terminus. 11. The method of claim 9, wherein the antagonist encoded by said nucleic acid has an amino acid sequence defined by sequence number 1, 2 or 3. 12. A method for inhibiting the biological activity of human IL-10, the method comprising: contacting cells with human IL-10 receptors with an effective amount of a human IL-10 antagonist, the antagonist comprising a lysine due to 157 Mature human IL-10 modified by substitution of amino acid residues with acidic amino acid residues or by deletion of one or more amino acid residues in a region containing about 12 carboxy-terminal residues. 13. The method of claim 12, wherein 1-11 amino acid residues have been deleted from the amino terminus of the antagonist. 14. The method of claim 12, wherein the antagonist has an amino acid sequence defined by sequence number 1, 2 or 3. 15. An antagonist of human IL-10 comprising mature human IL-10 modified by deletion of 1-11 amino-terminal amino acid residues. 16. The antagonist of claim 15, wherein 7, 10 or 11 oxyacid residues have been deleted. 17. A nucleic acid encoding a human IL-10 antagonist comprising mature human IL-10 modified by deletion of 1-11 amino-terminal amino acid residues. 18. The nucleic acid of claim 17 encoding an antagonist in which 7, 10 or 11 amino acid residues have been deleted. 19. A recombinant vector comprising the nucleic acid of claim 17, capable of directing the expression of the nucleic acid. 20. A host cell comprising the recombinant vector of claim 19. 21. A method of producing a human IL-10 agonist comprising mature human IL-10 modified by deletion of 1-11 amino-terminal amino acid residues, said method comprising: under conditions of expressing a nucleic acid Culturing the host cell of claim 20. 22. The method of claim 21, wherein the nucleic acid encodes an agonist in which 7, 10 or 11 amino acid residues have been deleted. 23. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of (a) an agonist of human IL-10, the agonist comprising and obtain a modified mature human IL-10, or (b) an antagonist of human IL-10 comprising a lysine residue at position 157 replaced by an acidic amino acid residue, or containing about 12 Modified mature human IL-10 obtained by deletion of one or more amino acid residues in the region of carboxy-terminal residues. 24. The pharmaceutical composition of claim 23, wherein 1-11 amino acid residues have been deleted from the amino terminus of the antagonist. 25. The pharmaceutical composition of claim 23, wherein the antagonist has an amino acid sequence defined by sequence number 1, 2 or 3. 26. The pharmaceutical composition of claim 23, wherein 7, 10 or 11 amino acid residues have been deleted from the agonist. 27. A use of an antagonist of human IL-10 in inhibiting the biological activity of IL-10, the antagonist comprising the replacement of the lysine residue at position 157 by an acidic amino acid residue, or due to the presence of about 12 carboxy-terminal The mature human IL-10 modified by deletion of one or more amino acid residues in the region of residues. 28. A use of an antagonist of human IL-10 in the preparation of a medicament for inhibiting the biological activity of IL-10, the antagonist comprising the replacement of the lysine residue at position 157 by an acidic amino acid residue, or the Mature human IL-10 modified by deletion of one or more amino acid residues in a region containing about 12 carboxy-terminal residues.