Analysis of the culture media of p40-transfected COS cells indicated the presence of 40 kDa monomers and 80 kDa disulfide-linked homodimers. Examination of partially purified p40 recombinant proteins demonstrated that only the homodimer but not the monomer binds to the IL-12 receptor. Partially purified 80 kDa homodimer inhibited [.sup.125 I]IL-12 binding to PHA-activated human lymphoblasts with an IC.sub.50 of 80 ng/ml, which is similar to the IC.sub.50 value (20 ng/ml) for the human IL-12 heterodimer. Although neither the 40 kDa monomer nor the 80 kDa dimer could stimulate human PHA-blast proliferation, the 80 kDa dimer inhibited IL-12-induced proliferation in a dose-dependent manner with an IC.sub.50 of 1 .mu.g/ml. The IL-12 p40 subunit contains the essential epitopes for receptor binding, but they are only active when p40 is covalently associated with a second protein such as p35 or p40. When p40 is associated with the p35 subunit, the heterodimer acts as an agonist mediating biologic activity. When p40 associates with itself, the homodimer behaves as an antagonist.

FIELD OF THE INVENTION 
This invention relates to a protein of two p40 subunits of interleukin-12 
linked by at least one disulfide bond which acts as an interleukin-12 
receptor antagonist. 
BACKGROUND OF THE INVENTION 
Interleukin-12 (IL-12), formerly known as cytotoxic lymphocyte maturation 
factor (CLMF) or natural killer cell stimulatory factor (NKSF), is a 
cytokine that has pleiotropic activities including stimulation of the 
proliferation of activated T and NK cells (1, 2), induction of INF-.gamma. 
production by peripheral blood mononuclear cells (2, 3), and enhancement 
of the lytic activity of NK/LAK cells (2, 4). 
IL-12 is a heterodimeric molecule with an approximate molecular weight of 
about 75 kDa consisting of two disulfide-linked subunits: p35, having an 
approximate molecular weight of about 35 kDa, and p40, having an 
approximate molecular weight of about 40 kDa, (2, 4-6). The p40 subunit 
shares amino acid sequence homology with the interleukin-6 receptor 
(IL-6R) (7) and therefore belongs to the cytokine receptor superfamily, 
whereas p35 has a distant but significant relationship to the IL-6/G-CSF 
cytokine family (8). It has been speculated that the p35/p40 heterodimer 
could represent a cytokine (p35) and soluble cytokine receptor (p40) 
complex, with the cellular IL-12 receptor providing function analogous to 
the IL-6 signal transducing protein, gp130 (7, 8). 
The biological activity of IL-12 is mediated by the binding of the intact 
IL-12 molecule to plasma membrane receptors on activated T or NK cells 
(9,10); however, the contributions of the individual subunits to receptor 
binding and signal transduction remain unknown. Studies with neutralizing 
antibodies to human IL-12 (11) and site-specific chemical modification 
(12) suggested that the p40 subunit contains epitopes important for IL-12 
binding to its receptor. Also, studies with human/mouse chimeric molecules 
indicated that p35 is responsible for the species specificity of the 
heterodimer for biological activities (13). 
We investigated both the binding and biological activities of each IL-12 
subunit. COS cells transfected with only the p40 cDNA produced both p40 
monomer and p40 homodimer having an approximate molecular weight of about 
80 kDa, the latter capable of binding to the IL-12 receptor but unable to 
mediate cellular proliferation. The 80 kDa p40 homodimer acts as a 
receptor antagonist useful in regulating the biological activity of IL-12 
in immune responses. 
SUMMARY OF THE INVENTION 
The present invention is directed towards an isolated and purified protein 
comprising two p40 subunits of interleukin-12 which are associated 
together, preferably by at least one disulfide bond, having a molecular 
weight of about 80 kDa. The 80 kDa p40 homodimer acts as an interleukin-12 
receptor antagonist. The preferred p40 subunit is that of SEQ. ID NO:1.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention is directed towards an isolated and purified protein 
comprising two p40 subunits of interleukin 12 associated together, 
preferably by at least one disulfide bond, having a molecular weight of 
about 80 kDa. The 80 kDa p40 homodimer acts as an interleukin-12 receptor 
antagonist. The preferred p40 subunit is that of SEQ ID NO:1. 
The IL-12 p40 homodimer is useful as an IL-12 antagonist to block the 
biological activity of IL-12 in pathologic immune responses. Current 
evidence from both in vitro and in vivo studies suggest that IL-12 plays 
an important role in the development of Th1-type helper T cells which 
promote cell-mediated immune responses (22,27), in triggering gamma 
interferon production by mature T and/or NK cells (28), and in 
facilitating specific cytolytic T lymphocyte responses (29). Excessive 
activity of Th1 cells (30, 31) and/or excessive production of gamma 
interferon (32-34) may be involved in the pathogenesis of some autoimmune 
disorders and septic shock, indicating that IL-12 p40 homodimer should be 
useful in the treatment of disorders such as rheumatoid and other 
inflammatory arthritides, Type I diabetes mellitus, multiple sclerosis, 
systemic lupus erythematosus, septic shock, etc. In addition, IL-12 p40 
homodimer should be useful in preventing or delaying homograft rejection 
and graft versus host disease. In using IL-12 p40 homodimer to prevent or 
reverse pathologic immune responses, it can be combined with other 
cytokine antagonists such as antibodies to the IL-2 receptor, soluble TNF 
receptor, or the IL-1 receptor antagonist, and the like. 
The dose ranges for the administration of the p40 homodimer protein may be 
determined by those of ordinary skill in the art without undue 
experimentation. In general, appropriate dosages are those which are large 
enough to produce the desired effect, for example, blocking the binding of 
IL-12 to its receptor. The dosage should not be so large as to cause 
adverse side effects, such as unwanted cross-reactions, anaphylactic 
reactions, and the like. Generally, the dosage will vary with the age, 
condition, sex and extent of disease in the patient, counter indications, 
if any, immune tolerance and other such variables, to be adjusted by the 
individual physician. The p40 homodimer protein can be administered 
parenterally by injection or by gradual perfusion over time. It can be 
administered intravenously, intraperitoneally, intramuscularly, or 
subcutaneously. 
Pharmaceutically acceptable carriers and preparations for parenteral 
administration include sterile or aqueous or non-aqueous solutions, 
suspensions, and emulsions. Examples of non-aqueous solvents are propylene 
glycol, polyethylene glycol, vegetable oils such as olive oil, and 
injectable organic esters such as ethyl oleate. Aqueous carriers include 
water, alcoholic/aqueous solutions, emulsions or suspensions, including 
saline and buffered media. Parenteral vehicles include sodium chloride 
solution, Ringer's dextrose, dextrose and sodium chloride, lactated 
Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient 
replenishers, electrolyte replenishers, such as those based on Ringer's 
dextrose, and the like. Preservatives and other additives may also be 
present, such as, for example, anti-microbials, anti-oxidants, chelating 
agents, inert gases and the like. See, generally, Remington's 
Pharmaceutical Science. 16th Ed., Mack Eds., 1980. 
The invention also relates to method for preparing a medicament or 
pharmaceutical composition comprising the P40 homodimer protein of the 
invention. 
MATERIALS AND METHODS 
Cell lines. 
KIT225/K6, an IL-2-dependent subclone derived from the human T cell line 
KIT225 (14), was obtained from Dr. T. Waldmann, NIH/NCI (Bethesda, Md.). 
These cells were previously found to express IL-12 receptors (15). 
KIT225/K6 cells were cultured in RPMI 1640 medium (BioWhittaker, 
Walkersville, Md.) supplemented with 2 mM L-glutamine (Sigma, St. Louis, 
Mo.), 100 U/ml penicillin, 100 .mu.g/ml streptomycin (Gibco, Grand Island, 
N.Y.), 15% FCS (JRH Biosciences, Lenexa, Kans.), and 100 U/ml human rIL-2 
(Dr. F. Khan, Hoffmann-La Roche, Nutley, N.J.). COS cells were cultured in 
DMEM (Gibco) with 4500 mg/liter glucose, 2 mM L-glutamine, 50 U/ml 
penicillin, 50 .mu.g/ml streptomycin and 10% FCS (JRH Biosciences). 
Expression of IL-12 subunits. 
The IL-12 expression constructs for COS-expression were built in the 
pEF-BOS vector which contains the promoter of the human polypeptide chain 
elongation factor 1.alpha.(EF-1.alpha.) chromosomal gene (16a). The cDNA 
fragments containing the entire coding region of the human or mouse p40 or 
p35 cDNAs generated by polymerase chain reaction (PCR) using the primers 
complementary to the beginning and end of the subunit cDNA coding 
sequences as described previously (6, 13) were subcloned individually into 
the pEF-BOS vector at the Xba 1 cloning site by blunt end ligation 
(standard procedure). The ligation products were transformed into E. coli 
strain DH-5 alpha, and the resulting colonies were screened by PCR for the 
correct insert orientation by using a forward primer within the pEF-BOS 
promoter and a reverse primer within the subunit coding sequences. 
Positive clones were selected and amplified in E. coli strain MC 1161. 
Plasmid DNAs were prepared by using the QJAGEN plasmid kit (Ojagen, 
Chatsworth, Calif.) and transfected into COS cells by using the DEAE 
dextran/chloroquine method (17). The DNAs at a concentration of 2 .mu.g/ml 
were mixed with 10% Nutridoma-SP (Boehringer Mannheim, Indianapolis, 
Ind.), 0.5 mg/ml DEAE dextran and 0.05 mg/ml chloroquine in DMEM 
(Dulbelcco's modified essential medium) medium and applied to COS cells 
seeded for 16 h. After a 2.5-3 h incubation, the cells were treated with 
10% DMSO in serum free DMEM medium for 3 min followed by washing with DMEM 
medium, and then cultured in DMEM/10% FCS medium. Supernatant fluids were 
collected from the cultures of transfected COS cells after 72 h. 
Coexpression of p40 and p35 subunits was performed by mixing the two 
plasmid DNAs at a 1:1 (W/W) ratio in transfection reagents. The 
supernatant fluids derived from the COS cultures transfected with pEF-BOS 
wild-type plasmid DNA were used as controls. 
The human IL-12 p40 construct for expression in Baculovirus system was 
built in pACDZ-1 vector (16b) (obtained from R. Gentz, F. Hoffmann-La 
Roche Ltd, Basel, Switzerland) at BamH1 site by using same approach 
described above. A recombinant baculovirus expressing the p40 chain was 
generated by cotransfecting SF9 cells with wild type baculovirus DNA and 
the p40 expressing plasmid pACDZ-1. Limited dilution cloning in 
microtiterplates was used to isolate a single recombinant baculovirus 
expressing the human IL-12 p40 subunit. 
IL-12 receptor binding and proliferation assays. 
The binding of COS-expressed IL-12 molecules to IL-12 receptor-bearing 
cells was measured by FACS (fluorecense activated cell sorthing) analysis 
essentially as described by Desai et al.(10). Briefly, 1.times.10.sup.6 
KIT225/K6 cells suspended in 25 .mu.l of FACS buffer (PBS 
(phosphate-buffered-saline)/2% FCS/0.05% sodium azide) were incubated with 
IL-12 preparations (25.mu.l) at room temperature for 40 min, followed by 
incubation with biotinylated mAb 8E3 (5 .mu.g/ml, 50 .mu.l), (11) for 30 
min, and then with streptavidin-PE (1.5 .mu.g/ml, 50 .mu.l; FisherBiotech, 
Pittsburgh, Pa.) for 20 min. The stained cells were analyzed on a FACScan 
flow cytometer (Becton Dickinson). Specificity of binding was determined 
by preincubating the IL-12 preparations (0.5 .mu.g/ml) with 4A1 (25 
.mu.g/ml), a rat inhibitory anti-human IL-12 monoclonal antibody, prior to 
adding cells. Control samples were incubated with normal rat IgG (25 
.mu.g/ml). The receptor binding properties of the COS-expressed IL-12 
molecules were also evaluated in an [.sup.125 I]IL-12 competitive receptor 
binding assay performed essentially as previously described (11). 0.1 ml 
aliquots of serial dilutions of culture supernatant fluids or purified 
IL-12 were mixed with 0.05 ml aliquots of binding buffer (RPMI-1640, 5% 
FCS, 25 mM HEPES pH 7.4) containing [.sup.125 I]IL-12 (2.times.10.sup.6 
cpm). The mixture was added to 0.1 ml of activated blasts 
(1.times.10.sup.7 cell/ml) and incubated in a shaking water bath at 
25.degree. C. for 1.5 h. Non-specific binding was determined by inclusion 
of 20 .mu.g/ml unlabeled IL-12 in the assay. Incubations were carried out 
in duplicate. Cell bound radioactivity was separated from free [125I]IL-12 
by centrifugation of 0.1 ml aliquots of the assay contents in duplicate 
through 0.1 ml silicone oil for 90 sec at 10,000.times. g. The tip 
containing the cell pellet was excised and cell bound radioactivity was 
determined in a gamma counter. 
The biological activity of COS-expressed IL-12 molecules was evaluated in 
proliferation assays using 4-day PHA-activated human lymphoblasts 
previously described (4, 13). 
Anti-IL-12 antibodies and sandwich enzymatic immunoassay (EIA). 
Goat and rabbit anti-human IL-12 antisera were obtained from animals 
immunized with purified human rIL-12 that had been expressed in CHO cells 
(kindly provided by Dr. A. Stern, Hoffmann-La Roche Inc., Nutley, N.J.). 
The IgG fraction was isolated from 100 ml of the antisera by Protein-G 
Sepharose (Pharmacia LKB, Piscataway, N.J.) affinity chromtography 
according to the manufacturer's procedures. Anti-human IL-12 antibodies 
were purified from the IgG fractions on a human IL-12-conjugated hydrazide 
AvidGel F. (BioProbe International) immunoaffinity column (1.5.times.2.0 
cm, 0.55 mg protein per ml resin). Biotinylation of the antibodies using 
Biotin X-NHS (Calbiochem, San Diego, Calif.) was performed as described 
previously (18). Monoclonal antibodies 4A1 and 8E3 are rat antibodies 
specific for the p40 subunit of human IL-12 (11) (kindly provided by Dr. 
Richard Chizzonite, Hoffmann-La Roche Inc., Nutley, N.J.). 
The IL-12 sandwich EIA, using mAb 4A1 as a capture antibody and 
peroxidase-conjugated 8E3 as detection antibody, was performed as 
described previously (11). This assay detects IL-12 heterodimer and p40 
subunit but not p35 subunit. Therefore, a second IL-12 sandwich EIA using 
polyclonal antibodies was developed to detect both p40 and p35. In this 
assay, 96 well EIA plates (Nunc MaxiSorp, Thousand Oaks, Calif.) were 
coated with affinity-purified goat anti-human IL-12 antibody (2 .mu.g/ml, 
50 .mu.l/well) at 4.degree. C. overnight and blocked with 1% BSA in PBS pH 
7.4 for 1 h at RT. Serial dilutions of IL-12 and culture supernatant 
fluids were applied to the plates, and incubated at RT for 2.5 h. The 
plates were subsequently incubated with biotinylated, affinity-purified 
rabbit anti-human IL-12 antibody (500 ng/ml, 50 .mu.l/well), followed by 
peroxidase-conjugated streptavidin (1 .mu.g/ml, 50 .mu.l/well, Sigma, St. 
Louis, Mo.). Color was developed with 100 .mu.l of 1 mM ABTS 
(2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)/0.1% (v/v) H.sub.2 
O.sub.2, and the absorbance at 405 nm was determined with a Vmax Kinetic. 
Microplate reader (Molecular Devices, Palo Alto, Calif.). All values are 
based on an IL-12 standard curve with no corrections calculated for 
differences in molecular weights of monomers or dimers. 
Immunoprecipitation. 
Immunoprecipitation of COS-expressed IL-12 subunits and heterodimers was 
performed as described (17). Briefly, 0.5 ml supernatant fluids from 
transfected COS cultures were incubated with 5 .mu.g IgG protein isolated 
from goat anti-IL-12 antiserum at 4.degree. C. on a rotating mixer 
overnight. The immune complexes were adsorbed onto Protein G-Sepharose 
(50% suspension, 10 .mu.l, Pharmacia LKB) at 4.degree. C. for 2 h, and the 
beads were washed twice with 1 ml NET-Gel buffer (50 mM Tris-HCl, pH 7.5, 
150 mM NaCl, 0.1% (v/v) Nonidet P-40, 1 mM EDTA, 0.25% (w/v) gelatin and 
0.02% (w/v) sodium azide), and once with 1 ml of 10 mM Tris-HCl (pH 7.5) 
containing 0.1% (v/v) Nonidet P-40. The bound proteins were dissociated 
from the beads by heating for 3 min at 95.degree. C. in either reducing 
(10% 2-ME) or non-reducing SDS sample buffer. 
SDS-PAGE and Western blotting. 
SDS-PAGE was performed according to the method of Laemmli (19). Western 
blotting was performed by electrophoretically transferring proteins to a 
nitrocellulose membrane (0.2.mu.) (MSI, Westboro, Mass.). The transferred 
membranes were blocked by incubation in PBST buffer (PBS with 0.05% v/v 
Tween-20) containing 5% (w/v) non-fat dry milk, and then probed with 
anti-IL-12 rabbit antisera (1:500 dilution). After three washes with PBST 
buffer, the membranes were incubated at room temperature with 
peroxidase-conjugated donkey anti-rabbit IgG antibodies (1:1000 dilution) 
(Jackson Immuno Research, West Grove, Pa.). The color was developed by use 
of 4-chloro-1-napthol (BioRad, Richmond, Calif.) in 20 mM Tris-HCl buffer 
(pH 7.5) containing 0.1% (v/v) H.sub.2 O.sub.2. 
Purification of COS-expressed P40. 
One liter of conditioned media containing approximately 3 .mu.g/ml of human 
recombinant p40 (rp40) was applied to a mAb 4A1-conjugated NuGel (NITS) 
immunoaffinity column (2.5.times.10 cm, containing 1.6 mg antibody per ml 
gel, kindly provided by Dr. A. Stem, Hoffmann-La Roche Inc., Nutley, N.J.) 
at a flow rate of 2 ml/min, and the column was washed extensively with PBS 
containing 0.5M NaCl and 0.2% Tween 20 until absorbance monitoring at 280 
mn was less than 0.01. The bound proteins were then eluted with 100 mM 
glycine/150 mM NaCl (pH 2.8) at a flow rate of 2 ml/min, and 20 ml 
fractions were collected and immediately neutralized with 1/10 vol. of 1M 
Tris-HCl (pH 8.0). The EIA-positive fractions were pooled, dialyzed 
against PBS overnight at 4.degree. C., concentrated by ultrafiltration 
using YM 10 membranes (Amicon, Beverly, Mass.) to 5 ml, and applied onto a 
HiLoad Superdex 75 (Pharmacia LKB) column (1.6.times.60 cm) equilibrated 
with Dulbecco's PBS buffer. The column was eluted at a flow rate of 1 
ml/min with the same buffer, and 1 ml fractions were collected. Proteins 
from each fraction were examined by EIA, SDS-PAGE and Western blot 
analysis. 
Deglycosylation. 
500 ng of pure human IL-12 or immunoprecipitated rp40 protein was denatured 
by heating at 95.degree. C. for 5 rain in 0.25M Na.sub.2 HPO.sub.4 (pH 
7.2), 0.5% SDS with or without 1% 2-ME. The samples were cooled to room 
temperature, adjusted to 1% Nonidet P-40, 20 mM EDTA, and then treated 
with 0.1 U of N-glycosidase F (Boehringer Mannheim, Indianapolis, Ind.) at 
37.degree. C. for 24 h. The deglycosylated proteins were examined by 
SDS-PAGE and Western blot analysis. 
Amino-terminal sequence analysis of COS-expressed p40. 
The immunoaffinity purified rp40 proteins were separated on 10% 
non-reducing SDS gel and transferred electrophoretically to an 
Immobilon.TM. PVDF membrane (Millipore, Bedford, Mass.). The bands at 
.about.80 and .about.40 kDa identified by Coomassie blue staining were 
subjected to automated Edman degradation on an Applied Biosystems Model 
470A gas-phase sequencer with on-line analysis of phenylthiohydantoin 
(PTH) amino add derivatives as described previously (20). 
RESULTS 
Expression and characterization of human IL-12 subunits. 
Human IL-12 subunits p35 and p40, or human IL-12 p35/p40 heterodimer were 
expressed by transfecting either subunit cDNA independently or 
cotransfecting both cDNAs at a 1:1 (w:w) ratio in COS cells. Secretion of 
the recombinant proteins was evaluated by two different EIA's. The 
p40-specific monoclonal antibody-based EIA was capable of detecting the 
p40 subunit and the p40/p35 heterodimer. The IL-12 specific polyclonal EIA 
was also capable of detecting the p35 subunit. Using human IL-12 as a 
standard, the concentration range of rp40 and rp35/rp40 proteins in the 
conditioned media was 0.5-3.0 .mu.g/ml, whereas the expression of rp35 
alone was approximately 0.2 .mu.g/ml. It remains unclear whether the p35 
expression was low or the sensitivity of the polyclonal EIA in detecting 
p35 was poor. 
The COS-expressed human IL-12 recombinant proteins were initially examined 
for their ability to inhibit the binding of [.sup.125 I]human IL-12 to 
PHA-activated human lymphoblasts. The rp40 supernatants at a 1:2 dilution 
exhibited 30-40% inhibition of [.sup.125 I]IL-12 binding in three 
independent experiments, whereas the rp35 supernatants were inactive. The 
binding of rp40 to the IL-12 receptor was further characterized by flow 
cytometry using KIT225/K6 cells which constitutively express IL-12 
receptors (IL-12R) (15). Dose-dependent binding of human IL-12 and rp40 to 
KIT225/K6 was observed in the range of 2.5-500 ng/ml (FIGS. 1A and 1B). 
Specificity of the binding was demonstrated by achieving greater than 80% 
inhibition of the binding by preincubation of IL-12 or rp40 with an 
inhibitory rat anti-human p40 monoclonal antibody, 4A1 (FIGS. 2A, 2B, 2C 
and 2D). Normal rat IgG had no effect on IL-12 or rp40 binding. 
Conditioned media containing the COS-expressed IL-12 subunit proteins were 
evaluated in the human PHA-blast proliferation assay (FIG. 3). The 
rp35/rp40-containing medium supported T cell proliferation in a 
dose-dependent manner with an apparent EC.sub.50 of 8 ng/ml. The rp40 
supernatants did not induce proliferation at concentrations equivalent to 
the rp35/rp40 supernatant. 
Characterization of the rp40 40 kDa and 80 kDa species. 
The recombinant human IL-12 subunits were immunoprecipitated with 
anti-human IL-12 goat antiserum and characterized by SDS-PAGE and Western 
blot analysis. Analysis of the rp40 expressed by COS cells transfected 
with only the p40 cDNA revealed two sets of multiple bands under 
nonreducing conditions with heterogeneous molecular weights of 70-85 kDa 
and 35-45 kDa (FIG. 4A). Under reducing conditions, only three closely 
spaced bands at approximately 38-49 kDa were identified suggesting that 
the 80 kDa proteins are disulfide-linked rp40 homodimers (FIG. 4B). 
Treatment of the rp40 immunoprecipitates with N-deglycosidase-F shifted 
both molecular weight species down to smaller products under nonreducing 
conditions (FIG. 5A), and converted the reduced triple bands to a single 
36 kDa product similar to p40 subunit of the deglycosylated human IL-12 
(12) demonstrating that the multiple bands of rp40 expressed in COS cells 
are due to glycosylation heterogeneity. 
In contrast, the immunoprecipitation of rp35 protein revealed only a single 
band with a molecular weight of 35 kDa under reducing conditions (FIG. 
4B). Under nonreducing conditions, a set of lightly stained bands were 
found at 60-70 kDa suggesting that rp35 may also partially form dimers. 
However, the polyclonal goat anti-IL-12 antibody poorly recognized the 
rp35proteins (FIG. 4A). Coexpression of p35 and p40 gave a pattern of 
bands which was a mixture of those seen when each subunit was expressed 
independently (FIGS. 4A and 4B). It is unclear whether the p35/p40 
heterodimer and the p40/p40 homodimer were produced simultaneously by COS 
cells cotransfected with the p35 and p40 cDNAs. Unfortunately, currently 
available reagents do not distinguish the p35/p40 heterodimer from the 
p40/p40 homodimer. 
To confirm the identity of the two rp40 species, the rp40 proteins were 
partially purified by 4A1 immunoaffinity chromatography. Only 60% of EIA 
positive material was recovered by elution with 100 mM glycine containing 
150 mM NaCl at pH 2.8. The 4A1 affinity-purified proteins were then 
separated by SDS-PAGE, electrophoretically transferred to a PVDF membrane, 
and subjected to amino acid microsequencing. One broad band at .about.80 
kDa and two bands at 35-40 kDa gave NH.sub.2 -terminal sequences identical 
to that of native human IL-12 p40 purified from NC-37 cells (4, 12) (Table 
I). No trace of p35 sequences as identified with the rp40 species. This 
result confirmed that the 80 kDa protein is a p40 homodimer. 
TABLE I 
______________________________________ 
Amino-terminal Sequences of COS-expresssed Human p40 Monomer, 
p80 Homodimer and Native Human IL-12 p40 subunit 
Protein Sequence 
______________________________________ 
Native Human p40 
I W E L K K D V Y V.sup.a [SEQ ID NO: 2] 
rp40 Dimer I W.sup.b E L k k D V Y V [SEQ ID NO: 2] 
rp40 Monomer I w E L k k D V Y V [SEQ ID NO: 2] 
(band 1) 
rp40 Monomer I W E L k k D V Y V [SEQ ID NO: 2] 
(band 2) 
______________________________________ 
.sup.a From Podlaski et al., 1991 
.sup.b Small case letter represents a signal with a recovery less than 2 
pmol. 
The immunoaffinity purified p40 proteins were further fractionated by 
Superalex-75 gel filtration chromatography. Two EIA positive protein peaks 
were identified at molecular weights corresponding to 80 kDa and 40 kDa 
(FIG. 6A). SDS-PAGE and Western blot analysis of the fractions confirmed 
the separation of dimer from monomer rp40 (FIG. 6B). The ratio of the 
monomer to dimer varied from experiment to experiment, but, on the 
average, approximately 30% of the COS-expressed rp40 was p40 homodimer. 
The Superdex 75 column fractions were tested for binding to KIT225 cells by 
FACS analysis. Binding activity correlated only with the 80 kDa p40-EIA 
positive protein (FIGS. 6A and 6B). The 80 and 40 kDa peak fractions were 
pooled separately, concentrated and examined in the competitive 
radioligand receptor binding assay (FIG. 7). The 80 kDa protein pool 
inhibited [.sup.125 I]human IL-12 binding to PHA-blasts with an IC.sub.50 
of 80 ng/ml, which is similar to the IC.sub.50 of human IL-12 heterodimer 
(20 ng/ml). However, the slope of the competition curve by the 80 kDa 
homodimer differed from that of IL-12 heterodimer suggesting a different 
binding interaction with the receptor. The 40 kDa protein pool inhibited 
[.sup.125 I]human IL-12 binding with an IC.sub.50 about one hundred times 
higher, which was probably due to a small amount of contamination with the 
p40 homodimer (FIG. 6B). 
The abilities of the rp40 monomer and dimer to support PHA-blast 
proliferation were also examined (FIG. 8). No proliferative response was 
observed with either rp40 species even at concentrations 10,000 times 
higher than that of human IL-12 required to elicit a 50% maximum response. 
The rp40 dimer was tested for its ability to neutralize IL-12-dependent 
proliferation of PHA-blasts. The 80 kDa protein at varying concentrations 
was mixed with 0.1 ng/ml of human IL-12 and added to PHA-blasts. 
Dose-dependent inhibition of IL-12-induced proliferation of PHA-blasts was 
achieved with an IC.sub.50 of .mu.g/ml (FIG. 9). 
IL-12 is unique among the lymphokines and cytokines in that it is a 
heterodimeric protein. Previous studies suggested that the p40 subunit is 
important for receptor binding (11, 12), and that the p35 subunit is 
primarily responsible for the species specificity observed in biological 
assays (13). 
To further clarify the functional role of the individual subunits and 
localize the epitopes mediating biological and binding activities, we 
expressed the individual subunits alone or in combination with each other 
in COS cells and tested the expressed proteins in binding assays and 
bioassays and by Western blot analysis. The rp35 protein was inactive at 
concentrations as high as 100 ng/ml in the binding and bioassays; however, 
the rp40 protein reproducibly exhibited binding activity without 
bioactivity. Analysis of the conditioned media from cultures of COS cells 
transfected with only the p40 cDNA revealed that such media contained both 
monomeric p40 and an 80 kDa molecule reactive with anti-p40 antibodies. 
Partial purification of the rp40 by immunoaffinity chromatography and HPLC 
gel permeation chromatography revealed that the 80 kDa protein, but not 
the 40 kDa protein bound to the IL-12R. 
Unfortunately, no reagents were available to distinguish an 80 kDa p40 
homodimer from the 75 kDa IL-12 heterodimer. The possibility that the 80 
kDa protein was not a homodimer of p40 but a heterodimer consisting of one 
IL-12 p40 subunit and a second 35-40 kDa exogenous COS-derived protein was 
investigated. In particular, reports that many cell lines constitutively 
express IL-12 p35 mRNA (21) raised the possibility that the 80 kDa protein 
could be human IL-12 p40 associated with COS-derived IL-12 p35. Western 
blot analysis by using p35 specific antibody and deglycosylation 
experiments (FIGS. 5A and 5B) supported the notion that the 80 kDa protein 
could be reduced to a p40 monomer. The lack of bioactivity despite good 
binding activity further suggested that the second protein was not a 
COS-derived p35 IL-12 subunit (assuming no species restriction in the 
activity of monkey IL-12 on human cells). Also, expression of p40 in a 
baculovirus system yielded a biologically inactive 80 kDa form of p40 
capable of binding to the receptor. It seems unlikely that insect cells 
produce an IL-12-like p35 protein. Most importantly, confirmation of the 
identity of the 80 kDa protein as p40 homodimer was provided by amino acid 
microsequencing of the protein demonstrating a single N-terminal sequence 
corresponding to the IL12 p40 subunit. 
In competitive binding analysis, the p40 homodimer was found to bind to the 
IL-12R nearly as strongly as heterodimeric IL-12, suggesting that the key 
binding epitopes of IL-12 are localized in the p40 subunit. Though the 
IC.sub.50 values for the heterodimer and the homodimer were similar, 20 
and 80 ng/ml respectively, the slopes of the competition curves were 
different. This suggests a difference in the interaction of the two 
ligands with the receptor. It is most likely that the p40 binding epitopes 
are conformational and induced by association with a p35 or a second p40 
subunit. 
The IL-12 p40 subunit has been previously reported to be produced in excess 
of heterodimeric IL-12 both by activated B lymphoblastoid lines and by 
human PBMC stimulated to produce IL-12 (12, 23). It is possible that the 
p40 homodimer is formed in cells expressing p40/p35 heterodimers. 
Based on our observations on the roles of the IL-12 subunits in binding and 
signaling, a model of IL-12 binding to its receptor is illustrated in 
FIGS. 10A and 10B. The p40 subunit contains the receptor binding epitopes 
that, however, are active only when p40 associates with a second protein, 
i.e. p35 or another molecule of p40. Both dimeric molecules bind to the 
IL-12R specifically, but only the dimer containing p35 acts as an agonist 
to mediate cellular transduction signals (FIG. 10A). In contrast, the 
p40/p40 dimer behaves as an antagonist to suppress IL-12 mediated 
responses (FIG. 10B). Gearing and Cosman (7) have suggested that IL-12 is 
analogous to a complex of cytokine and soluble receptor based on the 
homology between p40 and the interleukin 6 receptor (IL-6R). They also 
proposed that the cellular IL-12R probably is a gp 130-like 
signal-transducing protein. Our model of the interaction between p35, p40 
and the IL-12R proposes that the IL-12/IL-12R system may function 
similarly to the IL-6R system. In the latter system, gp130 does not bind 
IL-6 (24), and neither IL-6 nor IL-6R alone stimulated proliferation of 
cells transfected with gp130 (25). Rather, the binding of IL-6 to IL-6R 
triggers the association of the receptor and gp130 (26), and in addition, 
complexes of IL-6 with soluble IL-6R can bind to gp130 to initiate signal 
transduction (26). Similarly, it appears that the association of IL-12 p40 
with p35 results in an alteration in p40 that permits binding to IL-12R 
with subsequent initiation of p35-dependent, IL-12R-mediated signal 
transduction. 
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__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 306 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
IleTrpGluLeuLysLysAspValTyrValValGluLeuAspTrpTyr 
151015 
ProAspAlaProGlyGluMetValValLeuThrCysAspThrProGlu 
202530 
GluAspGlyIleThrTrpThrLeuAspGlnSerSerGluValLeuGly 
354045 
SerGlyLysThrLeuThrIleGlnValLysGluPheGlyAspAlaGly 
505560 
GlnTyrThrCysHisLysGlyGlyGluValLeuSerHisSerLeuLeu 
65707580 
LeuLeuHisLysLysGluAspGlyIleTrpSerThrAspIleLeuLys 
859095 
AspGlnLysGluProLysAsnLysThrPheLeuArgCysGluAlaLys 
100105110 
AsnTyrSerGlyArgPheThrCysTrpTrpLeuThrThrIleSerThr 
115120125 
AspLeuThrPheSerValLysSerSerArgGlySerSerAspProGln 
130135140 
GlyValThrCysGlyAlaAlaThrLeuSerAlaGluArgValArgGly 
145150155160 
AspAsnLysGluTyrGluTyrSerValGluCysGlnGluAspSerAla 
165170175 
CysProAlaAlaGluGluSerLeuProIleGluValMetValAspAla 
180185190 
ValHisLysLeuLysTyrGluAsnTyrThrSerSerPhePheIleArg 
195200205 
AspIleIleLysProAspProProLysAsnLeuGlnLeuLysProLeu 
210215220 
LysAsnSerArgGlnValGluValSerTrpGluTyrProAspThrTrp 
225230235240 
SerThrProHisSerTyrPheSerLeuThrPheCysValGlnValGln 
245250255 
GlyLysSerLysArgGluLysLysAspArgValPheThrAspLysThr 
260265270 
SerAlaThrValIleCysArgLysAsnAlaSerIleSerValArgAla 
275280285 
GlnAspArgTyrTyrSerSerSerTrpSerGluTrpAlaSerValPro 
290295300 
CysSer 
305 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
IleTrpGluLeuLysLysAspValTyrVal 
1510 
__________________________________________________________________________