CA2186854A1 - Human interleukin variants generated by alternative splicing - Google Patents

Abstract

Novel splice mutants of interleukins-2 and 4 are disclosed, which contain exons 1, 3 and 4 of the full-length mRNAs, but have exon 2 deleted. The proteins resulting from the expression of these splice mutants are useful in regulating the activity of the full-length interleukins.

Description

` 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 ~

2 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 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 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 ~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 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 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~

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~

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~

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 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.

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 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~

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 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~ &GTTCT-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 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 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~

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).

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~

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.

~ 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 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 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~

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).

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 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 ~ ~ 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$NG
(Lii) N~MBE~ OF ~yu~ S: 22 (iv) CORPFCPONDENCE AnDP~.CS:
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B STREET: P.O. Box 1404 C CITY: Al qY~ ia D~ STATE: Virginia E) COuh~: Unlted States ,F) ZIP: 22313-1404 (v) CU~ui~ pR~n~Rr.~ FORM:
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~iii) A.lO~N~/AGENT INFORMATION:
(A) NA~E: Crane-Feury, Sharon E
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(A) TELEP~ONE: (103) 536-6620 (B) TFA~FAX: (103) 836-2021 (2) ~r~ATION FOR SEQ ID NO:l:
(i) S~u~ ~A~ TS$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 (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 (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

Claims (16)

WHAT IS CLAIMED IS:

1. An isolated nucleic acid comprising exons 1, 3 and 4 of human

2. The isolated nucleic acid of Claim 1, wherein the nucleic acid is RNA.

3. The isolated nucleic acid of Claim 1, wherein the nucleic acid is DNA.

4. An expression vector comprising the isolated nucleic acid of Claim 3.

5. A transformed cell comprising the vector of Claim 4.

6. The polypeptide expressed by the expression vector of Claim 4.

7. An antibody directed to the polypeptide of Claim 6.

8. An isolated nucleic acid comprising exons 1, 3 and 4 of human interleukin-2.

9. The isolated nucleic acid of Claim 8, wherein the nucleic acid is RNA.

10. The isolated nucleic acid of Claim 8, wherein the nucleic acid is DNA.

11. An expression vector comprising the isolated nucleic acid of Claim 10.

12. A transformed cell comprising the vector of Claim 11.

13. The polypeptide expressed by the expression vector of Claim 12.

14. An antibody directed to the polypeptide of Claim 13.

15. A method of regulating the activity of interleukin-4, comprising administering to a human an amount of the polypeptide of Claim 6 effective to decrease the biological effects of interleukin-4.

16. A method of regulating the activity of interleukin-2, comprising administering to a human an amount of the polypeptide of Claim 13 effective to decrease the biological effects of interleukin-2.