Note: Descriptions are shown in the official language in which they were submitted.
` W 095127052 21 86~54 PCT/US~5~ C3~
HUMAN INTERLEURIN VARIANTS GENERATED BY ALTERNATIVE SPLICING
BACKGROUND OF THE lNVkhllON
Field of the Invention
This in~ention relates to novel splice mutants of
interleukins, which contain deletions of one or more
exons, the expression of which results in truncated
proteins which are useful in regulating the action of
their full-length counterparts.
DescriDtion of the Related Art
Interleukin-4 is a 15 kDa glyc~protein secreted by
activated T cells, (~oward et al. (1982) J. ~xp. ~ed.
155:gl4), mast cells (Brown et al. (1987) Cell 50:809)
and basophils tSeder et al. (lg91) Proc. Na~l. Acad. Scl.
~SA 88:2835) which regulates a wide spectrum of cellular
functions in hematopoietic and nonhematopoetic cells.
The sequence of I~-4 is ~isclosed in U.S. Patent No.
5,017,691.
Recently the 3 dimensional structure of IL-4 has
been sol~ed (Powers et al. (1992) Sciencs 256:1673). The
protein contains 4 left hand ~-helices and two B sheets.
This structural motif is shared by a growing group of
~ ~ ~ 6 ~
WO 95/27052 PCI~/US35J'O lC91
growth factors which do not share prlmary sequence
homology. Powers et al (Powers et al. (1992) Science
- 256:1673) spe~l~ted that I~-4 contains two bin~ sites
for its receptor, based upon analogy to the growth
hor~one/growth hormone receptor system tDe Vos et al.
(1992) Sclence 255:306). The first bin~;ng site is
predicted to involve IL-4 helices o~ and QC~ whereas the
second site is predicted to involve helix ~D~ strand-B~,
and the connecting loop between strand B~ and helix ~3
(Powers et al. (1992) Science 256:1673). One predicted
IL-4/I~-4 receptor interaction site, Asp3l, lies within
the strand BA f exon 2. Exon 2 also contains Cys~,
which forms an intramolec~ ~ disulfide bond with Cys~.
Disruption of this disulfide bond, which would occur in
IL-4~2, is not critical for the biologic acti~ity of
mutant IL-4 molecules (Rruse et al. (1991) FEBS Letters
286:58).
IL-4 belongs to a multigene family of cytokines that
share chromosomal location and mol~c~ r organization and
structure (Boulay et al. (1992) J. Biol. Chem.
267:20525). M~m~er5 of the family include IL-2, IL-3,
IL-4, IL-~, and GM-CSF.
SLmilar to the IL-4 gene, the IL-2 gene is c~osed
of 4 exons, with exon 2 the shortest at 60 bp (Fujita et
al. (1983) Proc. Natl. Acad. sci. ~SA 80:7437). It has
been suggested that the IL-2 molecule has a configuration
WO 95/2~052 2 ~ 3 6 ~ 5 4 PCT~Sss/0l03~
of left-h~n~ alpha-helices and B sheets similar to that
of IL-4 (Bazan (1992) Sclenc~ 257:410). Exon 2 of IL-2
(amino acid residues 31 to 50) ~nco~s a B sheet, a short
~ helix, and the loop con~cting helices Q~ and ~9 (Bazan
(1992) Sciencs 257:410), a region which is si~ r to
that ~co~e~ by exon 2 of IL-4 (Powers et al. (1992)
Science 256:1673). Exon 2 of IL-2 Pnco~s the portion of
the IL-2 molecule that binds the ~ chain (pS5) of the IL-
2 receptor (SauYe et al. (1991) Proc. Natl. Acad. Sci.
~SA 88:4636).
I~-4 has been shown to co-stimulate proliferation of
resting B cells with anti-IgM antiho~ies (Howard et al.
(1982) J. Exp. Med. 155:914), rescue resting B cells from
apoptosis (Illera et al. (1993) J.Trm7~n~ :3521),
15 ~ Ig production by activated B cells (Defiance et
al. (1988) J. Immunol. ~ 2000), and regulate isotype
swit~ to IgGI and IgE in mice (Coffman et al. (1986)
J. Immunol. 136:4538) (Vitetta et al. (1985) J. Exp. Med.
162:1726), and IgG4 and IgE in humans (T~ ren et al.
(lg89) Eur. J. r~ nOl . 13:131). IL-4 exposure has been
demonstrated to increase the number of IgM (Shields et
al. (1989) Immunology 66:224), CD23 (10-12), N~C class II
molec~les (~ ss~t et zl. (1988) J. J~mtttlol. 140:2625)
(Roehm et al. (1984) J. EYP. ~ed. 160:679), LFA-l and
~FA-3 (Rousset et al. (1989) J. Immunol . 143:1490), and
lL-4 Le~eyLor tIL-4R) (Renz et al. (1991) J. Tm~
146:3049) molecules on the surface of B cells. In T
wos~70~2 ~ 3 8 6 8 ~ ~W~9;/0~91
cells, IL-4 has been shown to ~ru~Le proliferation
tFern~ ez-Botran et al. (1986) J. Exp. Ned. 164:580)
(Nosmann et al. (lg86) Proc. Natl. Acad. Sci. ~S~
83:5654) (Mitchell et al. (1989) J. T~?~nol. 142:1548),
generation of the Th2 phenotype (Fer~n~ez-'otran et al.
(1986) J. ~YP. ~ed. 164:580) (Le Gros et al. (1990) J.
Exp. Med. 172:921) and expression of IL-4R (Renz et al.
(1951) J. Immunol. 146:3049).
IL-4 exhibits a synergistic effect with IL-3 in
~ru~oLing the growth of mast c~lls (M~5~ 1 et al. (1986)
Proc. Natl . Acad. Sci . ~SA 83: 5654). IL-4 activates
macrophages to increase tumoricidal acti~ity, N~C class
II expression, and bin~i n~ of IgG immune complexes
(Crawford et al. (1987) J. T?"?n~nol . 139:135). Precursors
of erythroid cells, megakaryocytes, and granulocytes--
macrophages can be co-stimulated with IL-4 to increase
colony formation (Peschell et al. (1987) Blood 70:254).
IL-4 also stimulates proliferation (Feghali et al. (1982)
Cl~n . Immunol . Immunopathol . 63:182), chemotaxis
(Postlethewaite et al. (1991) J. Cl~n. I~vest. 87:2147),
extracellular matrix production (Postlethewaite et al.
(1992) J. Clin . In~st . 90:1479), and intercellular
- adhesion molecule-l (ICAM-1) expression (Piela-Smith et
al. (1992) J. Immunol . 148:1375) by fibroblasts.
Interleukin-2 (IL-2) is a T cell growth factor
secreted by amplifying T cells (T~), which stLmulate
prol~feration and differentiation of cytotoxic T cells
WO95/27052 ~ ~6~5~ Pcr/u~gs~ 9~
(Tc). Tc blast cells exprass surface re~ or for IL-2.
The IL-2 L e~ or (IL-2) is composed of 3 separate
proteins p55 (~ chain), p75 (B chain), and p65 (~ chain~.
In different combinations, these ~h~inc give~rise to
various forms of the IL-2R with different affinities and
capacity to tr~n~ proliferative signals (Taniguchi et
al. (1993) Cell 73 :5). Similarly, the I~-4R consists of
at least two rhA i nc . The first IL-4R chain which was
described shares significant homology to the B chain of
the IL-2R and other members of the growth factor receptor
superfamily (ldzerda et al. (1990) J. ~xp. ~ed. 171:861).
Very recently, a SD~n~ IL-4R chain was identified, which
is the ~c chain of the IL-2R (Russell et al. (1993)
Science 262:1877). IL-4~, like IL-2R, may have several
functional forms (Rigley et al. (1991) Int. Immunol.
3:197)-
8ecause of the widespread effects of IL-4, it is not
surprising that the regulation of IL-4 activity is
pivotal in determining the outcome of certain diseases
(Scott et al. (1988) J. Exp. Med. 168:1675) (Heinzel et
al. (1989) J. Exp. Med. 169:59) (Yamamura et al. (1991)
Science 254:277) (Zwingenberger et al. (1991) Scand. J.
Immunol. 34:243) (Wierenga et al. (1990) J. Immunol.
144:465). In murine leish~n;~is (~Pin~el et al. (1989)
J. Exp. ~ed . 169 : 59 ), human leprosy ~Yamamura et al.
(1991) Sci~nce 254:277~, and human schistosomiasis
(Zwingen~eryer et al. (1991) Scand. J. Immunol. 34:243),
woss/270s2 2 1 8 6 8 54
the production of IL-4 is ~cso~i~ted with chronic
infection. Increased production of IL-4 in response to
alle~y~-~ characterizes human atopic Le~L~v~a~c (Wierenga
- et al. (1950) J. Immunol. 144:465). Studies of the
mole~ll~r regulation of IL-4 activity have previously
focused on the effects of promoters, ~nh~nc~rs, and
negative regulatory elements within the IL-4 gene (~n~el
et al. (1992) J. Tm~-~nol. 149:3239) (Li-Weber et al.
(1992) J. ~m~ol. 148:1913) (Abe et al. (1992) Proc.
Natl. Acad. Sci. ~SA 89:2864) (Li-Neber et al. (1993) J.
r~ nOI. ~ 1371) (Szabo et al. (1993) Mol. Cell. Biol.
13:4793).
S~M~ARY OF T~ lN V N'l'lON
Accordingly, a major object of the present invention
is to provide an isolated nucleic acid cont~; n i n~ exons
1, 3 and 4 of human IL-4.
Another object of the present invention is to
provide an isolated nucleic acid cont~inin~ exons 1, 3
and 4 of human IL-2.
A further object of the present in~ention is to
provide an ex~Lession for the isolated nucleic acids
cont~i n in~ exons 1, 3 and 4 of human IL-2 and 4.
A still further object of the present invention is
to provide polypeptides resulting from the expression of
the isolated nucleic acids cont~ in i n~ exons 1, 3 and 4 of
human I1-2 and 4.
. ~
. -
WO95/27052 ~ 1 `8 6 8`5 ~ PCT/U~9S/OSO~q
Yet a further object of the present invention is to
provide anti hq~ ies to the palypeptides resulting from the
ex~ession of the isolated nucleic acids cont;ti n i n~ exons
1, 3 and 4 of human IL-2 and 4.
Another object of the present invention is to
provide a method of regulating the activity of hu~an IL-2
and 4 by ~m i n ictering an amount of the polypeptides
resulting from the expression of the isolated nucleic
acids cont~ exons 1, 3 and 4 of hu_an IL-2 and 4,
respectively, effective to decrease the biological
effects of human IL-2 and 4, respectively.
Nith the foregoing and other objects, advantages and
features of the invention that will become hereinafter
apparent, the nature of the invention may be more clearly
understood by reference to the following detailed
description of the preferred ~mho~iments of the invention
and to the app~t~e~ cla; m.c .
8~TEF DESCRIPTION OF THE ~RAWINGS
Figure 1 shows the detection of two IL-4 mRNA
species. Total cellular RN~ was extracted from human
peripheral blood monotlltrlear cells tPB~C) stimulated for
6 hours with the anti-CD3 MAb, ORT3, then subjected to
reverse transcriptase-polymerase chain reaction (RT-PCR)
using oligonucleotide primers specific for exons 1 and 4
of human IL-2, exons 1 and 4 of ht~a~ IL-4, and
interferon-~ (IFN-~). IL-2, IL-4, and IFN-~ cRNA
woss/270s2 2 1 8 6 3 5 ~
internal s~ rds were co-amplified in the same reaction
tubes. The RT-PCR amplification products were sub~ected
to gel electrophoresis in a 6% polyacryl ~m; ~P gel. The
5' PCR oligonucleotide primer in each pair was end-
la~eled with ~P, so that amplification products could bedetected on autoradioqrams. Lane l contains mol~ r
weight markers, lane 2 contains IL-2 amplification
products, lane 3 contains IL-4 amplification products,
and lane 4 contains IFN-~ amplification products.
Figure 2 shows the digestion of I1-4~2 DNA with PstI
but not ~incII. Total cellular RNA was extracted from
human PBMC stimulated for 6 hours with the anti-CD3 NAb,
ORT3, then subjected to RT-PCR using oligonucleotide
primers specific for exons l and 4 of human I~-4. The 5'
PCR oligonucleotide primer was end-labeled with ~P.
Aliquots of the RT-PCR mixture were undigested tlane l),
or digested with ~incII tlane 2) or PstI (lane 3)j which
digest IL-4 exons 2 and 3, respectively. The RT-PCR
amplification products were then subjerted to gel
electrophoresis in a 6% polyacry~ P gel. An
autoradiogram of the gel showed that ~incII cleaved the
362 bp I~-4 RT-PCR product, but left the 314 bp I~-4~2
RT-PCR product undigested. PstI cleaved both IL-4 and
I~-4~2 RT-PCR products.
Figure 3 shows the sequence analysis of IL-4 c~Na
and cDNA o~ I~-4 lac~ing exon 2 tI~-4~2). I~-4 and IL-
4~2 RT-PCR amplification products were cloned into the
WO 95127052 2 1 $ ~ 8 5 4 ~.,lIU~/04094
_9_
pC~II vector and their DNA se~l~n~eC determined using
the dideoxy-mediated chain ter~ination method (41).
Sequence analysis of IL-4~2 cDNA demonstrat`ed the
pr~ePn~ of IL-4 exons 1, 3 and 4, with exon I spliced
directly to exon 3, in frame. Sequence analysis of IL-4
cDN~ isolated, cloned, and se~enc~ in parallel with IL-
4~2 cD~A demonstrated the expected presence of exons 1,
2, 3 and 4. An autoradiogram of the sequencing gel at
the region of the IL-4~2 exon 1-exon 3 splice junction is
shown.
Figure 4 shows RNase protection of IL-4 and IL-4~2
RNA. A radiolabeled IL-482 probe cont~i n j n~ an IL-4 exon
l-exon 3 junction was purified and hy~ridized to 15-20 ~g
of denatured total cellular RNA from activated PBMC or
yeast tRNA. ~nhy~ridized RNA was digested with RNase TI,
and the protected RNA fragments were size separated in a
6% denaturing polyacrylamide gel and subjected to
autoradiography. Lane 1 shows mole~l~r weight markers,
lane 2 shows the purified IL-4~2 probe, lane 3 shows
protection of total cellular RNA from activated P3NC, and
lane 4 shows protection of t~NA as a negative ~ol.LLol.
The 342 bp band in lane 2 ~ e~ents protected IL-4~2 RNA
~ and the faint 279 bp band represents protected IL-4 RNA.
Figure 5 shows expression of IL-4 and IL-4~2 mRNAs
in different ratios in different healthy donors. P8MC
~rom ~ healthy indi~iduals were stimulated with anti-CD3
MAb for 6 hours. Ex~ression of IL-4 and IL-4~2 ~RNAs was
WO 9S127052 2 1 8 6 ~ 5 4
--10--
tested with RT-PC~ using IL-4 exon l-and exon 4-specific
oligonucleotide primers. The 5' PC~ oligonucleotide
primer was end-labeled with ~P, so that amplification
products could ~e detected on autoradiogr~m-c. The RT-PCR
amplification products were then subjected to gel
electrophoresis in a 6% polyacrylamide gel. An
autoradiogram of the gel showed that the ratio of IL-
4:IL-4~2 mRNA was approximately 2:1 in individual 1 (lane
1), 1: 1 in individual 2 (lane 2), and to 1:2 in
individual 3 tlane 3). Lane 4 contains molec~ r weight
markers, and lane 4 contains the negative co,.LLol RT-PC~
products.
Figure 6 shows the expression of IL-4 and IL-4~2
mRNAs by human T cell clones. The ~/~ T cell clone GIL
and the ~/~ CD4+ T cell clone CAS were each stimulated
for 6 hours with anti-CD3 mAb. Expression of IL-4 and
IL-4~2 mRNAs by each clone was tested with RT-PCR using
IL-4 exon 1 and exon 4-specific oligonucleotide primers.
RT-PCR products were detected by ethidium bromide
20 s~in;n~ of agarose gels. Both clone GIL (lane l) and
clone CAS (lane 2) pro~t~re~ IL-4 and IL-4~2 mRNAs,
although at different ratios.
Figure 7 shows the kinetics of the expression of IL-
4 and IL-4~2 mRNAs by activated PBMC. PBMC were
stimulated with OKT3 ~Ab, the RNA extracted at the times
indicated. EX~L e,sion of IL-4 and IL-4~2 mRNAs by each
clone was tested with RT-PCR using IL-4 exon l-and exon
,
WO 9SJ27052 PCT/U~3510 1~91
21 ~6~54
4-sp ifiC oligonucleotide primers. The 5' PC~
oligonucleotide primer was end-labeled with ~P. The RT-
PCR amplification products were subjected to gel
electrophoresis in a 6~ polyacrylamide gel. An
autoradiogram of the gel is shown, in which lane 1 = O
hours, lane 2 = 3 hours, lane 3 = 6 hours, lane 4 = 8
hours, lane 5 s 12 hours, and lane 6 = negative control
RT-PCR products.
Figure 8 shows that mice do not produc~ IL-4~2 mRNA.
Spleen cells from B~rR/c mica were stimulated with PMA
and ionomycin for 24 hours. RN~ was extracted and
subjected to RT-PCR using murine IL-4 exon 1- and exon 4-
specific primers. ~uman IL-4 and IL-4~2 mRNA expression
was assayed in parallel from anti-CD3 MAb stimulated PBMC
with human I~-4 exon 1 and exon 4-specific primers. The
RT-PCR products were subjected to agarose gel
- electrophoresis and detected with ethidium bromide
st~ n~. IL-4, but not IL-4~2, ~RNA expression was
obser~ed in the murine spleen cells (lane 2), whereas
human PBMC expressed both IL-4 and IL-4~2 mRNA (lane 2).
Lane M contains mol~lar weight mar~ers.
Figure 9 shows the detection of two IL-2 mRNA
species. Total cellular RNA was extracted from human
P~MC stimulated for 6 hours with the anti-CD3 NAb, O~T3,
then subjected to RT-PC~ using oligonucleotide primers
- specific for exons 1 and 4 of human I~-2. In panel A,
the 5' PCR oligonucleotide primer was end-labeled with
wossl270s2 PCT~SsS/040s4
2 1 8635~
~P, and the RT-PC~ amplification products were subjected
to gel ele~-Lu~horesis in a 6% polyacrylamide gel. Two
RT-PC~ products were identified. In panel B, the RT-PC~
products were size separated by polyacrylamide gel
S electrophoresis, transferred to a nylon membrane by
blotting, and hybridized with an I~-2 exon 3-specific
probe (first autoradiogram) or an IL-2 exon 2-specific
probe (second autoradiogram). Lane M contains molec~ r
weight markers in each gel. Lanes 1 and 3 contain RT-PC~
products, and lanes 2 and 4 contain negati~e ~o~.L~ol RT-
PCR products. Two bands hybridized with the exon 3-
specific probe (first autoradiogram), whereas only the
larger band hybridized with the exon 2-specific probe
tsecond autoradiogram). In a S;mi l~r expériment shown in
panel C, the RT-PCR products were hybridized with an I~-2
exon l/exon 3 junction specific probe. Lane M contains
mole~~ weight mar~ers, and lane 2 contains RT-PCR
products. Two bands hybridize with this probe, and the
relative intensity of the smaller band (IL-2~2) compared
to the larger band (native IL-2) is much greater than is
seen in p~nol S A or B.
Figure 10 shows the complete sequence of the IL-4
gene (SEQ ID N0:23) (Arai et al, J. Immunol ., Vol. 142,
pp. 0274-0282 (1989)). The I~-4~2 (SEQ ID NO:24) of the
present invention contains the se~le~c~s encoded ~y exons
1, 3 and 4, but not 2.
woss/270s2 2 1 8 6 8 5 4 PCT/u~s~ I091
Figure ll shows the complete sequence of the IL-2
gene (SEQ ID NO:25) (Fujita et al, Proc. Natl. Acad.
Sci., Vol. 80, pp. 7437-7441 (1983)). The I1-2~2 (SEQ rD
NO:26) of the present invention contains the sequences
~co~ by exons l, 3 and 4, but not 2.
DEr~lr~n ~ESCRIPTION OF TXE PREFERRED EMBODIMENTS
OF THE lN V ~:N l loN
The present invention ~mo~ctrates the
expression of IL-4~2, a second mRNA isoform transcribed
from the IL-4 gene by alternative splicing. Alternative
splicing is an efficient me~h~is~ by which multiple
protein isoforms may be generated from a single genetic
locus. Protein isoforms generated by this regulatory
me~h~nis~ may vary in function, cellular localization, or
pattern of developmental expression (Smith et al. (1989)
Annu . Rev. Genet . 23: S27) . Alternative splicing is used
in terminally differentiated cells to ~e~,ibly modify
20 protein expression without changing the genetic content
of the cells (Smith et al. (1989) Annu. Rev. Genet.
23: 527) .
IL-4 ~2 was first obser~ed as an additional RT-PCR
amplification product during analysis of cyto~in~ gene
25 expression. Cloning and sequencing of the cDNA
demonstrated that IL-4~2 consists of exons l, 3 and 4 of
the IL-4 gene, but not exon 2. Splicing of exon l to
exon 3 o~ s in IL-4~2 mgNA without changing the re~
woss/270s2 2 1 868 S4 PCT~Sss/04094
-14-
frame; exons 1 and 3 are directly opposed at the splice
junction without using splico donor or acceptor sites
different from those used by IL-4 mRNA. Other than the
omission of exon 2, no other changes in the entire
protein ~n~ ing region are observed when IL-4~2 and IL-4
mRNAs are ~o~r~red. To date, all h~ nc tested express
both IL-4 and IL-4~2 mRNAs. Both IL-4 and IL-4~2 mRNAs
increase with T cell acti~ation, and the ratio of IL-
4:IL-4,~2 mRNA increases. A few healthy humans expressed
more IL-4~2 than IL-4 mRNA on occasion, but this fin~i~g
was not maintained over time in these same individuals.
The present invention also demonstrates that external
events can change the ratio of IL-4 to IL-4~2 mRNA.
The IL-4~2 of the present invention can be isolated
from any human immune cell, preferably peripheral blood
mononuclear cells (PBMC) and T cells. The cells obt~;
from a human donor can be separated from blood and other
cells using any method known in the art, preferably by
density gradient centrifugation, and preferably using a
medium such as, but not limited to, Histopaque.
Cells with the a~o~iate surface mar~ers,
includinq subsets of T cells, preferably CD4+ ~/~ T cells
and ~/~ T cells, can be isolated using any te~h~i~ue
known in the art to separate such cell subsets. A
particularly preferable method is using positive
selection ~ia specific monoclonal ant;ho~ies. Especially
preferable monoclonal antiho~ies include anti-Leu3a
-
wossl270s2 2 1 8 6 8 5 4 pcT~ssslol-91
--15--
~p~cific for CD4, and ~TCSl, specific for V~l - J~l and
J~2.
Following bi n~ i n~ of the NAb to the cells, the cells
can be treated with a s~co~ antibody specific for the
first antibody, which is either coupled to a separation
medium, or which can be coupled to a separation medium
via a particular linkage, such as a biotin-a~idin
linkage. Particularly preferable for the present
invention is a sheep - anti-mouse IgG coupled to a
10 ~ OL L such as Dy~h~s N-450 (Dynal).
Onco the cells are separated, they are cloned in the
presence of mitogens, growth factors and/or feeder cells.
Preferable mitogens include but are not limited to
phytoh~m~gglutinin (P~A) at a cQnc~tration of l-l00
~g/ml, preferably at about l0~g/ml. Preferable growth
factors include but are not limited to IL-2, at a
co~ca~tration of l-l00 U/ml, preferably about 50 U/ml.
Preferable feeder cells include but are not l~mited to
allogeneic P8MC, preferably irradiated at l000-l0,000
rad, preferably at about 3,000 rad. The cells may also
be treated with supernatant from a hybridoma cell line,
preferably OKT3, which may stinulate T cell
proliferation.
The cells can be grown in any suitable medium, but
RPNI is preferable. The medium is preferably
supplemented with serum, such as human serum, preferably
humRn male AB serum, and/or fetal calf serum (FCS). The
woss/270s2 PCT~sss/o4os4
21 ~36854
-16-
serum content is 3-12%, most preferably 10% total serum.
It is pAFticularly preferable to use a comkination of
human male AB serum and FCS, most preferably a mixture cf
5% of each serum.
The calls are then ~Yp~n~, preferably by bi-weekly
stimulation with mitogens, feeder cells and growth
factors. The expression of surface mar~ers can be
confirmed using flow cytometry, fluorescence activated
cell sorters (FACS), im~ nh~istochemistry and the like.
Preferably, the cells are treated with FITC-conjugated
ant;ho~;es using st~n~7rd t~chn;ques.
RNA can be extracted from the cells by any means
known in the art, preferably using guanidinium
thiocyanate. The RNA can then be reverse transcribed
into cDNA using known methods, pre~erably with M-N~V
reverse transcriptase and random hexamer primers.
The cDNA generated by reverse transcription of the
RNA can then be amplified for further use. Such
amplification schemes include but are not limited to
polymerase chain reaction (PC~), ligase chain reaction
(LCR) and ~ariants thereof. Conditions for such
proced~es are well known in the art. The amplification
products so generated can then be isolated by any
t~hnique known in the art. A particularly preferable
method is by separation on an agarose gel and
electroelution o~ the product onto DEAE paper followed by
phenol/chloroform extraction.
woss/270s2 PCT~S9~10~9~
21 86~54
-17-
The ampli~ied isolated DNA can then be ligatéd into
a vector suitable for se~ncin~, transformed into
competent cells, and DNA prepared therefrom. Isolation
of such plasmids is by te~hni~ues well known in the art.
~he DNA inserts can then be se~t~ce~ using any method
known in the art, including ~axam-Gilbert se~uencing, or
preferably by the dideoxy chain termination reaction of
Sanger et al.
The RNA of interest can be identified using any
means known in the art, but particularly preferable is an
RNA protection assay. According to this method, a
radiol~h~lled probe is made which will bind to the RNA of
interest. The radio~helled probe is incubated with
total cellular RNA, and unhybridized RNA is digested
using RNase. Vpon hybridization of the l~h~lled probe to
the RNA of interest, the RNA of interest is protected
from the RNase and can be identified by electrophoresis
on a polyacrylamide gel, with subsequent autoradiography.
~ikewise, the cDNAs prepared can be characterized by
Southern blot wherein the DNA of interest is run on an
agarose gel, the nucleic acids on the gel are transferred
to a nylon or nitrocellulose ~embrane, and the membrane
is hybridized with a probe which will aid in the
characterization of the DNA. Particularly preferable for
the present invention is a probe which spans the
exon/exon junctions of an interleukin. Such probes are
then able to identify alternative splice mutants.
wossl27os2 21 86354 PCT/U~g~/O~
-18-
The above-described methods are suitable for use in
detectinq expression in various donors and various cells
obt~inP~ therefrom. In addition, the kinetics of
expression can be analy2ed to determ~ne whether splica
variants are erpressed to the same extent as the wild
type polypeptides upon stimulation of c~lls.
The alternative splice variants of the present
invention find use in treating ~arious conditions,
exemplified but not limited to (1) allergic reactions,
including, but not limited to anaphylactic shock, asthma,
and eczema; (2) infectious conditions, including, but not
limited to leishmania, and for delaying the clinical
transition from human ;rm~no~ficiency virus (HIV)
antibody positivity to ac~uired immune deficiency
syndrome (AIDS); (3) autoimm-lne disorders, including but
not limited to systemic sclerosis and diabetes; (4)
fibrotic diseases, including, but not limited to
~Y~Pq-si~e scar tissue formation, excessive extracellular
matrix formation, ~Yressive wound healing, and for
treating burns; and (5) disorders involving endothelial
cells, as IL-4 has been shown to alter the morphology of
such cells. In addition, the splice variants of the
present invention may be useful in the treatment of any
condition which arises from over-expression of the full-
length polypeptides.
The present invention not only amplifies a ceco~and using RT-PCR with IL-4 primers, but also
woss/270s2 2 1 8 6~ 54 PCT~59~ 5~
-19-
demonstrates that the second band is related to IL-4
using an in~p~n~nt method, an RNase protection assay.
The present invention also provides se~uence data ~or the
entire protein Dn~o~ing region to definitively show that
the molecule is identical to IL-4, except for the
omission of exon 2.
The sequence data disclosed herein show that IL-4
exon 2 functions as a cassette exon (Smith et al. t19~9)
Annu . Rev. Genet . 23:527), and that no shift in the
rp~ing frame o~ when it is omitted. The RNase
protection assay demonstrates that the IL-4~2 transcript
is expressed in the same sense orientation as IL-4
transcripts, because an anti-sense probe was used for
protection.
Also determined was whether the alternative splicing
of exon 2 was unique to IL-4 mRNA or part of a more
general regulatory me~h~nism for cytok;nes. The
cytokin~s tested were IL-2, -3, -5, and GM-CSF, which
share protein folding motifs, genomic organization, and
~e~-y~or extracellular bin~ing domains with IL-4 t8Oulay
et al. (1992) J. Biol. Chem. 267:20525). The present
- invention also demonstrates that IL-2, but not IL-3, IL-
5, or GM-CSF, also uses alternative splicing of exon 2.
Both IL-2 and IL-4 splice variants omit exon 2, which
~o~ sLmilar regions of s~con~ry structure and
participate in ~e~-y~or bi~in~ for each mole~ule.
wossn70s2 21~6~54 PCT/u~gs/01C9~
-20-
Alternative splicing can be used in ht-~-nc to
provide variants of IL-4 and IL-2 which function as
agonists or antagonists of the native cyto~;~oc~
~opatl~ing upon the ~tl~hPrs and types of receptors on the
cells. 8y analogy to IL-2 mole~ules with defined amino
acid substitutions (57), IL-2~2 will still bind to the
inter~ediate affinity IL-2R tB/~ rh~inC) and generate a
cellular response. Where loss of the ability to bind to
the ~ chain reduces the capacity of IL-2~2 to activate
cells through the high affinity trimolecular ~/B~
complex, the cause is either ineffective triggering or
reduction of the assembly of the complex. In these
cases, IL-2~2 is a competitive inhibitor of IL-2
activation through high affinity IL-2R. Similarly, IL-4R
has at least two form_ with lower (the conventional IL-4R
chain alone) and higher (the conventional IL-4R chain
plus ~c) affinities (Rt1CSP11 et al. (1993) Science
262:1877) (Kondo et al. (1993) Science 262:1874). I~-4~2
will bind to the conventional IL-4R chain and serve 2S an
agonist through the lower affinity IL-4R, yet will
antaqonize cellular activation through the high affinity
IL-4R by bloc~ing heterodimerization of the conventional
IL-4R chain and~c.
A se-on~ species of IL-4 mRNA can be identified
using both the reverse transcriptase polymerase chain
reaction and an RNase protection assay. This novel IL-4
~RNA is 48 base pairs smaller than IL-4 mRNA, which is
wossn7052 21 8 68 54
-21-
the size of IL-4 exon 2. Se~uence data of cloned cDNA
demonstrates that this variant contains IL-4 exons 1, 3
and 4, with exon 1 spliced directly to exon 3 in an open
r ~i ng frame. The entire protein ~n~oA i ng region of
this variant, named IL-4~2, is identical to IL-4, except
for the omission of exon 2. IL-4~2 mRNA is detected in
all human PBMC and T cell clones tested, but is absent
from mouse spleen cells. Amounts of both IL-4 and IL-4~2
mRNAs increase upon T cell activation, although IL-4 mRNA
increases to a greater extent than does IL-4~2 mRNA.
Similar experiments suggest that h~ nc also express a
variant of IL-2 mRNA, in which exon 2 is deleted by
alternative splicing. Human IL-3, IL-~, and GM-CSF do
not use alternative splicing to delete exon 2. Thus,
variants of both human IL-4 and IL-2 exist in which
similar structural regions of each molecule are omitted
by alternative splicing of mRNA.
The following examples are presented in order to
more fully illustrate the preferred embo~ nts of the
invention. They should in no way be construed, however,
as limiting the broad scope of the invention.
EXAMPLE 1-
C-ll 8~p~s~tio~ ~d T C~ll Cloning.
~uman PBMC were isolated from healthy donors by
density gradient centrifugation using ~istopaque 1077
(Sigma Chemical Co., St Louis, M0). A CD4+ ~/~ T cell
woss/270s2 ~1 8 6 8 5 4 PCT~S95/04094
-22-
clone, CaS, and a ~/~ T coll clone, GI~, were isolated
from human P~MC t~rough positive selection using MA~
anti-Leu 3a (~ecton Dir~;nCo~, Mountain View, CA),
specific for CD4, and NAb ~TCS1 (T Cell Sciences,
Cambridge, NA), specific for V~l-J~l- (36) and V~1-J~2-
(Ronig et al. (1~89) Eur. J. rm~T~nOl. 19:2099) encoded
epitopes. Su~sequent treatment with sheep anti-mouse IgG
coupled to Dyn~he~s M-450 (Dynal Inc., Great Nec~, ~Y)
and magnetic bead separation were carried out according
to the manufacturer's instructions.
Positively selected cells were ;~ ;ately cloned by
limiting dilution in the presence of 10 ~g/ml P~A (Sigma
Chemical Co.), 50 ~/ml r human IL-2 (Hoffmann-La Roche
Inc., Nutley, NJ), and irradiated (3000 rad) allogeneic
P3MC as feeder cells. Complete tissue medium was RPNI-
1640 cont~i ni ~ 5% heat-inactivated human male AB serum,
5% heat-inactivated FCS, 10 mM Hepes, p~ 7.4, 2 mM L-
glutamine, 1 mM sodium ~yr~v~te, 0.1 mM non-essential
amino acid mix, 5 x 10 5 N 2-~E, and 5 ~g/ml gentamicin
sulfate. The T cell clones were ~ in 2 ml
cultures by biweeXly stimulation with PHA and additional
feeder cells. Additional r human IL-2 at the same
con~ntration was added every 4 d. Expression of CD4 and
V~l by T cell clones CAS and GIL, respectively, was
confirmed using two-color flow cytometric analysis with
FITC-conjugated Leu 3a MAb or FITC-conjugated ~TCS1 MAb
WO 951270S2 2 1 8 6 8 ~4 P~-l/U~SI~4C9~
-23-
and PE-conjugated anti-human Leu-4 (CD3) MAb (Becton
Di~;neon), using st~n~rd te~h~iques.
EXAMP$E 2
S ~ C~ll 8timul~t~on.
PBMC (S x 106~ or 5 x 106 cloned T calls plus 2.5 x
106 irradiated (3000 rad) allcgeneic PBMC were stimulated
in 2 ml cultures in complete tissue culture media
supplemented to a final co~ tration of 10% with
supernatant of the anti-CD3 MAb secreting hybridoma, OKT3
(American Type Culture Collection, Roc~ville, MD). This
co~r^ntration of ORT3 supernatant had previously been
deter~; n~ to optimally st; m~ te T cell proliferation.
~MPLE 3
R~a ~olat~on ~nd R~-PC~.
Total cellular RNA was isolated from PBNC, T cell
clones, and BALB/c spleen cells by acid ~An;~in;um
thiocyanate-phenol chloroform extraction tChomczynski et
al. (1987) Anal. ~ h~m. 162:1S6). One ~g of RNA was
denatured ~or 5 minutes at 65C and then reverse
transcribed into cDNA using in a 15 ~1 reaction mixture
cont~ g 200 U of M-~LV reverse transcriptase tBe~h~s~A
R~Q~ArCh La~S (BRL), Beth~s~A, MD], 50 mM Tris-HCl, pH
8.3, 7S m~ XCl, 8 mM DTT, 3 ~ MgC~, 0.5 mM each dATP,
dCTP, dGTP, dTTP (Pharmacia LKB Biotec~nology~
Piscataway, NY), 1 U/ml RNasin (Promega, MA~;-O~ WI),
wogst270s2 ~ ~ 8 6 8 5 ~ PCT~Sgs/040s4
and random hPx~-r primers (BRL). This reaction mixture
was i"~lh~ted at 37C for l hour.
A 25 ~l PCR reaction mixture was made cont~;nin~ 2.5
~1 c~NA mixture, 50 m~ Tris-~Cl, pH 8.8, 50 mM KCl, 4 mM
MgCl2, O.2 mM each dATP, d P, dGTP, dTTP, 0.4 mM each 3'
and 5' PC~ oligonucleotide primers, and 0.625 ~ Taq
polymerase (Perkin Elmer Cetus, Norwalk, CT). The 5' PC~
oligonucleotide primers were 5' end-la~eled with ~-~Pj-
ATP (Amersham Corporation, Arlington Heights, IL) and T4
polynucleotide kinase tUnited States Bio~h~ical (USB),
Cleveland, 0~;, following the USB protocol. The PCR
mixture was amplified as follows: denaturation at g5C
for 3Q ~D~0~5, primer annealing at 60C for 2 minutes,
and primer extension at 72C for 3 minutes (15-30
cycles), followed with a final 7 minute 72C extension.
Ten PCR products were subjected to gel electrophoresis
though 2.5% agarose or 6~ polyacrylamide gels. Products
of a moc~ reverse transcriptase reaction, in which H20 was
added in place of RNA, were used as negative cGl~L~ol
amplifications in all experiments.
The PC~ oligonucleotide primer pairs used in these
experiments were: human IL-2 exon 1 forward 5'-
ATGTACAG&~TGCAA~ -3' tSEQ ID NO: l~ and exon 4
reverse 5'GTTA~ AGATGATGCTTTGAC-3' ~SEQ ID NO: 2~ ;
human I~-3 exon l forward 5' TCCTGCTCCAACTCCTGG-3'
~SEQ ID NO: 3] and exon 4 reverse 5'-GCTCAAA~ L~
3' tSEQ ID No: 4~; human IL-4 pair A exon 1 forward
. ;.
wos~270s2 2 1 8 6 8 5 4 pcT~sss~ J~1
-Z5-
5'-TCTTCCTGCTAGCATGTGC-3' ~ SFQ ID N0: 5] and exon 4
reverse 5'-CGTACTCTGGTTGGCTTTCC-3' tSEQ I~ N0: 6~; human
IL-4 pair B exon 1 forward 5'-AAGCTTATGGGTCTCAC~
3' tSEQ ID NO: 7] and exon 4 L ~V~ ~e 5t-
S GGA~ L~ATCAG C~ ArTTTGA-3' tSE~ ID N0: 8~; murine
IL-4 exon 1 forward 5'-AGCr~TA~CCaCGGATGCGAC-3' tS~Q ID
N0: 9~ and exon 4 Leve~ae 5'-CTCAGTACT~C~ÇTAATCCAT- 3'
tSEQ ID NO: lOJ; human IL-5 exon l forward 5'- .
~ A~CCTTGGC~ 'PAA~A~çC-3' ~SEQ ID NO: ll] and
exon 4 reverse 5'-CCATTCTCCGCC~AÇ&CTGACTAATTTTT-3~ ~SEQ
ID N0: l2~; human GM-CSF exon l forward 5'-
ATGTGGCTGCAGAGCCTGCTGCTC-3' t SEQ ID N0: 13] and exon 4
reverse 5'TCACTCCTGGACTG&CTCCCAGCA-3' tSEQ ID NO: 14~;
and human IFN-~ forward 5'CAGCTCTGCATCG~ >TCT-3'
tSEQ ID NO: 15] and re~erse 5'-TG~ C~ACCTT~-~A~GCAT-
3' tSEQ ID NO: 16]. Bam~I and ~indIII restriction enzyme
r~Co~nition se~l~nres are underlined in the human IL-4
pair B prLmers. Construction o~ the IL-2, IL-4 and IFN-
~c~NA internal st~ rds are described in (Alms, W.J. et
al. which is hereby inco~oLated by reference in its
entirety).
.
EXAMP~E 4
~lOn~ o~ RT-PC~ Product-~ a~d DNa 8e~A~ci~g.
Complementary DNAs for IL-4 and IL-4~2 were
generated and amplified by RT-PCR using IL-4 exon l and 4
specific primers cont~i~;ng digestion sites for Bam~I and
-
WO 95/2~052 2 1 8 6 8 5 4 PCT/U~55i~ ~C9 1
--26--
dIII restriction ~ n~rleases. Amplification
products for I1-4 and IL-4 ~2 were isolated from 2.5~
agarose gels using DEAE paper (Sam~rook, J. et al. (198g)
Molecular cloninq: a laboratorv manual Cold Spring Har~cr
Laboratory Press, New York) (incorporated herein by
referenca in its entirety). After two phenol/chloroform
extractions, the c~NA products were ligated into the
pCRT~ II vector (In~itrogen Corp., San Diego, CA) and then
used to transform INV~F' competent cells, according to
the manufacturer's instructions. Plasmids cont~ g IL-
4 and IL-4~2 cDNA inserts were isolated by conventional
t~chn;ques (Sambrook, J. et al. (1989) Molecular cloninc:
a laboratorY manual Cold Spring ~arbor Laboratory Press,
New York) (incorporated herein by reference in its
entirety) and used in sequence analyses. IL-4~2 cDNA
inserts were se~nr~ by the dideoxy-mediated chain
termination method (Sanger et al. (1977) Proc. Natl.
Acad . sci . ~SA 74: 5463) (incu~v~ated herein by reference
in its entirety), using the Ml3 (-20) forward primer (5'-
2 0 G~rp~ Ac~ t`GG~::cAGT-3 ' ) t SEQ ID NO: 17 ] and Seguenasen'
(USB), and analyzed by electrophoresis in a 7~ Long
RangeSM (AT Biochem, Malvern, PA) qel. IL-4 and I~-4~2
c2NA inser~s without ~aq polymerase-induced sequenco
~LO' S were then used for RNase prote~ion assays.
woss/270s2 ~ 1 8~:54 PCT~SsS/o4os4
EXAMPLE 5
R~ase Protection A~s~y~.
A 362 bp IL-4~2 RT-PCR fragment that c~n~P~ IL-4
exon l to exon 4 with an exon 1-3 junction was cloned
into the pCRT~ II vector. The insert orientation was
determined by sequence analysis. An RNase protection
assay was performed using Ambion RPA II~U (Ambion Inc.,
Austin, TX~, according to the manufacturer's protocol.
Briefly, radiolabeled IL-4~2 probe was generated by
i~th~ting 100 ng of Spe~ linearized IL-4~2-con~inin~
plasmid with 5 units T7 RNA polymerase (BRL), 0.5 mM each
A~P, P, and GTP, 12 ~M ~T~ and 6 ~M 400 Ci/mmol 5't~-
~P]-~TP (~u~o~L NEN, Boston, MA) for 45 min at 37C. The
final specific activity of the IL-4~2 probe was l x 109
cpm/~g DNA. The radiolabeled probe was subjected to gel
electrophoresis in a 6% denaturing polyacrylzmide gel,
and the full length IL-4~2 probe was identified by
autoradiography. The band con~ img the probe was
excised from the gel, and the IL-4~2 probe was eluted at
37C in 400 ~l buffer cont~ g 2 M ammonium acetate, l~
SDS and 25 ~g/ml yeast transfer RNA (t~NA). The
radiolabeled IL-4~2 probe (1 x 106 cpm) was hybridized
with 15-20 ~g of denatured total cellular R~A or tRNA for
16 hours at 37C in 80% formamide, 40 mM PIPES, pH 6.4,
400 ~M NaCl, and 1 mM EDTA buffer. ~nhy~ridized RNA was
digested at 30C for 30 minutes with 200 ~l RNase
woss/270s2 2 1 8 ~ 8 5~4 PCT~Ss~/o~~g~
-28-
digestion buffer (Ambion Inc.) con~ini~g 4000 ~/ml RNase
Tl (BR~). RNases were inacti~ated, and the protected RNA
fragments were size separated in a 6% denat~ring
polyacrylamide gel and subjected to autoradiography.
The RNase protection analysis was used to verify the
prPsP~rP of IL-4~2 mRNA in human PBMC. A 464 bp IL-4~2
probe cont~i n; ~g IL-4 exons 1, 3, and 4, including the
exon l-exon 3 splice junction, was radiolabeled. This
probe would be expected to hybridize with and protect a
342 bp fragment of IL-4~2 mRNA tnucleotides +136 to +198
of exon 1 plus nucleotides +247 to +525 of exons 3 and
4]. In addition, the probe should protect a 63 bp
fragment of exon 1 tnucleotide +136 to +198] of IL-4 mRNA
and a 279 bp fragment of exons 3 and 4 (nucleotides +247
to +525] of I~-4 mRNA, because I~-4~2 and IL-4 share
these exons. RNase protection of total cellular RNA from
anti-CD3 stimulated P~MC verified the presence of both
IL-4~2 (342 bp) and IL-4 (279 bp and 63 bp) fragments
(Fig. 4).
EXAMPLE 6
Oligo~ucleotide ~ybridis~tion.
RT-PCR amplification products were size separated by
agarose gel electrophoresis. The gel was so~P~
se~uentially for 30 minutes each in denaturation solution
(1.5 N NaCl, 0.5 M NaOH) and neutralization solution (1.5
M NaC1, 1 M Tris-~Cl, pH 7.4) for 30 minutes. The RT-PCR
woss~70s2 2 ~ 8 6 8 5 4 pcT~ss~lo1~s4
-2~-
amplification products were next transferred to nylon
membranes by blotting overnight in 20x SSC buffer. The
DNA samples were cross-l;~ke~ to the membrane by ~V light
irradiation. N~m~ranes were prehybridized in 6x SSC, lOx
~nh~rdt's solution, 0.1% SDS and 50 ~g/ml sper~ DNA for
at least 1 hcur at 42C and then hybridized overnight
with 0.2 ~g ~P 5' end-labeled oligonucleotide probe at
49OC in 6x SSC and 1% S~S. The membrane was washed ~hree
times in 6x SSC and 1% SDS for 10 minutes at room
temperature, followed by a final 49C wash. Membranes
~- were then subjected to PhosphorImager analysis (Molec~ r
Dynamics, S~yvdler CA) or subjected to autoradiography.
Cytokine specific oligonucleotide probe sequences were:
human IL-2 exon 2-specific 5'-CTr~rr~r~-~TGCTCACA-3' tSEQ
ID N0: 18]; human IL-2 exon 3-specific 5'-
C~ GAGGAAGTG A-3' tSEQ ID N0: 19]; human IL-3 exon
1/exon 3 junction-specific 5'-C~ -LGCTGGPAA~T~CC-3~ tSEQ
ID NO: 20]; human IL-5 exon l/exon 3 junction-specific
5'-GCCAATGAGr~rr~T~-3' tSEQ ID NO: 21]; and human GM-
CSF exon 1/exon 3 junction-specific 5'-
GCTGAGATGGAGCC~ACC-3' tSEQ ID N0: 22].
Two IL-4 mRNA species were consistently detected
from all donors tested (Fig. 1). The larger IL-4 RT-PCR
amplification product was 362 bp, correspqn~ing to the
predicted size of IL-4 mRNA. The ~Dcon~, smaller RT-PC~
amplification product, designated IL-4~2, migrated with
an apparent size of 314 bp. Changes in the PCR buffer
woss/270s2 21 868~ PCT~S9~/01^3~
-30-
MgC~ con~ntration, primer an~ i ng temperature, and
pairs of I~-4 exon 1- and 4-specific PCR primers were
sful in elLminating the s~aller RT-PC~ product
(data not shown).
The consistent expression of the smaller 314 bp
fragment when total cellular RNA was subjected to RT-PCR
and the lacX of a corresponding product when an IL-4 cRNA
was Si~ rly subjected to RT-PCR (Fig. 1) suggested that
this fragment was a specific RT-PCR amplification product
resulting from alternative splicing of the IL-4 gene
transcript. The IL-4 gene contains 4 exons and 3 introns
(Arai et al. (1989) J. Tr~?-nol. 142:274). The apparent
size difference between the IL-4 m~NA RT-PCR product and
the IL-4~2 RT-PCR product was 48 bp, which is the size of
IL-4 exon 2. To test whether the 314 bp IL-4~2 RT-PC~
product did not contain IL-4 exon 2, whereas the larger
362 bp IL-4 RT-PCR product did, both products were
digested with ~LncII and PstI, which digest IL-4 exons 2
and 3, respectively. ~incII cleaved the IL-4 RT-PCR
product, but left the IL-4~2 RT-PCR product undigested
(Fig. 2). In ~ol.LLast, PstI cleaved both IL-4 and IL-4~2
RT-PC~ products (Fig. 2).
~XAMPLE 7
8egue~ce A~ly~is of I~ 2.
The IL-4 and IL-4~2 RT-PCR amplification products
were then cloned into the pCRT~ ector and their DNA
woss1270s2 2f ~68~54 PCT~S9;1~S~g~
se~ncoC determined (Fig. 3). Sequence analysis of
IL-4~2 cDNA demonstrated the presence of IL-4 exons l, 3
and 4, with exon 1 spliced directly to exon 3. Se~uence
analysis of IL-4 c~NA isolated, cloned, and se~nc~ in
parallel with IL-4~2 c~NA demonstrated the expected
pr~s~ of exons l, 2, 3 and 4, with a exon 2 to exon 3
in-frame splice junction. Of note, both IL-4 and IL-4~2
contain gaa residues 5' at exon 2-exon 3 and exon l-exon
3 splices, respectively. No other sequence changes were
- lO observed throughout the entire protein-o~co~ region of
IL-4~2.
EXAMPBE 8
~ 2 mRXa Espr~ssion i~ ~e~lthy ~um~n~ ~d in ~um~n T
C~ll Clo~
IL-4 and IL-4~2 mRNA expression were analyzed in
PBMC from 25 healthy humans. IL-4 and IL-4~2 mRNA were
co-expressed in all donors tested, but varied in relative
ratio from individual to individual. Examples of this
variability are shown in Fig. 5. In this experiment,
PBMC from 3 indiv~ c were stimulated with anti-CD3 NAb
for 6 hours. The relative expression of IL-4 to IL-4~2
mRNA was measured by RT-PCR using conditions under which
the PCR products were being exponentially amplified (25
cycles). The ratio of IL-4:IL-4~2 mRNA varied from
approxi~ately 2:l in individual l to l:2 in individual 3.
Individual 2 expressed approximately e~ual amounts of IL-
WO 95/27052 P~,l/U:,9S,'~ 1~3 1
21 86~4
-3Z-
4 and IL-4~2 mRN~s- The ~XyL ~~sion of greater or equal
levels of IL-4 than IL-4~2 mRNA was the predominant
phe..oLy~a and was present in 22 of 25 indivi~lt~c tested,
with a range of 16:1 to 1:1. Three indivi~ s~ however,
expressed greater levels of IL-4~2 mRNA than IL-4 mRNA,
on at least one occasion.
To confirm that T cells were the source of IL-4~2
mRNA expression among the PBMC, cloned T cells were
tested. The a/B CD4+ T cell clone CAS and the Q/B T cell
lo clone, GIL, were each stimulated for 6 hours with anti-
CD3 NAb. Both cloned T cells proAt~c ~ IL-4 and IL-4~2
mRNAs (Fig. 6).
~MPLE 9
~inetics of I~-4~2 EsprQ~ion.
Experiments were done to determine if stimulation of
T cells ~y an anti-CD3 MAb results in the u~Le~lation of
both IL-4 and IL-4~2 mRNA levels and if IL-4~2 mRNA is
regulated in~opon~o~tly of IL-4 mRNA. PBNC were
stimulated with ORT3 MA~, and the ratio of IL-4~2 mRNA to
IL-4 mRNA was measured at different times (Fig. 7). 80th
IL-4~2 and IL-4 mRNAs were expressed spontaneously in
these PBMC, with 3.5 times more IL-4 than IL-4~2 mRNA in
this part~ r experiment. Both IL-4 and I~-4~2 mRNAs
increased with PEMC activation, but IL-4 mRNA increased
more than IL-4~2 mRNA. At 8 hours, 7 times more IL-4
than IL-4~2 mRNA was present, but by 12 hours, the ratio
:
woss/270sz ~l 8~ PCT~Sg~0109~
had LeLuL-~ed to h~ . At 24 and 4~ hours, ratios of
IL-4 to IL-4~2 mRNA remained at the hacPI in~ of
a~L~imately 4 to 1 (data not shown).
5~AMprl~ 10
3 o~ 2 ~ $n x~c~.
The human and murine IL-4 genes are each cnm~osed of
4 exons and 3 introns, both with a 48 bp exon 2. To.
determine whether mice also express an altornatively
spliced variant of IL-4 with exon 2 deleted, spleen cells
from B~rR/c mice were stimulated with PMA and ionomycin
for 24 hours. RNA was extracted and subjected to RT-PCR
using murine IL-4 exon 1- and exon 4-specific primers.
Human IL-4~2 ~RNA expression was assayed in parallel from
anti-CD3 MAb stimulated PBMC. IL-4, but not IL-4~2, mRNA
expression was observed in stimulated murine spleen
cells, whereas human P3MC expressed both IL-4 and IL-4~2
mRNA (Fig. 8).
20~ pr~ 11
Alt~rnati~e 8pl~c$ng of E~on 2 $~ Also Observed for ~uman
IL-2 ~RNa but not ~uman I~-3, I~-~ and G~-CSF mRN~.
Because IL-4 belon~s to a multigene family of
CytQ~ ines, IL-2, IL-3, IL-5, and GM-CSF ~RNAs were
examined to determine whether alternative splicing is
used to produce ~ariants that are missing exon 2. Total
RNA isolated from human PBMC stLmulated for 6 hours with
' WO9S/27052 ~1 86~54 PCT/U5~3101-94
-34-
the anti-CD3 ~b OKT3 was subjected to RT-PCR
amplification using exon l- and exon 4-specific PC~
primers for the cyto~in~s of interest. Two RT-PCR
amplification products were identified for IL-2 (Fig.
9A). The larger amplification product was 458 bp, which
COL~eSPOn~ to the size of native I~-2 mRNA (Fuiita et
al. (1983~ Proc. Natl. Acad. Sci. ~SA 80:7437). The
smaller amplification product was approximately 398 bp, a
size consistent with an alternatively spliced variant of
IL-2 that omitted exon 2. In ~On~LaSt to the fin~i~g5
with IL-2, only one RT-PCR amplification product each was
identified for I~-3, IL-5, and GM-CSF (data not shown).
To further test for the presence of alternative
splice variants involving exon 2, ~L-2 RT-PCR products
lS were size separated by gel ele~ro~horesis, transferred
to a nylon membrane, and hy~ridized with I~-2 exon 2- or
exon 3-specific oligonucleotide ~L obe3. Two IL-2 RT-PCR
products hybridized with the IL-2 exon 3-specific
oligonucleotide probe (Fig. ~B). In ~.Llast, the
smaller 398 bp product did not hy~ridize with an exon 2-
specific oligonucleotide, whereas the larger 458 bp
product did. This suggests that the smaller 398 bp
product is an alternative splice variant of IL-2 that is
missing exon 2. In all experiments, the ratios of
IL-2~2:IL-2 mRNA were much lower than the usual ratios of
IL-4~2:IL-4 mRNA, mzking IL-2~2 mRNA difficult to detect.
To improve detection of IL-2~2 mRNA, RT-PCR products were
W095/270~2 2 1 ~ 6 ~ 5 4 PCT/u~3s ~409~
-35-
hybridized with an IL-2 exon l/exon 3 junctional probe
(panel C). R~c~uc~ portions of the probe were homologous
to exon 1 or exon 3, native IL-2 c~NA was detected with
this probe as a larger 458 bp band on the autoradiogram.
~owever, because this probe cont~i n~d the exon l/exon 3
junction, IL-2~2 mRNA was easily discerned as a smaller
398 bp band.
In Si~;~Ar studies, the RT-PCR products for IL-3,
IL-5 and GM-CSF were size separated by gel
electrophoresis, transferred to a nylon m~mhrane, and
hybridized with oligonucleotide probes encoding an exon
l/exon 3 junctional sequence for IL-3, IL-5 and G~-CSF,
respectively. No RT-PC~ products hybridized with the
IL-3, IL-5 or GM-CSF exon l/exon 3 specific probes (data
not shown).
EXAMPLE 12
Rabbit antisera s~ecific for IL-4~2 ~rotein
A synthetic 16-mer peptide LNSLl~KNL~ L~ (SEQ ID
NO:27) was made. This peptide is specific for the exon
l-exon 3 junction in IL-4~2 and is not present in IL-4.
This peptide was made multimeric through coupling to MAPs
resin. Purified multimeric peptide was used to immunize
and boost two rab~its, a total of three injections. The
post-immunization, but not pr~im~ni~tion sera from each
rabbit binds the IL-4~2 synthetic peptide, but not
recombinant human IL-4 or IL-2, in Western blots.
woss/270s2 PCT~S95/04094
2 1 8685~4
-36-
EXAMP~E 13
AnalYsis of su~ernatants from activated human T cell
clones for ~resence of IL-4~2 protein.
Supernatants from activated human T cell clones were
S obt~in ~, and the proteins therein were run on SDS-PA OE .
Western blots were perfor~ed using the antisera ob~; n~
in Example 12 on the proteins separated by SDS-PAGE. IL-
4~2-specific antisera bound to IL-4~2 found in some,-but
not all of the supernatants tested.
While the invention has been described and
illustrated herein by references to ~arious specific
material, procedures and examples, it is understood that
the invention is not restricted to the particular
material combinations of material, and proc~ res
selected for that ~L~ose. Numerous variations of such
details can be implied as will be appreciated by those
skilled in the art.
WO95l27052 2 ~ S 6 ~ ~ 4 PCTIu~lo ~^94
-37-
~ ~ yU ~-N~ BISTSNC
(1) G~an INFORMATION:
(~) APP ICANT: Alms, William Qt al
(ii) T~T~ OF l~v~h~SON: ~MAN INTTT~r~ T~ V~RIA~TS ÇT~T~A~Rn BY ~T-T~N~IVE
SPEIC$NG
(Lii) N~MBE~ OF ~yu~ S: 22
(iv) CORPFCPONDENCE AnDP~.CS:
'A' AnDpT.~SsFT.~: Burn~, Doana, Sw6~^~or & ~athiJ
B STREET: P.O. Box 1404
C CITY: Al qY~ ia
D~ STATE: Virginia
E) COuh~: Unlted States
,F) ZIP: 22313-1404
(v) CU~ui~ pR~n~Rr.~ FORM:
'A) XEDI~M TY2E: Floppy diJk
B) COMP~TEP~: IBH PC c^mpatihle
C) OPERATING SYSTE~: PC-DOS/~S-DOS
~D) SOFTWaRE: Pat_ntIn p-le~Q ~1.0, Version ~t.25
(~i) ~u~R~ APPLICATION DATA:
(A) APPTICATION N~BER: To be ~ ^d
(B) FITING DASE: E~en date herewith
(C) C~ASSIFICATION:
~iii) A.lO~N~/AGENT INFORMATION:
(A) NA~E: Crane-Feury, Sharon E
(B) REGISTRaTION N~MBE~: 36,113
(ix) TE~ECO~XUNICATION INFORMATION:
(A) TELEP~ONE: (103) 536-6620
(B) TFA~FAX: (103) 836-2021
(2) ~r~ATION FOR SEQ ID NO:l:
(i) S~u~ ~A~ TS$ICS:
~A' ~ENGT~: 25 ba~e pairs
B SYPE: n~cleic acid
C ST~^NDFnNFCS: single
,DI TOPO~CGY: lin~ar
( ii ) YnT ~C~T ~ TYPE: DNA (g- io)
(xi) S~-yu~N~ DB-Cr~TPTION: SEQ ID NO:l:
ATGT~ ^r~ T~CAACTCCT GTCTT 25
(2) ~NrOR~AsION FOR SEQ ID NO:2:
yu~ CaiUaCTE~ISTICS:
a~ LENGT~: 25 bas_ pairs
B TYPE: nucleic acid
C STP~NnFnNESS: singl~
~D, TOPOLOGY: lin ar
W095/27052 2 1 ~ 6 ~ J 4 PCTrUS9S/04094
-38-
(ii) XOL3C~3 $YPE: DNA (gs~ c)
(xi) S~yu~-~ D~SrQTPTSON: SEQ ID NO:2:
C$$AGTG$$G AGATGa~GCr ~5GAC 25
(2) rNFORKaTION FOR SEQ ID NO:3:
yU~ s ~T A,~S~llT-9$ICS:
~A) ~ENG$~: 18 ~ase palr8
8) TYP~: nuclQic acid
C ) ST~2ANT~T~T~NT-C S: ~ingl~
,D) TOPOLOGY: linear
(ii) ~ODEC~L~ TYPE: DNA (~Pn~-~c)
(xi) S~YU~NL~ DESCRIPTION: SEQ ID NO:3:
T~G~C~A A~ & 18
(2) INFOR~ATION FOR SEQ ID NO:4:
(i) a~yu~ CaARACrE~IS$ICS:
'A) LENG$~: 18 ~as~ pa~r~
B) TYPE: nucleic ac~d
C) STp~ T~T~ e;s ~ingl~
,D) TOPOLOGY: line~r
(ii) ~OLEC5L~ TYPE: DNA (sencmic)
(~i) ~QU~L~ D~-SrQTPTION: SEQ ID NO:4:
GC~CaaAG$C G~ .G 18
(2) INFOR~aTION FOR SEQ ID NO:5:
(i) S~u~ C~ARAC~S~S$ICS:
'A' LENGT~: 19 ~as- pa~ss
B TYPE: nucl-ic ~cid
C ST~ T~ s ~ingl~
~D $OPOLOGY: linear
(ii) ~T,~C~T~ TYPE: DNA (9~~ ;c)
yu~5 DT'~Sr~TP$~0N: SEQ ID NO:5:
,1 AGCA5G$GC 19
(2) ~ ATION FOR SEQ ~D NO:6:
i ) SlSyUlS~I L:5 ~ ~ T S$ICS:
IA) rENG$~ 19 ba~c paiss
B) TYPE: nucl-ic ~c~d
C) ss~n~nN~ss ~ingl~
,D) TOPO~OGY: lincar
WO 95/27052 2 1 ~ 6 ~ 5 4 PcT/u~gsm ,-9~
~3g--
( il ) ~nt ~CBT-~ TYPE: DNA (genomic)
(xi) a~u~A~ Dr~'SrPTPSrON: SEQ rD NO:6:
CCTACSCTCC ~.G~ C 19
~2) ~ ~R~AT~ON FOR SEQ ID NO:7:
(i) s~u~ C~ARACT~RrSSlCS:
'A LENG~: 25 ~a~ pairs
8 TYPE: nucleic acid
C STR~ SS: ~ingle
D, sopaLocy: linear
(ii) ~OIEC~2 TYPE: DNA (genomic)
(Xi) S~yu~N~ DESCRrPTION: SEQ ~D NO:7:
AACCrTATGG CT CAC~SC CCaAC 2S
(2) ~N~OR~AS~ON FOR SEQ ID NO:8:
( i ) ~ b'yU ~ C~ARACTERrSTrCS:
'A) ~2NGS~: 27 ~ase pairs
3) SYPE: nucle$c acid
C) STR~ SS: single
~D) SOPOEOGY: linear
(ii) ~nr~C~r~ SYPE: DNA (genomic)
(xi) s~Qu~ DESrPTPTlON: SEQ lD NO:8:
G&ATCCSCA$ CACCSCCAAC A~ TGA 27
(2) 1~v~A$~0N FOR SZQ lD NO:9:
(i) 5~Q~ENOE r~CT~TSS~CS:
'A' ~ENGT~: 21 kaso paira
~B SYPE: nucleic acid
, C S~ n~ ~iSS: ingle
D, SOPC~OGY: lin-ar
(ii) ~nr~C~T~ $YPE: DNA (g~n~mi~)
(Xi) C~jy~ DF crp TPS~ON: S2Q rD NO:9:
AGCraT~TCC ACC&ATGCGA C 21
2 ) ~N ~. ~ TION FOR SEQ rD NO:10:
( i) S~iyu A~ S r~TsTrcs:
'A ~ENG$~: 22 ba~e pair~
B TYPE: nucl-ic acid
,C sT~n~n~FSS: in~le
.D, SOPO~CGY: linear
W O95/27052 PCTrUS9SJ0~091
2 1 ~6~5~
-4~-
il ) MOt ~Cnt 2 TYPE: DNA (, ~
~xi) S~yu~ DFSrPtPT~ON: SEQ ID NO:15:
C'TCAGTA A CG~GTaATCC AT 22
(2) ~N~ATION FOR SEQ ~D NO:ll:
( ~ ) S~ yu~s r~RP~~ 'rICS:
'A) LENG$~: 32 base paLrs
B) TYPE: nucleic acid
C) ST~AN~nN~SS: ingle
D) TOPO~OGY: llnear
(ii) MQr~C~ TYPE: DNA (gen ic)
(xi) s~Qu~c~ DESr~T2TION: SEQ ~D NO:11:
C~A AAG~ C c$cr~A~ GC 32
(2) INFORMAT~ON FOR SEQ ~D NO:12:
(i) ~Qu~ CaARAC$ERlSTlCS:
'A) LENGT~: 31 base pa~ru
B) TY2~: nuclelc ac~d
C) S~N~n~SS: ~lngle
~D) TOPOLOGY: linear
(ii) H~t~C~T~ TYPE: DNA (gsnl ic)
(Xi) S~:yU~NC~' DESC~PTION: SEQ ID NO:12:
CCA~,~,CCC ccc~r~cs GaC$AATT~T T 31
(2) rNFORMATION FOR SEQ ID NO:13:
( i ) S~U~ STICS:
A' LENGT~: 24 base pa~r~
B TYPE: ~uclqic acid
C ST~ SS: ~ingle
~D TOPO~OGY: l~near
(ii) u~t~C~T2 TYPE: DNA (~n. ic~
(xi) ~ DECrQTPSION: SEQ ID NO:13:
A~ &~CC AGAGCC$GCS GC$C 24
(Z) ~FOR~ASION FOR SEQ ~D NO:14:
) s ~ s~u~s ~ T-ST~CS:
IA~ LE~GT~: 24 base pairs
B TYPE: nucleic acld
C ST~NI~I':IINI. SS ingle
D TOPOLOGY: linear
W O95/27052 2 1 86B~ 4 PCTrUS95/04094
-41-
(il) N~CnT~ ~YPE: DNA ~ gF- i)
(xi) SZQ~ZN OE D~erP~PTION: SZQ ID NO:14:
SCA~ CG A~GG~-C~C ACCa 24
( 2 ) LN~ AT~ FOR SEQ ID NO:15:
(i) ab:yU~iN~ 5~TST~CS:
(Al ~ENC~: 24 ba~ pair~
(8 TYPE: ~cloic ac~d
(c STRAr~ SS: uingle
(D TO~OLOCY: linear
($i) ~T-~C~T-~ TYPE: DNA (g~ ic)
(xi) a~yu~N OE O~-Cr~TPTIoN: SEQ lD NO:~5:
CaGC.~G~A .C~L.~G&G TTCT 24
(2) ~N~ ~hMATION FOR SEQ ID NO:16:
(1) S~yubN~ CE~RAC5ZRISTICS:
,A) ~ENGT~: 24 ba~e pairs
B) m E: ~ lei~ acid
I C) S~V~ NI~-CS: ~i lgl~
D) TOPOLOGY: linear
(ii) M~T~C~T~ SYP~: DN~ (9l - ~c)
(xi) SEQ~ZNOE DFSr~PTION: SEQ ID NO:16:
lG ~ CCTTCAAASa CCAT 24
(2) lNr ORnASION FOR SEQ ID NO:17:
(i) SEQ~ENCB r~T7~TSTICS:
tA~ LENG~: 17 base pairs
B SYP~ cloic acid
~C ST~R~NnFnNFcs singlo
~D~ SOPOLCGY: lin-ar
(ii) ~tFC~T.2 m E: DNa (9~
(xi) SEQ~E~CE DFCr~TpTIoN SEQ ID NO:17:
C~ " CGrCaCT 17
2 ) lNr ~K~ATION FOR SEQ ID NO:18:
(i) SEQ~E~ OE rU~R~rT~Rr-CTICS:
'A' ~ENC~: 18 ba~o pairs
IB m~ n~cl~ic acid
,C S~RP~llbl~cs: ingl-
~D, SOPOLOGY: linoar
WO 95/27052 2 1 8 ~ ~ 5 ~ PCI/US9S/04094
--42 ~
( 11 ) YrT ~ T 2 mr: DNA ( ~
~xi) s~u~ D~SrPIPSION: SEQ ID NO:18:
csr'~ ' SGcscaca 18
(2) ~O~ASION FOR SEQ ID NO:lg:
Eyu~ 5 C~MAC~S$ICS:
'Al ~ENGT~: 18 base pairs
Bl m E: nucleic acid
C I S ~ P N I I I I I ~N I- ~ S singl~
~DI SOPOLOGY: linear
( ii ) 2~nT ~ P~ mE: DN~ ( g~ c )
(xi) S~:yU~h~ D~SrPTP~ION: SEQ ID NO:l9:
C~.~AG& AAG~GCTA 18
(2) INFOR!~SaSION FOR SEQ ID NO:20:
(1) s~yu~ C9ARACTERISTICS:
A ~E~GT~: 19 ~ase pairs
B SYPE: nucl-lc acid
C 5~UUNU~ UN.~ 55 single
1, D, SOPOI,OGY: linear
( li ) YnT-~c~T ~ mE: DN~ (genomic)
( Xi ) Sh~U ":hW! DESC~IIPSION: SEQ ID No: 20:
(2J INFORNaT~ON FOR SEQ ID NO:21:
t$) y~",~ ~s2TsTICS:
~'A ~ENGT~: 18 base pa$rs
B mE: nuclelc acid
C S~ CS: $ngl~
~D SOPOLOGY: linear
(~i) YnT~c~T~7 TYPE: DNA (genomlc)
~xi) ~u~w DFSr~2TPTION: SEQ lD NO:21:
C~ASGaGC ~ '~rTG 18
( 2 ) INFORNaS~ON FOR SEQ ID NO: 22:
(i) c,.ylJl~ . r~, ~ TSTICS:
A l ~2NCT~1: 18 ~as- pais
B m~ nuclsic acid
I C S~21~ N ~1~ 1 I N 1.. .C S ingl~
~D, 50POLOCY: linear
WO 95/Z7052 2 ~ ~ 5 8 ~ ~ PCTIUS95/04094
~43 ~
( ~ ) ~r ~ DNA ( ~
(~ci) SEQ~ENOE Cl rS~tt r~ ON: SEQ SD NO:22:
GCSCAGATG~ C~'` ~C 18
