CA2185352C - Novel muteins of ifn-.beta. - Google Patents

Abstract

A IFN-.beta. mutein in which phe (F), tyr (Y), trp (W) or his (H) is substit uted for val (V) at position 101, when numbered in accor dance with wild type IFN-.beta., DNA sequences encoding these IFN-.beta. muteins, recombinant DNA molecules containing those DNA sequences operatively linked to expression control sequences and capable of inducing e xpression of an IFN-.beta. mutein, hosts transformed wit h those recombinant DNA molecules, pharmaceutical compositions containing IFN-.beta. muteins and methods of using those compositions to trea t viral infections, cancer or tumors or for immunomodulation.

Description

NOVEL MUTEINS OF IFN-~
TECHNICAL FIELD OF THE INVENTION
This invention relates to muteins of interferon-beta ("IFN-~") in which val (V) at position 101, when numbered in accordance with wild type IFN-~, is substituted with phe (F), trp (W), tyr (Y) or his (H) .
BACKGROUND OF THE INVENTION
Interferons are single chain polypeptides secreted by most animal cells in response to a variety of inducers, including viruses, mitogens and polynucleotides. Interferons participate in regulation of cell function, and have antiviral, antiproliferative and immunomodulating properties. Native human interferons are classified into three major types:
a-IFN (leukocyte), IFN-~ (fibroblast) and y-IFN
(immune). Native IFN-~ is produced primarily by diploid fibroblast cells and in lesser amounts by lymphoblastoid cells.
IFN-(3 is a glycoprotein. Its nucleic acid and amino acid sequences have been determined.
(Houghton et al., "The Complete Amino Acid Sequence of Human Fibroblast Interferon as Deduced Using Synthetic Oligodeoxyribonucleotide Primers of Reverse WO 95/25170 ~ ~ PCT/US95/03206 Transcriptase," Nucle~id Acids Research, 8, pp. 2885-94 (1980); T. Taniguchi et al., "The Nucleotide Sequence of Human Fibroblast DNA," Gene, 10, pp, 11-15 (1980)), Recombinant IFN-~ has been produced and characterized, IFN-~B exhibits various biological and immunological activities. One of IFN-~'s biological activities is its antiviral activity. This antiviral activity can be neutralized by antibodies to IFN-,B.
See EP-B1-41313. Preparation and purification of antibodies to IFN-~ is described in EP-B1-41313 and the references cited therein. IFN-~ is also able to bind to cells that express interferon receptors, such as Daudi cells or A549 cells.
As a result of these activities, IFN-(3 has potential applications in immunotherapy, antitumor and anticancer therapies, and antiviral therapies.
Numerous investigations and clinical trials have been and continue to be conducted into the antitumor and anticancer properties of both wild type and recombinant IFN-~. These include treatment of several malignant diseases such as osteosarcoma, basal cell carcinoma, cervical dysplasia, glioma, acute myeloid leukemia, multiple myeloma and Hodgkin's disease. In addition, IFN-~ has been shown to cause local tumor regression when injected into subcutaneous tumoral nodules in melanoma and breast carcinoma-affected patients.
IFN-,8 (wild-type and recombinant) has been tested clinically in a variety of viral infections, including papilloma viruses, such as genital warts and condylomata of the uterine cervix; viral hepatitis, including acute/chronic hepatitis B and non-A, non-B
hepatitis (hepatitis C); herpes genitalia; herpes zoster; herpetic keratitis; herpes simplex; viral enc~.ephalitis; cytorre.<~a:lovir~us pn~.umorz:i~c; and in the prophylaxis of :rh=uno-rirta=, . C':l..i.rna.~~al t:~:.~rai s using rec~ombi.nant zFN-,Q in the tx~eatmerrt:. of ruu.l.tiple sclerosis haz a also been condLrc~ted and IFIV--~3 i;~ GaL~I:>r awed for s<rle in thE~ United Stat;e.s for. the t:re:~t:.rrw:rnt~ ca a': r~wa.Z.tiple sc:le:r-osis.
SUMMARY OF' THE :INVENTION
This invent: ion p~-ov:ic.~es rnui.r~:~i.n5 of. IFN-,a wherein the val (V) at po sition 1()_~, wh.~~rr r~urnl»:~;recl in accord,v~ne:e with wild type :L FN--,C>', is subst:i.t:l..rted w~.t~hx plus (F) , t:yr (Y) , trp (W) , or his (I-i) . 'I"his inv~rent:.:ion ~a~Lsc~ provides DI'JA
sequences encodincf these I1~'P~-11 rru..at ce:irm:~, rec:ombinanl_ :.)NA
molecules containing thc~~:e sec~ueru.~es <:sl:.~e.r.ati.-~relT~,~ l:in:hced to expression contro:l_ ~;equerrcE,s anc,~i c-~~.~pal>1.e c~f induciazg, in an appropriate luost, the expr~assic~rv c~:~ t:h~. I~'N_.~; mute:in;a, hosts transformed with t:ho;~e recomb.i.rrar:atu DN~~ molecules and pharmaceut:ical comps>~~iti.~~rr:~ c:~rat<:x:ir-~i:rnc~ t:.lue IFN-,~3.
Ti:ae:>e compositions are useful irn iminr~arlc>tlrer<.~l,~y as well as i.n ant icancaz.~, antitumor~ and a:arzt:iv ix~cr1 t::lr~ara~ i.a s .
In particular, t: he inv~.erzt;:ic>r~ prc>v:i.des an I:E~'N-,Q
mutein wherein the v~i1 ('f) c:or~~.e:_.pc~nc~irug to position 101 in wild type IFN-~3, as numberE~d .ira ~~~~c,~arcn~~rzc.e with w.i_Lc~ type IFN-~3, is subst:.~tuteci wi.tln ph;~a (F") ; t:~~Tz ('~°) , trh (W) , c>r his (I-I) , wherein said mutein dispaay, ant.~..~r~.ral act:iv:ity and wherein that activrit.y caAo lie r.~t 'L~:l~-rst: ~-artially nc~ut:e~al.ized by antibodies to wild type IF"t~J--~3.
In one aspect, therfe is. de~;c:x:ibed a rc=cc~mb:i.nant DNA molecule crraractE:rized by flee I:)NA ;~ec~uenc~e of t~hc, invention, the sequence being ~:>p~vrate iv~l y l inked to a~n expression control. se.que}:xc~.~: ir1 t:lvAr_ re c::ombinant TINA nu:alecule.

61(709-284 In another aspect:, t~er~e is r:~e sc.~x~ibed a host cell tra.nsforme~d wit=.rr a recombinant ,::az~dA mo ~f_~ci.u~e of the invention.
In another aspect , t:ht-~~:e i s cie~~c~ribed a method of prcvducing the IFN--,C~ mutei.r~ of thr: inv~~rnt:ican, compri:~inc~ the steps of rwlturinc~ t2~e host: cell abovre_y arena collecting t=he IFN- f3 mutein.
In another aspec:ø:: , tr~.e2-e is c,le~~c:x~ibed a pharmaceutical cornpa:::,iti.oru <~ofnp:c::.~ sine ~:m ~~.ntiviral, an t:ic:aneer, antitv.rmo:r ox~ _~.rr~rr~u.nc~c~~c.>c~ul,:~t.°tor~
effective amount of the IFN-,~ mut.e-.~n c>f t:xie iro~.rc:~mt.:i.on a.r~c~ a pharrnaceut:ic:ally acceptable carr_i.er.
In anothex:~ aspect " ~,::rrere is c~~:s c:r.z.bed use ~:~f the IFN-~3 mutein of true unvent~:~on f~:~x- thf~~ i>re~=aration of a medicament. for they tlmeatment ~::~f ~e,~:~.ra:d.. ::.r~.fE~ct:a.ons, ca:rrcer or tumors, ox for imn:mnc~mndulat.-i~::~r:E.
In another aspecet:, t~~c.exe i~.~ cv:e:~cribed use ~:af a DNA
seauence of the inveryr_ior~. t:~or the p:rep~~rat iarr of a medicament: for the t.x-eatment ~~f vi.ra:l irn:ECt:ion~~, cancers, tumors, undesired ce:~_1 pr~o:l.:if~-~xvat:.:i«zrr, c:~x~ fox, i mrnunomodul at i on .
In anotrier aspect::, t~-rere i;~ c~.Eascribed a bac::terial, fur.~.gal or insect c:el.i.. tx~~~n;afox~rneyc.:T with a xc>c.vomb:in<~nt DI~1A
molecule of the irmernt:ion.
In another aspect: , t_hexve i:; ci.escri.bed an animal or plant cell. in cult:ux a tr~:~rl~fox_~rr~ec< wit::tr. a xe.r_~ombinant DICTA
molecule of the inverat~io::~.

BRIEF DESc~'RIPTION OF THE DL2.~WTN(~:~
Figure 1 depicts t.:he am.ir~o acid sequence of t:he preferred mut~ein of this _~nvexnt~c:axn Ik~'N--~.~ (phel,~) ( SE:Q ID NO : 1 ) .
Figure 2 depicts the px~ra:herz~~.~d degenerate C)NA
sequence encoding IFN-,G (pheloa) <xx:~<~ the. ~~..gna1 seqwen,.~e of.
native IFN-,~ (SEQ ID NO: 2.;I ..
Figure ~ slxows an anal.ys~.s c.~i T: FN-(3 (phelol) b~_nding to interferon receptor bwar:~nc~ <:e:~.ls.. t-~'<~rAel.s .A and ;3 ~~how receptor binding c~at.a ~~,x- :~.:sl _ L~,'~~._~, ~~>~~~:~.o~ ) and wi:lc3 type t'sI-IFN-~3, res ect:ive~l p y, a.n D;:~ucla. ~:wa_:1..- Panel>> c~' arid D
show receptor binciing da::a foa~ '''':C--:LFl~~--~~i (~;helo, ) axed wild type l2sI._I:FN-,~, respe~cti-~~ee~_~~r t::r:.. ~~~419 c-~---lis..

WO 95I25I70 ~ ~ g PCT/US95103206 Figure~4 shows an analysis of IFN-~(phe~D~) and wild type IFN-~ by peptide mapping by endoproteinase Lyse-C.
Figure 5 shows that antibodies to wild type IFN-8 neutralize the activity of IFN-~(phe~~~) and wild type IFN-~.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, "wild type IFN-~" means an IFN-~, whether native or recombinant, having the normally occurring amino acid sequence of native human IFN-~, as shown, e.g., in EP-B1-41313, Figure 4.
As used herein, "IFN-/3 mutein" means a polypeptide wherein the val (V) at position 101, when numbered in accordance with wild type IFN-~B, is substituted with phe (F), tyr (Y), trp (W), or his (H), preferably phe (F). Our most preferred IFN-~ muteins have an amino acid sequence identical to wild type IFN-J3 at the other residues. However, the IFN-~
muteins of this invention may also be characterized by amino acid insertions, deletions, substitutions and modifications at one or more sites in or at the other residues of the native IFN-~ polypeptide chain. In accordance with this invention any such insertions, deletions, substitutions and modifications result in an IFN-~ mutein that retains an antiviral activity that can be at least partially neutralized by antibodies to wild type IFN-~.
We prefer conservative modifications and substitutions (i.e., those that have a minimal effect on the secondary or tertiary structure of the mutein).
Such conservative substitutions include those described by Dayhoff in the Atlas of Protein Sequence and WO 95!25170 -Structure 5_ (1978), and by Argos in EMBO J., 8, 779-785 (1989). For example, amino acids belonging to one of the following groups represent conservative changes:
- ala, pro, gly, gln, asn, ser, thr;
5 - cys, ser, tyr, thr;
- val, ile, leu, met, ala, phe;
- lys, arg, his; and - phe, tyr, trp, his.
We also prefer modifications or substitutions that eliminate sites for intermolecular crosslinking or incorrect disulfide bond formation. For example, IFN-a is known to have three cys residues, at wild-type positions 17, 31 and 141. United States patent 4,588,585 refers to an IFN-~ mutein in which the cys (C) at position 17 has been substituted with ser (S).
This substitution can also be utilized in this invention. For example, this invention contemplates an IFN-~ mutein having ser (S) substituted for cys (C) at position 17 and val (V) at position 101 substituted with phe (F), trp (W), tyr (Y), or his (H), preferably phe (F), when numbered in accordance with wild type IFN-~.
By "numbered in accordance with wild type IFN-~B" we mean identifying a chosen amino acid with reference to the position at which that amino acid normally occurs in wild type IFN-~. Where insertions or deletions are made to the IFN-~ mutein, one of skill in the art will appreciate that the val (V) normally occuring at position 101, when numbered in accordance with wild type IFN-~, may be shifted in position in the mutein. However, the location of the shifted val (V) can be readily determined by inspection and correlation of the flanking amino acids with those flanking va1101 in wild type IFN-~.

The IFN-~ muteins of the present invention can be produced by any suitable method known in the art. Such methods include constructing a DNA sequence encoding the IFN-~ muteins of this invention and $ expressing those sequences in a suitable transformed host. This method will produce recombinant muteins of this invention. However, the muteins of this invention may also be produced, albeit less preferably, by chemical synthesis or a combination of chemical synthesis and recombinant DNA technology.

In one embodiment of a recombinant method for producing a mutein of this invention, a DNA sequence is constructed by isolating or synthesizing a DNA sequence encoding the wild type IFN-~ and then changing the 1 '.i codon f or va 1 ~ to a codon f or phe ( F ) , trp ( W ) , tyr.

(Y) or his (H), preferably phe (F), by site-specific mutagenesis. This technique is well known. See, e.g., Mark et al., "Site-specific Mutagenesis Of The Human Fibroblast Interferon Gene", Froc. Natl. Acad. Sci.

USA, 81, pp. 5662-66 (1984); United States patent 4,588,585.

Another method of constructing a DNA sequence encoding the IFN-S muteins of this invention would be chemical synthesis. For example, a gene which encodes ~!5 the desired IFN-~ mutein may be synthesized by chemical means using an oligonucleotide synthesizer. Such oligonucleotides are designed based on the amino acid sequence of the desired IFN-~ mutein, and preferably selecting those codons that are favored in the host cell in which the recombinant mutein will be produced.

In this regard, it is well recognized that the genetic code is degenerate -- that an amino acid may be coded for by more than one codon. For example, phe (F) is coded for by two codons, TTC or TTT, tyr (Y) is coded WO 95/25170 2 ~. 8 5 3 5 ~ pCT~S95/03206 for by TAC or TAT and his (H).is coded for by CAC or CAT. Trp (W) is coded for by a single codon, TGG.
Accordingly, it will be appreciated that for a given DNA sequence encoding a particular IFN-~ mutein, there will be many DNA degenerate sequences that will code for that IFN-~B mutein. For example, it will be appreciated that in addition to the preferred DNA
sequence shown in Figure 2, there will be many degenerate DNA sequences that code for the IFN-~8 mutein shown in Figure 1. These degenerate DNA sequences are considered within the scope of this invention.
The DNA sequence encoding the IFN-~i mutein of this invention, whether prepared by site directed mutagenesis, synthesis or other methods, may or may not also include DNA sequences that encode a signal sequence. Such signal sequence, if present, should be one recognized by the cell chosen for expression of the IFN-~ mutein. It may be prokaryotic, eukaryotic or a combination of the two. It may also be the signal sequence of native IFN-,B. The inclusion of a signal sequence depends on whether it is desired to secrete the IFN-~ mutein from the recombinant cells in which it is made. If the chosen cells are prokaryotic, it generally is preferred that the DNA sequence not encode a signal sequence. If the chosen cells are eukaryotic, it generally is preferred that a signal sequence be encoded and most preferably that the wild-type IFN-~3 signal sequence be used.
Standard methods may be applied to synthesize a gene encoding an IFN-~ mutein according to this invention. For example, the complete amino acid sequence may be used to construct a back-translated gene. A DNA oligomer containing a nucleotide sequence coding for IFN-~ mutein may be synthesized. For WU 95/25170 ~ ~ . PCT/US95/03206 - g example, several small oligonucleotides coding for portions of the desired polypeptide may be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.
Once assembled (by synthesis, site directed mutagenesis or another method), the DNA sequences encoding an IFN-~ mutein of this invention will be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the IFN-~ mutein in the desired transformed host. Proper assembly may be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.
The choice of expression control sequence and expression vector will depend upon the choice of host.
A wide variety of expression host/vector combinations may be employed. Useful expression vectors for eukaryotic hosts, include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus.
Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E.coli, including col E1, pCRl, pBR322, pMB9 and their derivatives, wider host range plasmids, such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g., NM989, and other DNA phages, such as M13 and filamentous single stranded DNA phages. Useful expression vectors for yeast cells include the 2~C

WO 95/25170 , ? PCT/US95/03206 plasmid and derivatives thereof. Useful vectors for insect cells include pVL 941. We prefer pBG311. Cate et al., "Isolation Of The Bovine And Human Genes For Mullerian Inhibiting Substance And Expression Of The Human Gene In Animal Cells", Cell, 45, pp. 685-98 (1986).
In addition, any of a wide variety of expression control sequences may be used in these vectors. Such useful expression control sequences include the expression control sequences associated with structural genes of the foregoing expression vectors. Examples of useful expression control sequences include, for example, the early and late promoters of Sv40 or adenovirus, the lac system, the trn system, the TAC or TRC system, the major operator and promoter regions of phage lambda, for example PL, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast a-mating system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
Any suitable host may be used to produce the IFN-~ muteins of this invention, including bacteria, fungi (including yeasts), plant, insect, mammal, or other appropriate animal cells or cell lines, as well as transgenic animals or plants. More particularly, these hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E.coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cells such as Snodoptera fruaiperda (SF9), animal cells such as Chinese hamster ovary (CHO) and mouse cells such as NS/0, African green monkey cells such as COS 1, COS 7, BSC 1, BSC 40, and BMT 10, and WO 95/25170 ~ PCT/US95/03206 human cells, as will as plant cells in tissue culture.
For animal cell expression, we prefer CHO cells and COS 7 cells in cultures and particularly the CHO-DDUKY-~1 cell line (infra, pp. 18-19).
It should of course be understood that not all vectors and expression control sequences will function equally well to express the DNA sequences described herein. Neither will all hosts function equally well with the same expression system. However, one of skill in the art may make a selection among these vectors, expression control sequences and hosts without undue experimentation. For example, in selecting a vector, the host must be considered because the vector must replicate in it. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered. For example, preferred vectors for use in this invention include those that allow the DNA
encoding the IFN-~ muteins to be amplified in copy number. Such amplifiable vectors are well known in the art. They include, for example, vectors able to be amplified by DHFR amplification (see, e.g., Kaufman, United States Patent 4,470,461, Kaufman and Sharp, "Construction Of A Modular Dihydrafolate Reductase cDNA
Gene: Analysis Of Signals Utilized For Efficient Expression", Mol. Cell. Biol., 2, pp. 1304-19 (1982)) or glutamine synthetase ("GS") amplification (see, e.g., United States patent 5,122,464 and European published application 338,841).
In selecting an expression control sequence, a variety of factors should also be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the actual DNA sequence encoding the IFN-~ mutein WO 95/25170 ~ PCT/US95/03206 of this invention, particularly as regards potential secondary structures. Hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for by the DNA sequences of this invention, their secretion characteristics, their ability to fold the polypeptides correctly, their fermentation or culture requirements, and the ease of purification of the products coded for by the DNA sequences.
Within these parameters, one of skill in the art may select various vector/expression control sequence/host combinations that will express the desired DNA sequences on fermentation or in large scale animal culture, for example, using CHO cells or COS 7 cells.
The IFN-~B muteins obtained according to the present invention may be glycosylated or unglycosylated depending on the host organism used to produce the mutein. If bacteria are chosen as the host then the IFN-~ mutein produced will be unglycosylated.
Eukaryotic cells, on the other hand, will glycosylate the IFN-~ muteins, although perhaps not in the same way as native IFN-~ is glycosylated.
The IFN-~ mutein produced by the transformed host can be purified according to any suitable method.
Various methods are known for purifying IFN-(3. See, e.g., United States patents 4,289,689, 4,359,389, 4,172,071, 4,551,271, 5,244,655, 4,485,017, 4,257,938 and 4,541,952. We prefer immunoaffinity purification.
See, e.g., Okamura et al., "Human Fibroblastoid Interferon: Immunosorbent Column Chromatography And N-Terminal Amino Acid Sequence", Biochem., 19, pp.
3831-35 (1980).

WO 95/25170 ~ ~ ~ PCT/US95/03206 -~ 12 -The biological activity of the IFN-~ muteins of this invention can be assayed by any suitable method known in the art. Such assays include antibody neutralization of antiviral activity, induction of protein kinase, oligoadenylate 2,5-A synthetase or phosphodiesterase activities, as described in EP-B1-41313. Such assays also include immunomodulatory assays (see, e.g., United States patent 4,753,795), growth inhibition assays, and measurement of binding to cells that express interferon receptors.
The IFN-~ mutein of this invention will be administered at a dose approximately paralleling that employed in therapy with wild type native or recombinant IFN-~. An effective amount of the IFN-~
mutein is preferably administered. An "effective amount" means an amount capable of preventing or lessening the severity or spread of the condition or indication being treated. It will be apparent to those of skill in the art that the effective amount of IFN-~
mutein will depend, inter alia, upon the disease, the dose, the administration schedule of the IFN-~ mutein, whether the IFN-~ mutein is administered alone or in conjunction with other therapeutic agents, the serum half-life of the composition, and the general health of the patient.
The IFN-~ mutein is preferably administered in a composition including a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" means a carrier that does not cause any untoward effect in patients to whom it is administered.
Such pharmaceutically acceptable carriers are well known in the art. We prefer human serum albumin.
The IFN-~ muteins of the present invention can be formulated into pharmaceutical compositions by well known methods. See, e.g., Remington's Pharmaceutical Sciences by E. W. Martin, describes suitable formulations. The pharmaceutical composition of the IFN-~ mutein may be formulated in a variety of forms, including liduid, gel, lyophilized, or any other suitable form. The preferred form will depend upon the particular indication being treated and will be apparent to one of skill in the art.
l~~ The IFN-S mutein pharmaceutical composition may be administered orally, intravenously, intramuscularly, intraperitoneally, intradermally or subcutaneously or in any other acceptable manner. The preferred mode of administration will depend upon the particular indication being treated and will be IS
apparent to one of skill in the art.
The pharmaceutical composition of the IFN-~B
mutein may De administered in conjunction with other therapeutic agents. These agents may be incorporated ZO as part of the same pharmaceutical composition or may be administered separately from the IFN-~ mutein, either concurrently or in accordance with any other acceptable treatment schedule. In addition, the IFN-~
mutein pharmaceutical composition may be used as an adjunct to other therapies.
2' Accordingly, this invention provides compositions and methods for treating viral infections, cancers or tumors, abnormal cell growth, or for immunomodulation in any suitable animal, preferably a mammal, most preferably human.
Also contemplated is use of the DNA sequences encoding the IFN-B muteins of this invention in gene therapy applications.
Gene therapy applications contemplated 3;j include treatment of those diseases in which IFN-B is WO 95/25170 ~ ~ g PCT/US95/03206 expected to provide an effective therapy due to its antiviral activity, e.g., viral diseases, including hepatitis, and particularly HBV, or other infectious diseases that are responsive to IFN-~B or infectious agents sensitive to IFN-~B. Similarly, this invention contemplates gene therapy applications for immunomodulation, as well as in the treatment of those diseases in which IFN-B is expected to provide an effective therapy due to its antiproliferative activity, e.g., tumors and cancers, or other conditions characterized by undesired cell proliferation, such as restenosis.
Local delivery of IFN-8 using gene therapy may provide the therapeutic agent to the target area while avoiding potential toxicity problems associated with non-specific administration.
Both in vitro and in vivo gene therapy methodologies are contemplated.
Several methods for transferring potentially therapeutic genes to defined cell populations are known. See, e.g., Mulligan, "The Basic Science Of Gene Therapy", Science, 260, pp. 926-31 (1993). These methods include:
1) Direct gene transfer. See, e.g., Wolff et al., "Direct Gene transfer Into Mouse Muscle In Vivo", Science, 247, pp. 1465-68 (1990);
2) Liposome-mediated DNA transfer. See, e.g., Caplen at al., "Liposome-mediated CFTR Gene Transfer To The Nasal Epithelium Of Patients With Cystic Fibrosis", Nature Med., 3, pp. 39-46 (1995); Crystal, "The Gene As A Drug", Nature Med., 1, pp. 15-17 (1995); Gao and Huang, "A Novel Cationic Liposome Reagent For Efficient Transfection Of Mammalian Cells", Biochem. Biophys.
Res. Comm., 179, pp. 280-85 (1991);

3) Retrovirus-mediated DNA transfer. See, e.g., Kay et al., "In Vivo Gene Therapy Of Hemophilia B:
Sustained Partial Correction In Factor IX-Deficient Dogs", Science, 262, pp. 17.7-19 (1993); Anderson, S "Human Gene Therapy", Science, 256, pp. 808-13 (1992).

4) DNA Virus-mediated DNA transfer. Such DNA
viruses include adenoviruses (preferably Ad-2 or Ad-5 based vectors), herpes viruses (preferably herpes simplex virus based vectors), and parvoviruses 1(l (preferably "defective" or non-autonomous parvovirus based vectors, more preferably adeno-associated virus based vectors, most preferably AAV-2 based vectors).
See, e.g., AIi et al., "The Use Of DNA Viruses As Vectors For Gene Therapy", Gene Theranv, 1, pp. 367-84 15 (1994); United States Patent 4,797,368, and United States Patent 5,139,941.
The choice of a particular vector system for transferring the gene of interest will depend on a 2p variety of factors. One important factor is the nature of the target cell population. Although retroviral vectors have been extensively studied and used in a number of gene therapy applications, these vectors are generally unsuited for infecting non-dividing cells.
In addition, retroviruses have the potential for oncogenicity.
Adenoviruses have the advantage that they have a broad host range, can infect quiescent or terminally differentiated cells, such as neurons or 30 hepatocytes, and appear essentially non-oncogenic.
See, e.g., Ali et al., supra, p. 367. Adenoviruses do not appear to integrate into the host genome. Because they exist extrachromosomally, the risk of insertional mutagenesis is greatly reduced. Ali et al., supra, p.
35 373.

R'O 95/25170 2, ~ ~ e~ ~ PCT/US95/03206 Adeno-associated viruses exhibit similar advantages as adenoviral-based vectors. However, AAVs exhibit site-specific integration on human chromosome 19. Ali et al., supra, p. 377.
In a preferred embodiment, the IFN-B
mutein-encoding DNA of this invention is used in gene therapy for vascular smooth muscle cell proliferation after arterial injury. Injury of the arterial wall results in the migration of smooth muscle cells into the intimal layer of the arterial wall, where they proliferate and synthesize extracellular matrix components. See, e.g., Chang et al., "Cytostatic Gene Therapy For Vascular Proliferative Disorders With A
Constitutively Active Form Of The Retinoblastoma Gene Product", Science, 267, p. 518 (1995). This proliferative response has been implicated in the pathogenesis of atherosclerosis.
One clinically significant setting for arterial injury follows percutaneous balloon angioplasty of the coronary arteries. Following mechanical dilation of the artery, in many cases a cellular proliferative response occurs, leading to regrowth of cells locally that impinges on the lumen and compromises blood flow. This response, known as restenosis, has not responded to conventional treatments including antiplatelet agents, angiotensin-converting enzyme antagonists, or cytotoxic drugs in humans. See, e.g., Ohno et al., "Gene Therapy For Vascular Smooth Muscle Cell Proliferation After Arterial Injury", Science, 265, p. 781 (1994).
According to this embodiment, gene therapy with DNA encodng the IFN-B muteins of this invention is provided to a patient in need thereof, concurrent with, or immediately after coronary balloon angioplasty.
This approach takes advantage of the antiproliferative WO 95125270 ~ ~ PCTIUS95I03206 activity of the IFN-~' muteins of this invention to prevent undesired SMC proliferation. The skilled artisan will appreciate that any suitable gene therapy vector containing IFN-$ mutein DNA may be used in accordance with this embodiment. The techniques for constructing such a vector are known. See, e.g., Ohno et al., su ra, p. 784; Chang et al., su ra, p. 522.
The coronary balloon angioplasty procedure is well known. Introduction of the IFN-B mutein DNA-containing vector to the target artery site may be accomplished using known techniques, e.g., as described in Ohno et al., supra, p. 784.
In order that this invention may be better understood, the following examples are set forth.
These examples are for the purpose of illustration only, and are not to be construed as limiting the scope of the invention in any manner.
Examples Expression Vector Containing Human IFN-~(phe~0~) We used plasmid pBG311 as the expression vector. A full description of pBG311 is given in Cate et al., "Isolation Of The Bovine And Human Genes For Mullerian Inhibiting Substance And Expression Of The Human Gene In Animal Cells", Cell, 45, pp. 685-98 (1986). The vector uses the SV40 early promoter, splice signal, and polyadenylation signal and was constructed using pAT153 as backbone.
A DNA fragment containing the DNA sequence shown in Figure 2 (SEQ ID NO: 2) was cloned into pBG311 and operatively linked to the SV40 early promoter through a DNA sequence encoding the signal sequence for native IFN-~ according to standard protocols. The resulting expression vector was designated pBeta-phe.

The IFN-~3 mutein DNA sequence (SEQ ID N0: 2) encodes an IFN-~3 mutein having an amino acid sequence identical to wild type IFN-(3 except that the val (V) at position 101, numbered in accordance with wild type IFN-~3, is substituted with phe (F). The mutein encoded by this sequence is designated IFN-(3 (pheloi) .
Competent Escherichia coli (SURETM, Stratagene) were transformed with the pBeta-phe plasmid according to standard procedures. Colonies containing the pBeta-phe plasmid (i.e., containing a DNA sequence encoding IFN-(3(pheiol) were identified by hybridization to a oligonucleotide probe specific for IFN-~i(phelol) using a standard protocol (M. Grunstein and D.S. Hogness, "Colon.y hybridization: a method for the isolation of cloned DNAs that contain a specific gene", Proc Natl Acad Sci USA, 72 (10): 3961-5 (1975)).
Amplification Vector We used plasmid pAdD26SV(A)-3 to amplify the IFN-(3(pheioi) gene in our ultimate transformants. This plasmid is described in Kaufman and Sharp, "Construction Of A
Modular Dihydrofolate Reductase cDNA Gene: Analysis Of Signals Utilized For Efficient Expression", Mol. Cell.
Biol., 2, pp. 1304-19 (1982) and in United States patent.
4,740,461. The plasmid expresses murine dihydrofolate reductase (DHFR) under the transcriptional control of the Adenovirus 2 (Ad2) major late promoter (MLP). A 5' splice site, derived from an immunoglobulin variable region gene, is located between the Ad2 MLP and the DHFR coding sequences. The SV40 polyadenylation site is present downstream of the DHFR gene. The plasmid contains the prokaryotic origin of replication (ori) and tetracycline resistance gene from pBR322.

R'O 95/25170 PGT/US95/03206 Transformation of a Cell Line The CHO-DUKX-Bl DHFR cell line was cotransformed with the pBeta-phe plasmid and plasmid pAdD26SV(a)-3. This cell line was derived from the wild type CHO-K1 cell line by ethyl methanesulfonate and UV irradiation induced mutagenesis. See Chasin and Urlaub, "Isolation Of Chinese Hamster Cell Mutants Deficient In Dihydrofolate Reductase Activity", Proc.
Natl. Acad. Sci. USA, 77, pp. 4216-20 (1980).
Dihydrofolate reductase catalyzes the conversion of folate to tetrahydrofolate. Cells without functional DHFR require exogenous ribonucleosides and deoxyribonucleosides for growth. Inhibition of growth can be induced by methotrexate, a folate analogue, which binds to and inhibits DHFR. Titration of methotrexate can lead to methotrexate resistance by amplification of the DHFR gene. (Kaufman & Sharp, 1982, su a). Amplification and increased expression of genes near DHFR often occurs when DHFR is amplified.
Therefore, cells resistant to high levels of methotrexate often demonstrate increased specific productivity of nearby genes.
The pBeta-phe plasmid (restricted with Xmni) and plasmid pAdD26SV(a)-3 (restricted with Stul) were mixed in a ratio of 10:1, respectively. The DNA was transformed into CHO-DUKX-B1 DHFR cells by electroporation. Cells were plated in non-selective a+
medium (a MEM base plus ribonucleosides and deoxyribonucleosides, 10% fetal bovine serum [FBS], 4 mM glutamine) and allowed to grow for 2 days. The medium was then exchanged for a- medium (a MEM base without ribonucleosides and deoxyribonucleosides, 10%
FBS, 4 mM glutamine) + 50 nM methotrexate (MTX). The cells were removed by trypsinization and plated at ca.

8 x 105 cells/10 cm tissue culture plate. After 14 days, clones were picked and grown in 96 well tissue culture plates. One clone was expanded into a 12 well tissue culture plate and then 7 days later put into a 6 well tissue culture plate in the presence of 250 nM
MTX. This clone was expanded into a T75 flask (grown in a' medium + 250 nM MTX) and then amplified in 750 nM
MTX. A subclone was picked into a 96 well tissue culture plate, expanded into a 48 well tissue culture plate, then a 6 well tissue culture plate and then a T75 tissue culture flask.
Purif ication Of IFN-~ (phs~0~ ) IFN-~(phe~0~), produced by culturing the above subclone (or others similar to it) and then secreted 1~~ into the culture medium, can be purified by immunoaffinity chromatography, substantially as described by Okamura et al., "Human Fibroblastoid Interferon: Immunosorbent Column Chromatography And N-Terminal Amino Acid Sequence", Biochem., 19, pp.
2y 3831-35 (1980).
CNBr-Sepharose 4B resin (2 g, 7 ml) is prepared by suspending in 1 mM HC1. The gel is washed with 1 mM HC1 for 15 min on a scintered glass filter.
Anti-IFN-~ mabs (such as 802, Yamasa, Japan) are 25 coupled to CNBr-Sepharose*4B resin by incubating in coupling buffer (100 mM NaHC03, pH 8.3, 500 mM NaCl) for 2 hours at room temperature on a rocker platform.
Typically, 1-2 mg IFN-S mab per ml of resin is coupled, but this amount can be varied. The unreacted CNBr is 3() blocked with 100 mM Tris-HC1, pH 8, 500 mM NaCl, overnight at 4°C. Alternately, the unreacted CNBr is blocked with 100 mM ethanolamine under substantially the same conditions.
* Trade-marls ~'1~0 95/25170 21 ~ 5.~ .~ ~L PCT/U895/03206 The coupled resin;i.s washed with three cycles of alternative pH. Each cycle consists of a wash with acetate buffer (100 mM, pH 4) containing 500 mM NaCl followed by a wash with Tris buffer (100 mM, pH 8) containing 500 mM NaCl.
A 1 cm X 3 cm column (2.3 ml bed volume) is prepared with the coupled resin. The column is equilibrated with PBS (greater than 5 column volumes).
IFN-~(phe~0~)-containing samples are diluted 1:3 in equilibration buffer, pH 6.8 and loaded. The load is chased with PBS, washed with 20 mM K2HP04, 1 M NaCl, pH

6.8, and eluted with 200 mM Na citrate, pH 2. The pH
of the eluate was adjusted to 6 by diluting the sample with 500 mM Mes, pH 6.
Characterization Hy Peptide Mapping An IFN-~(phe~0i), mutein that had been produced and purified in a different and less preferred manner than described above was characterized by peptide mapping. A 30,ug aliquot of IFN-~B (phel0~ ) or wild type IFN-~ sample was lyophilized, suspended in 200 ~1 of endoproteinase Lys-C digestion buffer (100 mM
TRIS, pH 9, 0.5 mM EDTA), incubated for 12 hours at 22°
with 1.5 ~g of endoproteinase Lys-C and subjected to mapping analysis on a C$ reversed phase HPLC column (.45 x 25 cm). The column was developed with a 30 minute, 0-70% gradient of acetonitrile in 0.1% TFA at 1.4 mls/min. The column effluent was monitored at 214 nm. Figure 4, Panel A shows a portion of the peptide map for IFN-~(phe101) with the arrowhead indicating the peptide TFLEEK (SEQ ID NO: 3). This peak did not occur in the peptide map for wild type IFN-~. Figure 4, Panel B shows the corresponding region of a peptide map for wild type IFN-~ with the arrowhead indicating the WO 95/25170 ~ ~ ' ' '~' PCT/U595103206 peptide TVLEEK (SEQ ID NO: ~,). The identity of the TFLEEK and TVLEEK were~~reFified by protein sequence analysis. We estim~~e~that the ~B-Phe101 and wild type ~-IFN were greater than 98% pure. Protein concentrations were estimated from absorbance at 280 nm using an extinction coefficient of 1.5 for a 1 mg solution. In order to stabilize the proteins for biological studies, they were diluted to 4 ~g/ml in PBS
containing 5% FBS and 5 mM HEPES, pH 7.5 Antiviral Activity Of IFN-~(phe~0~) In The CPE Assay The preparation of IFN-f3(phe~0~) that was characterized by peptide mapping was analyzed in a Cytopathic Effect (CPE) assay for antiviral activity.
A wild type recombinant IFN-~B standard was prepared in Dulbecco's Modified Eagle Medium (DMEM), i0% FBS, 4mM
glutamine at a concentration of 10,000 units/mL and stored in aliquots at -70°C. On day 1, standard, control and IFN-~Phe samples were diluted in DMEM, 10%
FBS, 4mM glutamine in three dilution series:
i) starting at 64 units/mL followed by 2-fold dilutions, ii) starting at 12 units/mL followed by 1.5-fold dilutions, and iii) starting at 6 units/mL
followed by 1.2-fold dilutions. Fifty microliters of the dilutions were then added in columns to the wells of 96-well microtiter plates. A549 cells were added to each well at 105 cells/ml, 50 uL per well, in DMEM, 10%
FBS, 4mM glutamine and the cells are incubated at 37°C, 5% C02 for 15 to 20 hours.
The plate contents were shaken into a bleach bucket and 100 uL encephalomyocarditis virus (EMC
virus) at appropriate dilution in media was added to each well. The virus and cells were incubated at 37°C, 5% C02 for 30 hours. The plate contents were then shaken into a bleach bucket and 0.75% crystal violet dye added to the plates. After 5 to 10 minutes, the plates were washed with distilled water and allowed to dry before being read visually.
Samples and standards were tested in duplicate on each assay plate, yielding two data paints per dilution series per assay day.
IFN-~(phe=0~) was tested in 14 assays in duplicate. Wild type recombinant IFN-~ was used as a 10~ standard. Based on these experiments, IFN-~B(phe~~~y had a specific activity of 4.8 x 108 U/mg with a 95%
confidence interval of 3.5 -6.7 x 108. Wild type IFN-~
had a specific activity of approximately 2.o X 108 units/mg with a confidence interval of 1.6-2.5 x 108.
15 The data in Figure 5 show a similar result.
The specific activity of recombinant IFN-~B(phe~Q~) is on average about 2.5 fold higher than that of recombinant wild type IFN-~, as measured in our antiviral assay.
2C1 llnalYeis of IFa1-~(phel0~) For Baceptar Binding The IFN-~(phe~0~) used in the CPE assay above was also analyzed for ability to bind to cells that express interferon receptors. For these studies we examined the binding of either wild type t25I-IFN-S or ~25I-IFN-~(phe~0~) to Daudi cells or A549 cells (Figure 3). Carrier-free IFN-~B was iodinated substantially according to the chloramine T method.
Unreacted iodine was removed by size exclusion chromatography on a Superdex*75 column that was ,0 equilibrated in PBS containing 1 mg/ml bovine serum albumin. The concentration of the iodinated IFN-S was determined by the CPE assay, assuming a specific activity of 2 x lob units/mg. Normally 5 ng (1 pL, * Trade-mark 300,000 cpm) of iodinated IFN-~ (either alone or in the presence of a 50 fold excess of non-iodinated interferon) was added to 1.7 mL eppendorf~tubes in a total volume of less than 10 ~rL. The labelled ligand was allowed to bind alone (-) or was competed with unlabeled IFN-S(phe101), a2-IFN (a2), y-IFN (y) or wild type recombinant IFN-S (WT).

Both Daudi cells and A549 cells (American Type Culture Collection) were used. The cells were suspended in DMEM/5% FBS at 2 x 106 cells/mL. To the samples of the IFN-~, 0.5 mL of the cell suspension was added. The tubes were mixed by inversion and incubated at ambient temperature for 45 minutes. The cells were then pelleted at 1000 x g for 2 min and washed two times with 0.5 mL DMEM/10% FBS. Each wash was followed by a centrifugation step at 1000 x g for 2 min. The cells were resuspended in 0.1 mL, transferred into tubes for counting and binding quantified in a Beckman gamma 407 counter.

The data suggest that the binding of IFN-S(phe101) is very similar to that of wild type IFN-S

on both cell types. Comparable amounts of wild type 1251-IFN-~' and 1251-IFN-~(phel0~) were bound and competed similarly by noniodinated a-IFN, wild type IFN-~ and IFN-~(phe101). The binding was not affected by the addition of recombinant human y-IFN.

Tha Antiviral Activity of I1~1-B (pheip 1) Is Substantially ~leutraliaed Bp Antibodies To gild Type IFH-8 ;SO A number of preparations of recombinant mutein IFN-B(phelQl) produced in CHO cells as described above were purified to approximately 90% purity using column chromatography. These samples were diluted to * Trade-mark 2~~85352 25 pg/ml. Wild type recombinant IFN-B, designated here as IFN-B(vallol)~ was produced and purified in substantially the same way.
Standard antiviral and antibody neutralization assays were used to demonstrate that antibodies to wild type IFN-B(valiol) at least partially neutralize IFN-B(phelol). The particular antiviral assay and neutralization assay that we used, detailed below, are substantially the same as the antiviral and antibody neutralization assays described in European patent EP B1 41313. See p. 26, lines 1-21; p. 29, line 48 - p. 32, line 3.
A. Preparation Of Cell-containing Plates A549 cells (ATCC CCL185) were seeded into 96 well plates at 3 x 104 cells/100 microliter media/well.
The media used was Dulbecco's Modified Eagle Medium (DMEM), 10% FBS, 4 mM glutamine. The cell containing plates were then incubated at 37°C, 5% COZ for approximately 24 hours.
B. Preparation Of Master Plates Master plates containing either sample or standard were then created with serially diluted wells (in duplicate). Sample wells contained either purified recombinant mutein IFN-B(pheloi) or wild type recombinant IFN-B(vallol) in the presence or absence of rabbit anti-IFN-B polyclonal sera. Control wells contained either buffer alone or recombinant wild type IFN-B (as a standard) in the presence or absence of an anti-LFA3 antibody (data not shown).
Serial dilutions were performed as follows.
200 microliters of control, or of wild type or mutein IFN-B sample (at a concentration of about 25 pg/ml), was added to each well in row A on each plate. The WO 95/25170 ~ ~ ~ ~ PGT/US95/03206 final concentration o~ sample in row A was 8.5 pg/ml.
Dilutions of 1:1.5 were then performed down each plate.
C. Antibodies To Wild Twe IFN-B
Antibodies to wild type IFN-B(valloi) were produced in rabbits immunized with recombinant wild type IFN-B(vallol). Rabbit anti-IFN-B polyclonal sera was collected from the immunized rabbits at appropriate intervals, pooled and stored until use (Rabbit IFN-B
serum pool 6/25/93; 5 ml/vial, 0.02% azide; ref.
0.01742.062).
D. Sample/Ab Incubation Rabbit anti-IFN-B polyclonal sera was added to the appropriate wells on the Master plates. The final dilution of antibody was 1:1000. The antibody/IFN-B mixture was incubated at room temperature for 45 minutes.
E. _Incubation of Cells With Control, Sample Or Sample/Ab The media was then aspirated from the cell-containing plates prepared and replaced with aliquots (100 ~.1/well) of control, IFN-B sample or IFN-B/Ab sample, as appropriate, from the Master plates prepared. The cell-containing plates were incubated at 37°C, 5% COZ for 16-24 hours.
F. Viral Challenve The next step was the viral challenge. The cell-containing plate contents were then aspirated and 100 ~1 of a solution of encephalomyocarditis virus (EMCV) at appropriate dilution was added to each well.
The virus and cells were incubated at 37°C, S% COz for 41-45 hours.

The cell-containing plates were developed using the XTT/PMS colorimetric method. A 1 mg/ml XTT

(3,3-[1-(phenylamino)carbonyl]-3,4-tetrazolium]-bis-(4-methoxy-6-nitro)-benzenesulfonic acid; Sigma) solution was prepared in phosphate buffered saline solution. A 1 mg/ml PMS (phenazine methnsulfate) solution was prepared in water. A PMS/X'TT solution was prepared at 1:50. The development solution was prepared by diluting the PMS/XTT 1:3 in phosphate buffered saline solution. The tetrazolium compound XTT

is reduced by living cells to form an orange colored formazon. Color formation correlates directly to viable cell number.

The cell-containing plates were aspirated and 1~; washed with 150 ;tl/well of phosphate buffered saline solution. Each well then received 150 ~ul of development solution. The plates were incubated at for 30-60 minutes.
5% C0 ~
, Absorbance at 450 nm was measured on a microplate reader with Molecular Devices*Thermo ~~

Softmax software. The results are displayed graphically in Figure 5. Absorbance is plotted against IFN-~ concentration.
Figure 5 shows that in the absence of rabbit anti-IFN-~ polyclonal sera, samples of mutein IFN-S(pheloi) (closed square; ~) and of wild type IFN-S(vahoi) (closed diamond; ~) protected the A549 cells from the EMCV. This is indicated by the increase in absorbance (indicating more living cells) with increasing mutein IFN-~ or wild type IFN-~
concentration.
Figure 5 also shows that in the presence of rabbit anti-IFN-S polyclonal sera, samples of mutein IFN-~(phelol) (open square; [~) and of wild type ?;5 IFN-~(vallol) (open diamond; O) failed to protect the * Trade-mark WO 95/25170 ~ ~ PGT/US95103206 A549 cells from the EMCV~ this is shown by the baseline absorbance value for any mutein IFN-B or wild type IFN-B concentration, indicating that almost all A549 cells were dead.
In sum, Figure 5 shows that the antiviral activity of mutein IFN-B(pheioi) was neutralized by antibodies to wild-type IFN-B (i.e., rabbit anti-IFN-B
antibodies).
Treatment of Restenosis With IFN-B Mutein Gene Therapy Initial testing of gene therapy for restenosis is conducted in a pig model using recombinant wild type porcine IFN-~ according to the following protocol.
Cell proliferation is measured by immunohistochemistry. All animals receive an intravenous infusion of BrdC (Sigma, St. Louis, MO), mg/kg total dose, 1 hour before death.
Immunohistochemistry with monoclonal antibody to BrdC
(1:1000 dilution, Amersham Life Sciences, Arlington 20 Heights, IL) is performed to label nuclei in proliferating cells as described in Goncharoff et al., J. Immunol. Methods, 93, p. 97 (1988). Identification of vascular smooth muscle cells is performed by immunohistochemistry with an antibody to smooth muscle 25 a-actin (1:500 dilution, Boehringer Mannheim, Germany) as described in Islk et al., Am. J. Pathol., 141, p.
1139 (1992).
Domestic Yorkshire pigs (12 to 15 kg) are anesthetized with zolazepamin-tiletamine (6.0 mg/kg) in combination with (2.2 mg/kg intramuscular) rompun with 1% nitrous oxide. The iliofemoral arteries are exposed by sterile surgical procedures, and a double-balloon catheter is inserted into the iliofemoral artery as described in Nabel et al., Science, 249, p. 1285 WO 95/25170 ~ ~ PCT/US95/03206 (1990). The proximal balloon is inflated to 300 mm Hg, as measured by an on-line pressure transducer, for 5 min. The balloon is deflated and the catheter is advanced so that the central space between the proximal and distal balloon now occupies the region of previous balloon injury. Both balloons are inflated, and the segment is irrigated with heparinized saline. The adenoviral inoculum is instilled for 20 min in the central space of the catheter. The catheter is removed and antegrade blood flow as restored.
The injured arteries of all pigs are infected with 101o plaque-forming units (PFU) per milliliter of an ADV-DE1 vector containing an insert encoding porcine IFN-~ or with an ADV-~E1 vector lacking the insert.
In each animal, both iliofemoral arteries are transfected with the same vector at a titer of 1 x lOlo PFU/ml and 0.7 ml is used in each animal (final dose of 7 x 109 PFU).
The vessel segments in these pigs are excised 21 or 42 days later. Each artery is processed in an identical manner. The region of instillation between the two double balloons is cut into five cross-sections of identical size. Sections 1 and 4 are fixed in methyl Carnoy and sections 3 and 5 are fixed in formalin, and all sections are paraffin-embedded and stained with hematoxylin-eosin. Additional antibody studies are performed on methyl Carnoy- or formalin-fixed arteries. Tissue from section 2 is flash-frozen in liquid nitrogen and stored at -80°C for DNA
isolation. Measurements of intimal and medial area are determined in four sections from each artery in a blinded manner by two independent readers, and the measurements for each artery are averaged. Slides of arterial specimens are studied with a microscope-based video imaging analysis system (Image-1 System, WO 95/25170 ~ ~ ~ PCT/US95/03206 Universal Imaging, Wesc~e,s~er, PA) as described in Nabel et al., Prac. Na~l. Acad. Sci U.S.A., 90, p.
10759 (1993).
As an alternative to the above-described adenoviral-based Ad-DE1 vector, direct gene transfer may also be used. One suitable construct is a plasmid derived from the RSV backbone, with the RSV-LTR
promoter driving expression of IFN-(3, with the SV-40 poly A' signal 3' to the IFN-~B DNA sequence. See, e.g., Gorman et al., Science, 221, pp. 551-53 (1983).
The above protocol is then modified so that the vector contains a DNA insert encoding the human IFN-~ muteins of this invention for gene therapy in humans.
Seguences The following is a summary of the sequences set forth in the Sequence Listing:
SEQ ID NO:1 -- Amino acid sequence of IFN-~E(phe~0~) SEQ ID N0:2 -- DNA sequence encoding IFN-~(phe~p1 ), including DNA sequence encoding the signal sequence of native IFN-~B
SEQ ID N0:3 -- Amino acid sequence of peptide TFLEEK.
SEQ ID No:4 -- Amino acid sequence of peptide TVLEEK.
Depos its E.coli K-12 containing plasmid pBeta-phe (which contains a DNA sequence encoding IFN-~(phet0l) and the native IFN-~B signal sequence) has been deposited. The deposit was made in accordance with the Budapest Treaty and was deposited at the American Type Culture Collection, Rockville, Maryland, U.S.A, on ~1~5352 - 3i -March ii, 1994. The deposit received the accession number 69584.
The foregoing description has been presented only for purposes of illustration and description.
S This description is not intended to limit the invention to the precise form disclosed. It is intended that the scope of the invention be defined by the claims appended hereto.

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(A) LEI: 187 aaaim acids (B) TAE: amirn acid (D) EDGY: linear (ii) ~.~E T1~E: ø~tein (~a) ~a~cN: s~ m rx~a:
Met ~ Asp L~ Cys Ieu Ieu Gln Ile Ala Leu Ieu Ieu C~ Phe Sir ~ ~' Ala Leu Ser I~t 9er Tirr Asi Ieu Ieu Gly Hoe Ieu Gln Arg Ser Ser Asn Hoe Gln Cys Gln Iys I~u IEU ~p Gln Ieu Ann Gly Ar9 I~u GlWyr G~ Ieu Iy5 AsP Azg Met Asn Hoe Asp ue Pty Glu Glu I1e I~ Gln Ieu Gln Gln Aye Gln I~ Glu Asp Ala Ala I81 'n'~' ue rn Glu Met Ieu Gln Asn ue Flee Ala ue F'he Azg Gln Asp Ser' Ser Sew ~ Gly~p Asn Glu ~ Ile Ual Glu Asn Ieu Isi Ala Asn Ual rn His Gln ue Asn His Ieu I~ ~S F3~e Ies Glu Glu Irk Ieu Glu I,~ Glu Asp Hoe ~ Arg Gly i~ Ieu Met 9e~r Ser Leu His Ieu Iy5 WO 95/25170 ~ ~ ~ PCT/US95/03206 ~lZYr'ZYr'~Y~l~huHisZyrIeuI~AlaIysGlnZyrS~r ~5 130 135 His C~ Ala ~p D~ Its Val Azg ~lal Glu ue Ieu Azg Asn Phe rn 14p 145 150 155 Pte ILe Asn Arg Ieu ~ Gly Zyr IEU Azg A~

(2) aT FLR 5~ ID I~:4:
(i) s~E C5:
(A) LEI: 6 ~ acirh (B) TAE: ~nirn acid (C~ SI~~: single (D) ~y: ~ ? r,~n-(ii) NL~.D31(CE 'I'VE: peptide (; ~; ) cue: 1x~
(iv) ANTI-SEE: r13 (xi,) I3'S~IICN: S~ ID I~:4:
~ Phe Ieu Glu Glo I~

(2) TIQd FLR SQ,2 ID PD:S:
(i) ~ Q~GS:
(A) Iii: 6 amim acids (B) TAE: amirn acid (D) EDGY: 1 ? ~"
(ii) 1~~E TAE: peptide (;;; ) cue.,: rp (iv) ANrI-~E: 1~
(,~) ~a~aJ: sQ2 m rx~:5:
~ Va1 Isi Glu Glu Iys ," PCT/US95/03206 Applicant's or agent's file Intemauonai application No.
reference number B17 9 CIP PCT
1NDICA1'IONS RELATING TO A DEPOSITED MICROORGANI~...
(PCt Rule 136is) A. 'lire indtcaUOns made below relate to the mtcroorgantsm referred to m the description E . COll K-12 , on page 31 ,lines 4-11 JA221/pBeta-phe I3. IDENTIFICATION OF DEI'USiT
Funkier deposits are identified on an additional sheet Q

Name of deposrtarv mstrtuuon American Type Culture Collection Address of deposoarv tnstUUUOn Irnclwlrnq postal codr and cauntrv) 12301 Parklawn Drive Rockville, Maryland 20852 United States of America Uate of depose Accession Number 11 March 1994 (11.03.94) 69584 C. ADDTfIONAL INDICATIONS IJra~,rhlaek iJnor oppJicobJr) This information is continued on an additional sheet In respect of the designation of the EPO, samples of the de-posited microorganisms will be made available until the pub-lication of the mention of the grant of the European patent or until the date on which the application is refused or with-drawn or is deemed to be withdrawn, as provided in Rule 28(3) of the Implementing Regulations under the EPC only by the is-sue of a sam le to an ex ert nominat d b D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (i/tbeiudicationrarena fwilJlai~tedStato) EPO

E. SEPARATE FURNISHING OF INDICATIONS
(!rout blaruE i/not applicable) The indications listed below will be submiued to the fnternauonal Bureau later (sped(ytluebatoulnarareo(tJteindi~otio~e.;., Ao~ion Na~nbn ojDepoait') For receiving Office use only For international Bureau use oaly 'this sheet was received with the international application Q This shut was received by the fntetnatiaml Buteay tra:

Authorized affixr Authorized otLcer ~

4~
D~tS L. BR~O~K

!!~TtPN~~TIONAL DIYtBO~t WO 95/25170 ~ ~ ~ PCTIUS95/03206 Appiicant'soragent'sfile B179 CIP PCT IlnternauonaiappiicationNo.
reterenx number 1NDICA1'IONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule l3bis) A. 'lhe mou:auutu mace below relate to ute mtaoorganism reierreu to m tee description E . CO11 K-12 r lines 4-11 JA221/pBeta-phe , on page B. IDENTIFICATION OF DEPOSIT
Further deposits are identified on an additional sheet Q

Name of depository mstituuun American Type Culture Collection Address of ocposoary mstrtuuon tixlrulinq postal cod, and country) 12301 Parklawn Drive Rockviller Maryland 20852 United States of America Uate of dcposu Acxssion Number 11 March 1994 (11.03.94) 69584 C. ADDITIONAL INDICATIONS (Iwvr bla~te if na applicobG_1 'Ibis information is continued on an sdditiami:beet a In respect of the designation of Finland, until the application has-been laid open to public inspection by the Finnish Patent Officer or has been finally decided upon by the Finnish Patent Office without having been laid open to public inspection, samples of the deposited microorganisms will be made available only to an expert in the art.

D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (i/tbei~tdicarionsaranor~osaUlaip~relStat~t) Finland E. SEPARATE FURNISHING OF INDICATIONS
(IvroveblomEi~notapplicabk) 'IbeindicationslistedbelowwillbesubmittedtothelnternatiotulBurrauiater(spaci/yt be6ateaJaarttreofrbeindinuoto~a., Aa~ioe Nratbn o/DepotsiY) ~ For receiving Off x use only For lnternatioml Bureau use only ~ 'Ibis:beet was reoerved with the international application Q 'Ibis:beet was received by the latetaatioml Bureau oa Aw6orized t~ L. ~..~ Authorized ofbtxr ~~ j-.~~JT~~(-P,~LTIOt~E,L DlVI9tOf~

Claims (34)

CLAIMS:

1. An IFN-.beta. mutein wherein the val (V) corresponding to position 101 in wild type IFN-.beta., as numbered in accordance with wild type IFN-.beta., is substituted with phe (F), tyr (Y), trp (W), or his (H), wherein said mutein displays antiviral activity and wherein that activity can be at least partially neutralized by antibodies to wild type IFN-.beta..

2. An IFN-.beta. mutein having an amino acid sequence identical to wild type IFN-.beta. except that the val (V) corresponding to position 101 in wild type IFN-.beta., numbered in accordance with wild type IFN-.beta., is substituted with phe (F), tyr (Y), trp (W) or his (H).

3. The IFN-.beta. mutein according to claim 2, wherein the val (V) is substituted with phe (F).

4. The IFN-.beta. mutein according to claim 2 having the formula:
Met-Ser-Tyr-Asn-Leu-Leu-Gly-Phe-Leu-Gln-Arg-Ser-Ser-Asn-Phe-Gln-Cys-Gln-Lys-Leu-Leu-Trp-Gln-Leu-Asn-Gly-Arg-Leu-Glu-Tyr-Cys-Leu-Lys-Asp-Arg-Met-Asn-Phe-Asp-Ile-Pro-Glu-Glu-Ile-Lys-Gln-Leu-Gln-Gln-Phe-Gln-Lys-Glu-Asp-Ala-Ala-Leu-Thr-Ile-Tyr-Glu-Met-Leu-Gln-Asn-Ile-Phe-Ala-Ile-Phe-Arg-Gln-Asp-Ser-Ser-Ser-Thr-Gly-Trp-Asn-Glu-Thr-Ile-Val-Glu-Asn-Leu-Leu-Ala-Asn-Val-Tyr-His-Gln-Ile-Asn-His-Leu-Lys-Thr-Phe-Leu-Glu-Glu-Lys-Leu-Glu-Lys-Glu-Asp-Phe-Thr-Arg-Gly-Lys-Leu-Met-Ser-Ser-Leu-His-Leu-Lys-Arg-Tyr-Tyr-Gly-Arg-Ile-Leu-His-Tyr-Leu-Lys-Ala-Lys-Glu-Tyr-Ser-His-Cys-Ala-Trp-Thr-Tle-Val-Arg-Val-Glu-Ile-Leu-Arg-Asn-Phe-Tyr-Phe-Ile-Asn-Arg-Leu-Thr-Gly-Tyr-Leu-Arg-Asn (SEQ ID NO:1).

5. A DNA sequence encoding an IFN-.beta. mutein of claim 1 or 2.

6. A DNA sequence encoding an IFN-.beta. mutein having an amino acid sequence identical to wild type IFN-.beta. except that the val (V) at position 101 in wild type IFN-.beta., as numbered in accordance with wild type IFN-.beta., is substituted with phe (F) , tyr (Y) , trp (W) or his (H) .

7. The DNA sequence according to claim 6, wherein the val (V) is substituted with phe (F).

8. The DNA sequence according to claim 7 encoding an IFN-.beta. mutein of the formula:
Met-Ser-Tyr-Asn-Leu-Leu-Gly-Phe-Leu-Gln-Arg-Ser-Ser-Asn-Phe-Gln-Cys-Gln-Lys-Leu-Leu-Trp-Gln-Leu-Asn-Gly-Arg-Leu-Glu-Tyr-Cys-Leu-Lys-Asp-Arg-Met-Asn-Phe-Asp-Ile-Pro-Glu-Glu-Ile-Lys-Gln-Leu-Gln-Gln-Phe-Gln-Lys-Glu-Asp-Ala-Ala-Leu-Thr-Ile-Tyr-Glu-Met-Leu-Gln-Asn-Ile-Phe-Ala-Ile-Phe-Arg-Gln-Asp-Ser-Ser-Ser-Thr-Gly-Trp-Asn-Glu-Thr-Ile-Val-Glu-Asn-Leu-Leu-Ala-Asn-Val-Tyr-His-Gln-Ile-Asn-His-Leu-Lys-Thr-Phe-Leu-Glu-Glu-Lys-Leu-Glu-Lys-Glu-Asp-Phe-Thr-Arg-Gly-Lys-Leu-Met-Ser-Ser-Leu-His-Leu-Lys-Arg-Tyr-Tyr-Gly-Arg-Ile-Leu-His-Tyr-Leu-Lys-Ala-Lys-GLu-Tyr-Ser-His-Cys-Ala-Trp-Thr-Ile-Val-Arg-Val-Glu-Ile-Leu-Arg-Asn-Phe-Tyr-Phe-Ile-Asn-Arg-Leu-Thr-Gly-Tyr-Leu-Arg-Asn (SEQ ID N0:1).

9. The DNA sequence according to claim 8 wherein the codon encoding the amino acid at position 101 is TTC.

10. A DNA having the formula of SEQ ID NO:2.

11. A recombinant DNA molecule characterized by the DNA sequence of any of claims 5 to 10, the sequence being operatively linked to an expression control sequence in the recombinant DNA molecule.

12. A host cell transformed with a recombinant DNA
molecule of claim 11.

13. A method of producing the IFN-.beta. mutein of claim 1, comprising the steps of culturing a host according to claim 12 and collecting the IFN-.beta. mutein.

14. The method according to Claim 13, wherein the IFN-.beta. mutein is encoded by a DNA sequence comprised by the formula of SEQ ID NO:2 and the host is an animal cell in culture.

15. A pharmaceutical composition comprising an antiviral, anticancer, antitumor or immunomodulation effective amount of the IFN-.beta. mutein of any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

16. The pharmaceutical composition of claim 15 for use in treating viral infections, cancer or tumors, or for immunomodulation.

17. A use of the IFN-.beta. mutein according to claim 1 for the preparation of a medicament for the treatment of viral infections, cancer or tumors, or for immunomodulation.

18. A use of the pharmaceutical composition of claim 15 for the treatment of viral infections, cancers or tumors, or for immunomodulation.

19. The use according to claim 17 or 18, wherein the val (V) is substituted with phe (F).

20. The use according to claim 17 or 18, the mutein having the formula:
Met-Ser-Tyr-Asn-Leu-Leu-Gly-Phe-Leu-Gln-Arg-Ser-Ser-Asn-Phe-Gln-Cys-Gln-Lys-Leu-Leu-Trp-Gln-Leu-Asn-Gly-Arg-Leu-Glu-Tyr-Cys-Leu-Lys-Asp-Arg-Met-Asn-Phe-Asp-Ile-Pro-Glu-Glu-Ile-Lys-Gln-Leu-Gln-Gln-Phe-Gln-Lys-Glu-Asp-Ala-Ala-Leu-Thr-Ile-Tyr-Glu-Met-Leu-Gln-Asn-Ile-Phe-Ala-Ile-Phe-Arg-Gln-Asp-Ser-Ser-Ser-Thr-Gly-Trp-Asn-Glu-Thr-Ile-Val-Glu-Asn-Leu-Leu-Ala-Asn-Val-Tyr-His-Gln-Ile-Asn-His-Leu-Lys-Thr-Phe-Leu-Glu-Glu-Lys-Leu-Glu-Lys-Glu-Asp-Phe-Thr-Arg-Gly-Lys-Leu-Met-Ser-Ser-Leu-His-Leu-Lys-Arg-Tyr-Tyr-Gly-Arg-Ile-Leu-His-Tyr-Leu-Lys-Ala-Lys-Glu-Tyr-Ser-His-Cys-Ala-Trp-Thr-Ile-Val-Arg-Val-Glu-Ile-Leu-Arg-Asn-Phe-Tyr-Phe-Ile-Asn-Arg-Leu-Thr-Gly-Tyr-Leu-Arg-Asn (SEQ ID NO:1).

21. The DNA sequence according to claims 5 or 6 for use in treating viral. infections, cancers, tumors, undesired cell proliferation, or for immunomodulation in a defined.
cell population or tissue in a patient.

22. A use of a DNA sequence according to claim 5 or 6 for the preparation of a medicament for the treatment of viral infections, cancers, tumors, undesired cell proliferation, or for immunomodulation.

23. A use of a DNA sequence according to claim 5 or 6 for the treatment of viral infections, cancers, tumors, undesired cell proliferation, or for immunomodulation.

24. The use according to claim 22 or 23, wherein the val (V) is substituted with phe (F).

25. The use according to claim 24, wherein the DNA.
sequence encodes an IFN-.beta. mutein of the formula:
Met-Ser-Tyr-Asn-Leu-Leu-Gly-Phe-Leu-Gln-Arg-Ser-Ser-Asn-Phe-Gln-Cys-Gln-Lys-Leu-Leu-Trp-Gln-Leu-Asn-Gly-Arg-Leu-Glu-Tyr-Cys-Leu-Lys-Asp-Arg-Met-Asn-Phe-Asp-Ile-Pro-Glu-Glu-Ile-Lys-Gln-Leu-Gln-Gln-Phe-Gln-Lys-Glu-Asp-Ala-Ala-Leu-Thr-Ile-Tyr-Glu-Met-Leu-Gln-Asn-Ile-Phe-Ala-Ile-Phe-Arg-Gln-Asp-Ser-Ser-Ser-Thr-Gly-Trp-Asn-Glu-Thr-Ile-Val-Glu-Asn-Leu-Leu-Ala-Asn-Val-Tyr-His-Gln-Ile-Asn-His-Leu-Lys-Thr-Phe-Leu-Glu-Glu-Lys-Leu-Glu-Lys-Glu-Asp-Phe-Thr-Arg-Gly-Lys-Leu-Met-Ser-Ser-Leu-His-Leu-Lys-Arg-Tyr-Tyr-Gly-Arg-Ile-Leu-His-Tyr-Leu-Lys-Ala-Lys-Glu-Tyr-Ser-His-Cys-Ala-Trp-Thr-Ile-Val-Arg-Val-Glu-Ile-Leu-Arg-Asn-Phe-Tyr-Phe-Ile-Asn-Arg-Leu-Thr-Gly-Tyr-Leu-Arg-Asn (SEQ ID NO:1).

26. The use according to claim 25 wherein the codon encoding the amino acid at position 101 is TTC.

27. The use according to claim 22 or 23 wherein the DNA sequence has the sequence of nucleotides 1-561 of SEQ ID NO:2 or nucleotides 64-561 of SEQ ID NO:2.

28. The use of any one of claims 22-27 wherein the viral infection is hepatitis.

29. The use according to claim 28 wherein the hepatitis is HBV.

30. The use of any one of claims 22-27 wherein the undesired cell proliferation is restenosis.

31. The use of any one of claims 22-27 wherein the cancer is glioma.

32. The use of any one of claims 22-27 wherein the cancer is melanoma.

33. A bacterial, fungal or insect cell transformed with a recombinant DNA molecule of claim 11.

34. An animal or plant cell transformed with a recombinant DNA molecule of claim 11.