Human Interleukin-3(Il-3) variants and their use to promote or antagonize IL-3-mediated processes

This invention relates to human IL-3 variants. The human IL-3 variants are used in pharmaceutical compositions, in methods for modulating proliferation, differentiation or functional activation of cells expressing the IL-3 receptor, and in methods of treatment.

This invention relates to variants and mutants of human interleukin-3 
(hIL-3), and in particular it relates to hIL-3 va sequence of wild-type 
hIL-3 is varied in order to obtain useful changes in activity, 
particularly in binding to IL-3 receptors and in biological function. 
Human IL-3 is a T-cell-derived glycoprotein of Mr 23-30 kd which promotes 
the proliferation and differentiation of haemopoietic progenitor cells, 
including megakaryocytes, eosinophils, basophils, neutrophils, monocytes 
and T-lymphocytes. It also induces the growth and the functional 
activation of more mature cells, including eosinophils, basophils and 
monocytes. The cDNA of hIL-3 has been cloned, and the mature protein of 
133 amino acids has been produced in recombinant form. The human IL-3 
receptor comprises at least two components, an a chain which binds IL-3 
with low affinity only, and a .beta. chain which allows high affinity 
binding when co-expressed with the .alpha. chain (Kitamura T, Sato N, Arak 
K-I and Miyajima A, 1991, Cell 66, 1165-1174). 
Subsequent structure-activity relationship studies of hIL-3 have been 
performed by functional analysis of hIL-3 deletion and substitution 
variants (Lokker et al, 1991a, J. Biol. Chem. 266, 10624-10631; 1991b, 
EMBO J. 10, 2125-2131) using recombinant hIL-3 variants generated by 
site-directed mutagenesis and expression in Escherichia coli. In this 
work, the variants were analysed for their ability to bind to the IL-3 
receptor and to induce the proliferation of the human IL-3 -dependent cell 
line M-07. These studies initially showed that hIL-3 residues Pro 33 and 
Leu 34 are essential for modulating the biological activity of hIL-3, and 
that certain substitution variants at residues 33 and 34, particularly the 
variant in which Pro 33 was substituted with Gly (Gly 33), showed an 
enhanced proliferation activity without a significant modification in its 
receptor binding capacity (Lokker et al, 1991a supra). Subsequent studies 
which extended the structure-activity- relationship studies showed that 
the hIL-3 residue Leu 111, and possibly also Lys 110, form part of an 
active site. Thus, substitution of Lys 110 with either Glu or Ala resulted 
in variants with substantially reduced activity in receptor binding and 
proliferation assays. Similarly, variants where Leu 111 was substituted by 
Pro or Met were totally inactive in these assays (Lokker et al, 1991b 
supra). 
It has now been discovered that variants or mutants of hIL-3 in which one 
or more amino acids in or adjacent to the predicted "D" or fourth 
predicted .alpha.-helix of hIL-3 is/are replaced with another amino acid 
show enhanced biological activity when compared with wild-type hIL-3. This 
enhanced biological activity is paralleled by enhanced binding to the 
specific a chain of the IL-3 receptor, and suggests that the variants or 
mutants may be used as therapeutic agents. 
According to a first aspect of the present invention, there is provided a 
human IL-3 variant or mutant, characterised in that one or more amino 
acids in or adjacent to the predicted "D" or fourth predicted 
.alpha.-helix of hIL-3 is/are replaced by another amino acid. 
In one embodiment of this aspect of the invention, there is provided a 
human IL-3 variant or mutant, characterised in that amino acid 101 (Asp) 
and/or amino acid 116 (Lys) is/are replaced by another amino acid. 
Particularly preferred variants or mutants in accordance with this aspect 
of the invention are: 
hIL-3 (Ala.sup.101) 
hIL-3 (Val.sup.116) 
hIL-3 (Ala.sup.101, Val.sup.116) 
In addition, it has also been found that replacement of one or more amino 
acids in the predicted "A" or first predicted .alpha.-helix with another 
amino acid, particularly replacement of amino acids 21, 22 and 25, results 
in loss of IL-3 activity to high affinity IL-3 receptors indicating that 
these residues form part of another IL-3 active part. It has, however, 
been shown that these biologically inactive mutants still retain binding 
ability to the .alpha. chain of the IL-3 receptor. The loss of biological 
activity suggests that these mutants may be used as antagonists. 
According to a second aspect of this invention, there is provided a human 
IL-3 variant or mutant, characterised in that one or more amino acids in 
the predicted "A" or first predicted .alpha.-helix of hIL-3 is/are 
replaced by another amino acid. 
In one embodiment of this aspect of the invention, there is provided a 
human IL-3 variant or mutant, characterised in that amino acid 21 (Asp), 
amino acid 22 (Glu) and/or amino acid 25 (Thr) is/are replaced by another 
amino acid. 
Particularly preferred variants or mutants in accordance with this aspect 
of the invention are: 
hIL-3 (Ala.sup.21, Leu.sup.22, Ala.sup.25) 
hIL-3 (Ala.sup.21, Leu.sup.22) 
hIL-3 (Ala.sup.21) 
hIL-3 (Arg.sup.21) 
hIL-3 (Leu.sup.22) 
hIL-3 (Arg.sup.22) 
hIL-3 (Ala.sup.25) 
In yet another aspect, this invention provides a human IL-3 variant or 
mutant which is characterised in that it combines the two sets of 
variations or mutations broadly described above, that is amino acid 
replacement is effected in both the "A" .alpha.-helix and in or adjacent 
to the "D" .alpha.-helix. These variants or mutants will combine the 
antagonist activity resulting from loss of biological activity with 
increased affinity, resulting in enhanced IL-3 antagonist potency. 
Particularly preferred variants or mutants in accordance with this aspect 
of the invention are: 
hIL-3 (Ala.sup.101, Val.sup.116, Arg.sup.22) 
hIL-3 (Ala.sup.101, Val.sup.116, Ala.sup.21, Leu.sup.22) 
The present invention also extends to the use of the mutants or variants as 
described above as therapeutic agents. Thus, these mutants or variants may 
be provided as active components in therapeutic compositions, together 
with one or more pharmaceutically acceptable carriers or diluents. 
The therapeutic use of the variants or mutants of this invention may 
include, for example, modulation of proliferation and differentiation of 
haemopoietic progenitor cells or of growth and functional activation of 
mature haemopoietic cells. This modulation may be as an agonist or an 
antagonist of IL-3 function. In its broadest sense, the therapeutic use of 
these variants or mutants extends to modulation of the function of all 
cells that express or are made to express IL-3 receptor, including both 
haemopoietic cells and non-haemopoietic cells such as non-myeloid cells 
expressing or made to express IL-3 receptor. 
Further details of the present invention are set out in the following 
Example, and in the accompanying Figures.

EXAMPLE 
Materials and Methods 
1. Site Directed Mutagenesis of Human IL-3 
Human IL-3 mutants were constructed using either site-directed mutagenesis 
or the polymerase chain reaction PCR. 
Substitution of amino acid residue 101 (aspartic acid) by alanine and amino 
acid 116 (lysine) by valine was performed by oligonucleotide site-directed 
mutagenesis. The method used was that of Zoller and Smith (1984, DNA, 3, 
479). 
The oligonucleotide sequences used were: 
(a) Asp(101)-Ala: 
##STR1## 
(b) Lys(116)-Val: 
##STR2## 
(note: altered residue(s) double underlined) 
Site-directed mutagenesis involved annealing a mutagenic oligonucleotide to 
a single stranded M13 vector containing a hIL-3 cDNA constructed 
synthetically (Phillips el. al., 1989, Gene, 84, 501-507). Addition of 
dNTPs and DNA polymerase (Klenow fragment) allowed extension from the 
mutant primer along the M13 template. A primer specific for the M13 
sequence (USP) was added to increase efficiency of the reaction. The 
resulting heteroduplex was transformed into an E.coli strain, JM101. 
Resulting plaques were lifted onto nitrocellulose filters and screened 
with the 32p-labelled mutagenic oligonucleotide. Single stranded DNA was 
prepared from positive plaques and sequenced to confirm the mutation 
(Zoller and Smith, supra). 
A two part polymerase chain reaction was used to create mutants in the 
double stranded IL-3 construct, pJLA.sup.+ IL-3 (Phillips et. al., 
supra). Three primers were involved. Two lay outside of the IL-3 gene and 
the third was the mutagenic oligonucleotide. In the first step the outside 
primer that binds to the antisense strand was used with the mutagenic 
oligonucleotide (binds to the sense strand). Twenty five cycles of PCR 
with these primers resulted in amplification of a portion of the gene. 
This portion contained the mutant sequence and was used as a primer 
together with the other outside primer (binds to the sense strand) for the 
second PCR reaction. 
After construction of the mutants by site-directed mutagenesis or PCR, the 
double stranded DNA was digested with BamHI and SacI and cloned with an 
SacI/EcoRI cut DNA fragment containing SV40 polyadenylation signals into 
BamHI/EcoRI pJL4 (Gough et. al., 1985, EMBO J., 4, 645). Plasmid DNA was 
sequenced to confirm the presence of the IL-3 mutant sequence. 
2. Transfection of IL-3 and Its Analogs. 
Transient transfections were carried out in COS cells. COS cells were grown 
to 50-70% confluence in Dulbecco's Modified Eagle's medium (DMEM) 
containing 20 mM Hepes, Penicillin, Gentomycin and supplemented with 10% 
fetal calf serum (FCS). Cells were harvested with trypsin/EDTA, 
centrifuged and immediately before use resuspended in 20 mM HEPES-buffered 
saline containing 6 mM glucose to 1.times.10.sup.7 cells/ml. 
DNA constructs were introduced into COS cells by electroporation (Chu et. 
al., 1987, Nucleic Acids Res., 15, 1311-1376). For each transfection, 20 
.mu.g of pJLA.sup.+ IL-3 plasmid DNA, 25 .mu.g sonicated salmon sperm DNA 
and 50 .mu.l FCS were mixed with 5.times.10.sup.6 COS cells. The mixture 
was electroporated using a Bio-Rad Gene Pulser before being plated out in 
DMEM + 10% FCS. After a 24 hour incubation period the medium was replaced 
with FCS-free DMEM and incubated for a further 72 hours before the 
conditioned medium was harvested and assayed for IL-3 protein. 
3. Visualisation of IL-3 Protein. 
COS cell supernatants containing IL-3 were size-fractionated by SDS-12.5% 
PAGE and then protein transferred to nitrocellulose. IL-3 protein 
detection was carried out by Western Blot analysis using anti-human IL-3 
antibodies and visualized by autoradiography after the addition of 
.sup.125 I-protein A. 
4. Quantitation of IL-3 Protein. 
The mount of IL-3 protein present in COS supernatants was quantitated by a 
radioimmunoassay (RIA). A competitive RIA was developed using .sup.125 
-I-labelled IL-3 and a polyclonal anti-IL-3 serum (gift from Dr S Clark, 
Genetics Institute). IL-3 was labelled with .sup.125 I by the iodine 
monochloride method as described (Contreras et. al., 1983, Meth. Enzymol. 
92, 277-292). COS cell supernatants (50 .mu.l) were incubated with rabbit 
anti-IL-3 serum (50 .mu.l of 1:10,000 dilution) in Eppendorf microtubes. 
After 4 hr incubation at 4.degree. C., 0.1 ng of .sup.125 I-IL-3 was added 
for a further 16 hr before adding 100 .mu.l of reconstituted anti-rabbit 
Immunobead reagent (Bio-Rad Laboratories, Richmond, Calif.) for 4 hr. The 
mixtures were then washed twice with PBS, and the pellet was resuspended 
in 200 .mu.l of PBS and transferred to 3DT tubes for counting in a 
gamma-counter (Packard Instrument Company, Meriden, Conn.). The amount of 
IL-3 protein was calculated from a standard curve constructed with known 
amounts of IL-3. 
Wild type IL-3 and IL-3 (Ala.sup.101, Val.sup.116) protein produced in E. 
coli were also quantitated directly by scanning densitometry (Fazekas de 
St. Groth et at., 1963, Biochim. Biophys. Acta., 71,377-391). Briefly, 
proteins were electrophoresed on 15% SDS PAGE and stained with Coomassie 
brilliant blue R250. Samples of wild type IL-3, IL-3 (Ala.sup.101, 
Val.sup.116) or RNAse standards were electrophoresed over a concentration 
range of 0.5-5 .mu.g in duplicate and the gel then analysed using an 
LKB-Pharmacia Ultrascan XL scanning laser densitometer. Data analysis was 
performed with GSXL densitometer software. The protein concentrations of 
the unknown samples were calculated using the area under the peak, 
relative to known amounts of RNase standards using the same absorbance 
coefficient. In some cases direct protein quantitation was also performed 
by HPLC peak integration by calculating the area under the IL-3 peak using 
the extinction coefficient of 0.83 AU.ml/mg. The values obtained with each 
method were very similar. An IL-3 preparation (gift from Genetics 
Institute) at 0.6 .mu.g/ml (by amino acid analysis) measured 0.59.+-.0.1 
(mean .+-. SD) lag/ml by scanning laser densitometry, and 0.6.+-.0.07 
.mu.g/ml by radioimmunoassay. In parallel, an IL-3 (Ala.sup.101, 
Val.sup.116) concentration of 1.45.+-.0.06 .mu.g/ml by scanning laser 
densitometry compared with 1.32.+-.0.2 .mu.g/ml by HPLC peak integration, 
and 1.35.+-.0.2 .mu.g/ml by RIA. 
5. Stimulation of Hemopoietic Cell Proliferation. 
Two types of assay were performed: 
(a) Colony assay: this assay measured the clonal proliferation and 
differentiation of bone marrow progenitor cells in semi-solid agar and was 
carried out as described (Lopez et. al., 1985, Blood 72, 1797-1804). 
Briefly, low density, macrophage-depleted bone marrow cells were cultured 
at a concentration of 0.5 to 1.times.10.sup.5 /mL in Iscove's modified 
Dulbecco's medium (IMDM, GIBCI, Grant Island, N.Y.), containing 0.33% agar 
(Difco, Detroit), 25% FCS (Commonwealth Serum Laboratories, Parkville, 
Victoria, Australia), and 20 .mu.mol/LK2-mercaptoethanol. Different 
dilutions of IL-3- containing COS cell supernatants were added to each 
plate. Plates were prepared in triplicate and scored after incubation at 
37.degree. C. in 5% CO2 in a humid atmosphere for 14 days. Clones 
containing 40 cells were scored as colonies. 
(b ) Proliferation of chronic myeloid leukaemic (CML) cells: Primarily CML 
cells from one patient were selected for their ability to incorporate 
[.sup.3 H] thymidine in response to IL-3 as described (Lopez el. al., 
supra ). Briefly, CML cells were placed at 2.times.10.sup.5 cells/mL fresh 
medium containing different concentrations of IL-3. Cells were incubated 
for 24 hours in a flat bottom 96-well NUNCLON plates (2.times.10.sup.4 
cells/well) before being pulsed with [.sup.3 H] thymidine (0.5 
.mu.Ci/well) for four more hours at 37.degree. C. The cells were then 
harvested onto glass filters with a Titertek automated cell harvester and 
counted into a Beckman liquid scintillation counter. Data are expressed in 
cpm, and each point is the mean of six replicates. 
6. Stimulation of Human Monocyte Function: 
(a) Monocyte purification. Monocytes were purified from the peripheral 
blood of normal donors, obtained from the Adelaide Red Cross Transfusion 
Service, as previously described (Elliott et. al., 1990, J. Immunol., 145, 
167-171). In brief, mononuclear cells were prepared by centrifugation of 
whole blood on lymphoprep cushions (Nyegaard, Oslo, Norway) and washed 
twice in HBSS, 0.02% EDTA, 0.1% heat inactivated FCS (Flow Laboratories, 
North Ryde, Australia) and monocytes were purified in a Beckman J-6M/E 
elutriator using the Sanderson chamber, a flow rate of 12 ml/min and a 
constant rotor speed of 2050 rpm. Cells remaining in the chamber after 30 
min were collected, washed twice in HBSS, and used immediately. Using 
these methods, monocyte purity as assessed by morphology and 
nonspecific-esterase staining was always &gt;90% and usually &gt;95%. The major 
contaminating cell types were lymphocytes and granulocytes (principally 
basophils). 
(b) Adhesion assay. Adhesion was measured by an isotopic method essentially 
as described (Elliott et. al., supra). In brief, purified monocytes (0.5 
to 1.times.10.sup.8) were resuspended in 1 ml RPMI 1640 with 0.1% FCS and 
antibiotics and incubated for 30 min at 37.degree. C. with 500 
.mu.Ci.sup.51 Cr in the form of sodium chromate (Amersham International., 
Buckinghamshire, England). Cells were washed thrice in RPMI 1640 and 
resuspended in culture medium consisting of RPMI 1640, 10% FCS, 
antibiotics, and 0.2% sodium bicarbonate. For measurement of adhesion, 1 
to 2.5.times.10.sup.5 monocytes were aliquotted per well in 96-well 
microtitre plates (Nunc, Karnstrup, Denmark) together with stimuli or 
control medium to a total volume of .mu.l, and incubated for the indicated 
periods. Monocyte settling under these conditions were observed to be 
complete within 10 min of incubation. At harvest, samples of supernatant 
were taken to assess spontaneous .sup.51 Cr release (usually &lt;10% of 
cell-associated radioactivity), wells were washed three times with RPMI 
1640 at 37.degree. C., and residual adherent cells lysed in 10 mM 
Tris-hydrochloride, and 1% Nonident p40 detergent (Sigma). Lysates were 
transferred to tubes and counted in a Packard auto-gamma 5650. Percent 
adherence was calculated according to the formula: 
##EQU1## 
7. Histamine Release Assay. 
This was carried out as previously described (Lopez et. al., 1990, J. Cell. 
Physiol., 145, 69-77). Briefly, basophils were obtained from the 
peripheral blood of normal individuals after dextran sedimentation and 
centrifugation over Lymphoprep. The percentage of basophils in these 
preparations varied between 0.2% and 10%. In 300 .mu.l 2.times.10.sup.4 
cells were incubated with 2 .mu.g/ml of purified human IgE. IgE-sensitised 
cells were mixed with a goat IgG antihuman IgE (Cappel 0101-0061) and 
rhIL-3, in a final volume of 500 .mu.l. After incubation for 60 min at 
37.degree. C., the cells were centrifuged and 350 .mu.l aliquots removed 
and stored at -20.degree. C. before assaying for histamine content. 
Histamine was assayed using a radioertzymatic method essentially as 
described (Shaff and Beavan, 1979, Anal. Biochem., 94, 425-430). Briefly, 
samples of 30 .mu.l were diluted with an equal volume of water and mixed 
with a 30 .mu.l solution comprising 27.5 .mu.l 0.1 M sodium phosphate, pH 
7.9, 1.5 .mu.l rat kidney histamine-N-methyltransferase, and 1.0 .mu.l 
(0.5 .mu.Ci).sup.3 H-methyl-S-adensoyl-L-methionine (Dupont Net 155, low 
SA). Tritium-labelled methyl-histamine was extracted into 
chloroform/ether, dried, and counted by scintillation spectrophotometry. 
The cells are expressed as nanograms of histamine per milliliter by 
extrapolation to a standard curve constructed with 10, 5 and 1 ng/ml of 
histamine (SIGMA). 
8. Radioreceptor Assay: 
(a) Radioiodination of hIL-3: rh IL-3 (gift from Dr. L. Park, Immunex 
Corporation, Seattle, Wash.) was radioiodinated by the ICI method as 
previously described (Contreras et. al., supra). Iodinated protein was 
separated from free .sup.125 I by chromatography on a Sephadex G-25 PD 10 
column (Pharmacia, Uppsala, Sweden) equilibrated in phosphate-buffered 
saline (PBS) containing 0.02% Tween 20, and stored at 4.degree. C. for up 
to 4 weeks. Before use, the iodinated protein was purified from Tween and 
nonprotein-associated radioactivity by cation exchange chromatography on a 
0.3-ml CM-Sepharose CL-6B column (Pharmacia) and stored at 4.degree. C. 
for up to 5 days. The radiolabelled IL-3 retained &gt;90% biological activity 
as judged from titration curves using noniondinated rh IL-3 as controls. 
(b) Competition Binding assay. Freshly purified monocytes were suspended in 
binding medium consisting of RPMI 1640 supplemented with 20 mmol/L/HEPES 
and 0.5% bovine serum albumin (BSA). Typically, equal volumes (50 .mu.l) 
of 4.times.10.sup.6 monocytes, 70 pM iodinated IL-3, and different 
concentrations of IL-3 and IL-3 analog were mixed in siliconised glass 
tubes for 16 hr at 4.degree. C. Cell suspensions were then overlaid on 0.2 
mL FCS at 4.degree. C. and centrifuged for 30 seconds at a maximum speed 
in a Beckman Microfugre 12. The tip of each tube was cut off above the 
visible cell pellet and counted in a Packard Auto-Gamma 5650 (Downer's 
Grove, Ill). The results are expressed as Percent competition where 100% 
is the competition observed in the presence of 100 fold excess native 
IL-3. 
9. Competitive Displacement Assay: 
Human peripheral blood monocytes were used in an assay to determine the 
ability of mutant M37 to compete for IL-3 binding sites with wild-type 
IL-3. These experiments show that M37 has 10-15 fold higher affinity for 
the high affinity receptor on these cells than wild-type IL-3. This is 
reflected in the calculated dissociation constants: 
WT: K.sub.d =9.4.times.10.sup.-12 
M37: K.sub.d =5.8.times.10.sup.-13 
10. High Affinity Binding to Cloned IL-3R .alpha. and .alpha. Chains 
PolyA+ RNA was isolated from the human cord blood cell line KMT2. Oligo dT 
primed double stranded cDNA was synthesised and used as template for PCR 
amplification. The PCR primers were designed to amplify the complete 
coding region of the IL-3R alpha chain and also to amplify the coding 
region of the IL-3R .alpha. chain. The PCR products were cloned into the 
vector pGEM-2 for sequence verification, and then into the eukaryotic 
expression vector, pCDM8. The IL-3R .alpha. chain-containing plasmid was 
transfected into COS cells by electroporation, either on its own or in 
conjunction with the IL-3R .alpha. chain-containing plasmid, and after two 
days the cells were used for binding studies. 
The binding of IL-3 (Ala.sup.101, Val.sup.116) produced in E. coli was 
compared to that of wild type IL-3 produced in E. coli and yeast in a 
competition assay using I.sup.125 -labelled IL-3. IL-3 (Ala.sup.101, 
Val.sup.116) was found to have 10 fold higher affinity for COS cells 
transfected with the IL-3R .alpha. chain cDNA and 15-fold higher affinity 
for COS cells transfected with the IL-3R .alpha. chain and .beta. chain 
cDNAs. 
RESULTS 
Mutations in the C-terminus of human IL-3 resulted in the production of 
three analogs: IL-3 (Ala.sup.101) (referred to as M6); IL-3 (Val.sup.116) 
(referred to as M9); and IL-3 (Ala.sup.101, Val.sup.116) (referred to as 
M37), with increased functional activity and binding (summarised in 
Table). The IL-3 mutant IL-3 (Ala.sup.101, Val.sup.116) showed the 
greatest increase in biological activity (15-20 fold) which correlated 
with increased binding affinity (16 fold). The likely location of the 
critical positions (101 and 116) are indicated in the predicted four alpha 
helical structure of IL-3 (FIG. 1 ) with residue 101 in a loop immediately 
before the predicted fourth alpha helix, and residue 116 within the 
predicted fourth alpha helix. 
The increased biological activity of mutants M6, M9 and M37 is demonstrated 
by the stimulation of CML cells (FIG.2) and of monocyte adherence (FIG.3) 
where these mutants were more potent than the wild type IL-3. An increase 
in the number of day 14 colonies as well as increased histamine release 
from basophils (FIG.4) was also observed for mutants M9 and M37. The 
increased ability to stimulate monocyte adherence correlated with their 
ability to bind to the IL-3 high affinity receptor of monocytes (FIG.5) 
where M37 bound with a K.sub.d of 0.58 pM compared to M9 (1.5 pM), M6 (3.1 
pM) and wild type IL-3 9.4 pM). The increased binding affinity was 
analysed on COS cells bearing the transfected IL-3 receptor .alpha. chain 
or both the .alpha. and .beta. chains. As shown in FIG. 6, M37 competed 
for binding more efficiently to the cells expressing only the .alpha. 
chain, thereby demonstrating that mutation in this part of the IL-3 
molecule results not only in increased potency but also in increased 
binding to a defined chain of the IL-3 receptor. FIG. 7 shows that M37 has 
higher affinity to cloned .alpha. and .beta. chains that are 
cotransfected, and IL-3 (Arg.sup.22) (referred to as M47) has less binding 
to the high affinity receptor obtained by cotransfecting the two chains. 
In contrast the IL-3 mutant, IL-3 (Ala.sup.21, Leu.sup.22, Ala.sup.25) 
(referred to as M25) showed lack of stimulation of CML proliferation 
(FIG.2) and of monocyte adherence (FIG.3). M25 was also negative at 
binding high affinity IL-3 receptors at the concentrations tested (FIG.5). 
These results show that M6, M9 and M37 enhance IL-3 binding as well as 
function, and that substitution of residues 21, 22 and 25 result in loss 
of agonistic function and high affinity binding. The contribution of the 
various mutations of M25 was analysed and the results shown in FIG. 8. It 
is evident that mutations at position 22 have the greatest influence on 
the loss of function of this mutant and mutation of glutamic acid at 
position 22 to arginine appears sufficient at abolishing IL-3 activity. 
This mutation, one that is likely to inhibit interaction with the .alpha. 
chain of the receptor (Lopez et al., 1992, EMBO J, 11:909-916), is a good 
potential basis of antagonists for IL-3 function. Furthermore combinations 
of M37 mutations with mutations in position 22 are likely to result in 
antagonists of increased affinity and therefore greater antagonist 
potency. Thus, the present invention includes this model of antagonist 
whereby two sets of mutations are introduced; one to functionally 
inactivate the molecule (e.g. position 22) and the other to increase its 
binding to one of the receptor chains (e.g. M37 mutant). 
TABLE 
______________________________________ 
Relative biological activity and binding affinity of IL-3 mutants 
BINDING 
C-TERMINAL 
PROLIFERATION MONO- Kd value 
MUTANTS COLONIES CML CYTE (pM) + 
______________________________________ 
IL-3 (Ala.sup.101) 
104 .+-. 35* 
205 .+-. 
153 .+-. 48 
2.7 
82 
IL-3 (Val.sup.116) 
420 328 .+-. 
312 1.8 
116 
IL-3 (Ala.sup.101, 
1700 1694 .+-. 
2100 0.58 
Val.sup.116) 281 
______________________________________ 
*Mean .+-. SD of several experiments where a full titration was carried 
out and the concentration of IL3-mutants giving 50% of biological activit 
compared to that of wild type IL3. 
+ K.sub.d of wild type IL3 = 9.4 pM. 
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SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
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TATAGTTCCGGCCACTGAC19 
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CAGTCACCGGCCTTGATAT19 
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