New multiclass hybrid interferon polypeptides, their corresponding encoding recombinant DNA molecules and transformed hosts which produce the new interferons are described. The amino acid sequences of these hybrids include at least two different subsequences, one of which has substantial homology with a portion of a first class of interferon (e.g., HuIFN-.alpha.) and the other which has substantial homology with a portion of a second class of interferon e.g., HuIFN-.beta.). Data indicates the interferon activity of .alpha.-.beta. hybrids may be substantially restricted to either cell growth regulatory activity or antiviral activity.

DESCRIPTION 
1. Technical Field 
This invention is in the field of biotechnology. More particularly it 
relates to multiclass hybrid interferon polypeptides, recombinant DNA that 
codes for the polypeptides, recombinant vectors that include the DNA, host 
organisms transformed with the recombinant vectors that produce the 
polypeptides, methods for producing the hybrid interferon polypeptides, 
pharmaceutical compositions containing the polypeptides, and therapeutic 
methods employing the polypeptides. 
2. Background Art 
Since the discovery by Isaacs and Lindenmann of interferon in 1957, many 
investigations have been conducted on the efficacy of interferon for 
treating various human diseases. Interferon is now generally thought to 
have three major clinically advantageous activities normally associated 
with it, namely, antiviral activity (Lebleu et al, PNAS USA, 73:3107-3111 
(1976)), cell (including tumor) growth regulatory activity (Gresser et al, 
Nature, 251:543-545 (1974)), and immune regulatory activity (Johnson, 
Texas Reports Biol Med, 35:357-369 (1977)). 
Interferons are produced by most vertebrates in the presence of certain 
inducers including viruses. Human interferons (HuIFN) thus far discovered 
have been divided into three classes: .alpha., .beta., and .gamma.. 
HuIFN-.alpha. is produced in human leukocyte cells or in transformed 
leukocyte cell lines known as lymphoblastoid lines. HuIFN-.alpha. has been 
purified to homogeneity (M. Rubenstein et al, "Human Leukocyte Interferon: 
Production, Purification to Homogeneity and Initial Characterization", 
PNAS, 76:640-44 (1979)). The pure product is heterogeneous in size and the 
various molecular species seem to have differences in cross-species 
antiviral activities (L.S. Lin et al "Characterization of the 
Heterogeneous Molecules of Human Interferons: Differences in cross-species 
antiviral activities of various molecular populations in human leukocyte 
interferons", J Gen Virol 39:125-130 (1978)). The heterogeneity of the 
leukocyte interferon has subsequently been confirmed by the molecular 
cloning of a family of closely related HuIFN-.alpha. genes from human 
leukocyte cells and from lymphoblastoid cell lines (S. Nagata et al, "The 
structure of one of the eight or more distinct chromosomal genes for human 
interferon-.alpha.", Nature, 287:401-408 (1980); D.V. Goeddel et al, "The 
structure of eight distinct cloned human leukocyte interferon cDNAs", 
Nature, 290:20-26 (1981)). However, a comparison of the DNA and amino acid 
sequences of the HuIFN-.alpha. interferons also reveals that many of the 
sequences exhibit homology at the nucleotide level, some in the order of 
70 percent, and that the related gene products of these homologous DNA 
sequences are also homologous. (D.V. Goeddel et al, "The structure of 
eight distinct cloned human leukocyte interferon cDNAs", Nature, 290:20-26 
(1981); N. Mantein et al, "The nucleotide sequence of a cloned human 
leukocyte interferon cDNA", Gene, 10:1-10 (1980); M. Streuli et al, "At 
least three human type .alpha. interferons: Structure of .alpha.-2", 
Science, 209:1343-1347 (1980)). 
HuIFN-.beta. is produced in human fibroblast cells. Although there is 
evidence that human fibroblast cells may be producing more than one 
HuIFN-.beta. (P. B. Sehgal and A. D. Sagar, "Heterogeneity of Poly(I) and 
Poly(C) induced human fibroblast interferon mRNA species", Nature, 
288:95-97 (1980)), only one species of HuIFN-.beta. has been purified to 
homogeneity (E. Knight, Jr., "Interferon: Purification and initial 
characterization from human diploid cells", PNAS, 73:520-523 (1976); W. 
Berthold et al, "Purification and in vitro labeling of interferon from a 
human fibroblast cell line", J Biol Chem, 253:5206-5212 (1978)). The amino 
terminal sequence of this purified HuIFN-.beta. has been determined (E. 
Knight, Jr. et al, "Human fibroblast interferon: Amino acid analysis and 
amino terminal amino acid sequence", Science, 207:525-526 (1981)). 
Molecular cloning by recombinant DNA techniques of the gene coding for 
this interferon has been reported (T. Taniguchi et al, "Construction and 
Identification of a Bacterial Plasmid Containing the Human Fibroblast 
Interferon Gene Sequence", Proc Japan Acad, 55 Ser B, 464- 469 (1979)). 
This well characterized human fibroblast interferon will be referred to as 
HuIFN-.beta.1 in the rest of this specification. 
Although interferons were initially identified by their antiviral effects 
(A. Isaacs and J. Lindenmann, "Virus Interference I. The Interferon", Proc 
Royal Soc, Ser B, 147:258-267 (1957)), the growth regulatory effect of 
interferons is another biological activity that has also been well 
documented (I. Gressor and M.G. Tovey, "Antitumor effects of interferon" 
Biochim Biophys Acta, 516:213-247 (1978); W. E. Stewart, "The Interferon 
System" Springer-Verlag, New York, 292-304 (1979); A. A. Creasey et al, 
"Role of GO-Gl Arrest in the Inhibition of Tumor Cell Growth by 
Interferon", PNAS, 77:1471-1475 (1980)). In addition, interferon plays a 
role in the regulation of the immune response (H. M. Johnsons, Texas 
Reports on Biology and Medicine, 35:357-369 (1977)), showing both 
immunopotentiating and immunosuppressive effects. Interferon may mediate 
the cellular immune response by stimulating "natural killer" cells in the 
spontaneous lymphocyte - mediated cytotoxicity (J. Y. Djeu et al, 
"Augmentation of mouse natural killer cell activity by interferon and 
interferon inducers", J Immun, 122:175-181 (1979)). 
Studies concerning the biological activities of interferons have been 
conducted by taking advantage of nucleotide and amino acid sequence 
homologies between HuIFN-.alpha.1 and HuIFN-.alpha.2. Hybrids of the two 
genes were constructed in vitro by recombinant DNA techniques such that 
the DNA sequence coding for the amino terminus of one gene was fused to 
the DNA sequence coding for the carboxy terminus of the other gene (M. 
Streuli et al, "Target cell specificity of two species of human 
interferon-.alpha. produced in Escherichia coli and of hybrid molecules 
derived from them", PNAS 78:2848-2852 (1981); P. K. Weck et al, "Antiviral 
activities of hybrids of two major human leukocyte interferons", Nucleic 
Acids Res, 9:6153-6166 (1981)). 
HuIFN-.alpha.1 has a lower specific activity on human WISH cells than on 
bovine MDBK cells while HuIFN-.alpha.2 behaves in the opposite manner. 
Also, HuIFN-.alpha.1 has some activity on mouse L cells while 
HuIFN-.alpha.2 has little activity on mouse cells. However, the 
HuIFN-.alpha.2-.alpha.1 hybrid (amino terminal sequence of HuIFN-.alpha.2 
fused to the carboxy terminal sequence of HuIFN-.alpha.1) has much higher 
activity on mouse L cells than on human cells (M. Streuli et al, "Target 
cell specificity of two species of human interferon-.alpha. produced in E. 
coli and of hybrid molecules derived from them", PNAS, 78:2848-2852 
(1981); N. Stebbing et al, "Comparison of the biological properties of 
natural and recombinant DNA derived human interferons", The Biology of the 
Interferon System, Elsevier/North-Holland, 25-33 (1981); P. K. Weck et al, 
"Antiviral activities of hybrids of two major leukocyte interferons", 
Nucleic Acids Res, 9:6153-6166 (1981)). Therefore, target cell 
specifications can be altered by making hybrid proteins. 
Although these .alpha.--.alpha. hybrids exhibited changes in target cell 
specificity as compared to the parent, it was not demonstrated that there 
was any attenuation or any restriction of any of the three interferon 
activities. 
Under some circumstances, the plural biological activity of interferon may 
be undesirable. For example, in the clinical treatment of patients who 
have received organ transplants and whose immune system has been 
suppressed because of anti-rejection drugs, administration of interferon 
to combat viral infection could result in undesirable stimulation of the 
immune response system and consequent rejection of the transplanted 
organs. Moreover, in clinical applications it is generally desirable in 
principle to focus drug therapy on a particular problem such as viral 
infection or tumor growth without the possibility of complicating factors 
resulting from other activities of the administered drug. In such 
treatment and applications it would be desirable to be able to use an 
interferon whose activity is limited to the desired activity. The present 
invention provides a novel group of hybrid interferons that have 
restricted interferon activity as well as changes in target cell 
specificity. 
DISCLOSURE OF THE INVENTION 
One aspect of the invention is a multiclass hybrid interferon polypeptide 
having an amino acid sequence composed of at least two distinct amino acid 
subsequences one of which subsequences corresponds substantially in amino 
acid identity, sequence and number to a portion of a first interferon and 
the other of which corresponds in amino acid identity, sequence and number 
to a portion of a second interferon of a different interferon class from 
the first interferon. 
A second aspect of the invention is DNA units or fragments comprising 
nucleotide sequences that upon expression encode for the above described 
multiclass hybrid interferons. 
A third aspect of the invention is cloning vehicles (vectors) that include 
the above described DNA. 
A fourth aspect of the invention is host organisms or cells transformed 
with the above described cloning vehicles that produce the above described 
multiclass hybrid interferons. 
A fifth aspect of the invention is processes for producing the above 
described multiclass hybrid interferons comprising cultivating said 
transformed host organisms or cells and collecting the multiclass hybrid 
interferons from the resulting cultures. 
Another aspect of the invention is pharmaceutical compositions comprising 
an effective amount of one or more of the above described multiclass 
hybrid interferons admixed with a pharmaceutically acceptable carrier. 
Another aspect of the invention is a method of regulating cell growth in an 
animal patient comprising administering to said patient a cell growth 
regulating amount of one or more of the above described multiclass hybrid 
interferons having interferon activity substantially restricted to cell 
growth regulatory activity. 
Still another aspect of the invention is a method of treating an animal 
patient for a viral disease comprising administering to said patient a 
viral disease inhibiting amount of one or more of the above described 
multiclass hybrid interferons having interferon activity substantially 
restricted to anti-viral activity.

MODES FOR CARRYING OUT THE INVENTION 
The hybrid interferons of the invention have an amino acid sequence 
composed of at least two distinct amino acid subsequences that are 
respectively substantially identical to portions of interferons from 
different classes. The term "substantially identical" means that a 
subsequence of the hybrid exhibits at least about 70%, preferably at least 
about 95%, and most preferably 100% homology with an amino acid 
subsequence of a given interferon. Lack of complete homology may be 
attributable to single or multiple base substitutions, deletions, 
insertions, and site specific mutations in the DNA which on expression 
code for the hybrid or given interferon amino acid sequences. When the 
hybrid is composed of more than two subsequences, the additional 
subsequence(s) may correspond to other portions of the interferons 
involved in the initial two subsequences (eg, if the initial two sequences 
are .alpha.1 and .beta.1, the other sequences are .alpha.1 or .beta.1) or 
correspond to portions of interferons different from those involved in the 
initial two subsequences. Hybrids composed of .alpha. interferon and 
.beta. interferon subsequences are preferred. Hybrids composed of only two 
subsequences (.alpha. and .beta.) are particularly preferred. Individual 
subsequences will usually be at least about 10 amino acid residues in 
length, more usually at least about 30 amino acid residues in length. 
Multiclass hybrid interferons of the invention exhibit activity that is 
different from the interferon activity exhibited by the parent interferons 
of which they are composed. The difference is manifested as a substantial 
reduction (relative to the parent interferons) or elimination of one or 
two of the three conventional interferon activities. Preferred hybrids are 
those whose interferon activity is substantially restricted to one of the 
three activities. Based on data developed to date the interferon activity 
of the .alpha.-.beta. interferons appears to be substantially restricted 
to either cell growth regulatory or antiviral activity. In some instances 
the hybrid interferons also have a host range (target) cell specificity 
different from that of the parent interferons from which they are derived. 
In other words hybrid interferons of the invention may exhibit a 
particular interferon activity in the cells of one but not another animal 
species in which the parent interferons also exhibit activity. 
The structural homologies between different classes of interferons (FIG. 1) 
permit construction of hybrid DNA molecules coding for the multiclass 
human hybrid interferon polypeptides. To construct the hybrid gene, it is 
preferred, although not required, that the gene donating the amino 
terminal end sequence be fused to some suitable promoter which directs 
expression of the gene and contains the appropriate promoter, operator and 
ribosomal binding sequence. The hybrids may be made by selecting suitable 
common restriction sites within the respective full genes for the 
different classes of human interferon. As an alternative, different 
restriction sites may be used for cleavage, followed by repair to blunt 
ends, followed by blunt end ligation. In either case, the proper reading 
frame must be preserved. Once the desired segments are ligated together, 
they are placed in a suitable cloning vector, which is used to transform 
suitable host organisms or cells. Where the amino terminal fragment 
carries the promoter, operator and ribosomal binding sequence, expression 
and biological activity of the resultant hybrids may be directly assayed. 
Fusions can be directed to different parts of the gene by choosing 
appropriate restriction enzyme sites. 
The following examples further illustrate the invention and are not 
intended to limit the scope of the invention in any way. 
EXAMPLE I: Construction of HuIFN-.alpha.1.beta.1 Hybrid 1. 
This example describes the construction of a hybrid interferon, containing 
sequences from HuIFN-.alpha.1 and HuIFN-.beta.1. It involves fusing the 
amino-terminal end coding region of the HuIFN-.alpha.1 DNA to the DNA 
coding for the carboxy-terminal end region of HuIFN-.beta.1 in such a way 
that the translational reading frame of the two proteins are preserved and 
the resulting protein being expressed from this hybrid gene will have the 
amino acid sequence of HuIFN-.alpha.1 at its amino terminal portion and 
the amino acid sequence of HuIFN-.beta.1 at its carboxy terminal portion. 
Purification and Isolation of HuIFN-.alpha.1 and HuIFN-.beta.1 DNA 
sequences. 
The plasmids used in the construction of the HuIFN-.alpha.1.beta.1 Hybrid 1 
are plasmids pGW5 and pDM1O1/trp/.beta.1 containing the genes coding for 
HuIFN-.alpha.1 and HuIFN-.beta.1 respectively. The structure of plasmid 
pGW5 is shown in FIG. 2 and that of plasmid pDM1O1/trp/.beta.1 in FIG. 4. 
The plasmid pGW5 was constructed from the plasmid pBR322 by substituting 
the region between the EcoRI site to the PvuII site with the E. coli trp 
promoter and the DNA sequence coding for the mature protein of 
HuIFN-.alpha.1 (FIG. 2). The DNA sequence between the HindIII site and 
EcoRI site of pGW5, encoding the mature protein of HuIFN-.alpha.1, is 
shown in FIG. 3. Also shown in FIG. 3 is the amino acid sequence of 
HuIFN-.alpha.1 (IFN-.alpha.D in FIG. 1). The plasmid pGW5 expressed 
HuIFN-.alpha.1 at high levels in E. coli. When grown in shake-flasks, 
about 2.times.10.sup.6 units of antiviral activity per ml of bacterial 
culture per A600 can be detected. 
The plasmid pDM1O1/trp/.beta.1 is a derivative of pBR322 with the E. coli 
trp promoter located between the EcoRI and HindIII sites (FIG. 4). The DNA 
sequences between the HindIII and BglII sites encode the mature 
HuIFN-.beta.1 protein sequence. The nucleotide sequence together with the 
amino acid sequence is shown in FIG. 5. When grown in shake-flasks, the E. 
coli strain carrying pDM1O1/trp/.beta.1 expresses HuIFN-.beta.1 at a level 
of 10.sup.6 units of antiviral activity per ml of bacterial culture per 
A600. 
The hybrid gene was constructed by taking advantage of the homologies 
between the HuIFN-.alpha.1 gene and the HuIFN-.beta.1 gene at around amino 
acid 70 of both proteins (FIG. 6). There is a HinfI restriction site 
(GATTC) present within this region of both genes. If both DNA sequences 
are digested with the enzyme HinfI and the DNA sequence 5'-proximal to the 
cutting site of the HuIFN-.alpha.1 DNA (the arrow in FIG. 6 depicts the 
cutting site) is ligated to the DNA sequence 3'-proximal to the cutting 
site of HuIFN-.beta.1, a fusion of the two genes is created while 
preserving the translational reading frame of both genes. 
Since there are several HinfI sites in the coding regions of both 
HuIFN-.alpha.1 and HuIFN-.beta.1, it is not possible to carry out a 
straightforward exchange of DNA sequences. In the case of HuIFN-.beta.1, a 
502 bp HindIII-BglII fragment containing the whole coding region from 
pDM1O1/trp/.beta.1 is first isolated. The plasmid DNA was digested with 
restriction enzymes HindIII and BglII (R. W. Davis et al, "Advanced 
Bacterial Genetics", Cold Spring Harbor Laboratory, pp. 227-230, 1980). 
(This reference will be referred to as "Advanced Bacterial Genetics" 
hereinafter), the DNA fragments were separated on a 1.5% agarose gel in 
Tris-Borate buffer ("Advanced Bacterial Genetics" p 148) and the DNA 
fragments visualized by staining with ethidium bromide ("Advanced 
Bacterial Genetics", pp 153-154). The appropriate DNA fragment, in this 
case a 502 bp fragment, is cut out of the gel, placed in a dialysis tubing 
with a minimum amount of 0.1X Tris-Acetate buffer ("Advanced Bacterial 
Genetics", p 148) and covered with the same buffer in an electroelution 
box and a voltage of 150-200 volts applied for 1 hour. The DNA is then 
recovered from the buffer in the dialysis tubing and concentrated by 
ethanol precipitation. The 502 bp HindIII-BglII fragment was then digested 
partially with HinfI to obtain the 285 bp partial HinfI fragment (denoted 
as .beta.-B) coding for the carboxy terminal end of HuIFN-.beta.1 (FIG. 
7). The partial digestion of the DNA fragment was accomplished by using 
one-tenth the amount of restriction enzyme required for complete digestion 
of the DNA ("Advanced Bacterial Genetics", p 227). The mixture was 
incubated at the appropriate temperature for the enzyme and aliquots of 
the digestion mixture were removed at 10-minute intervals for up to 1 
hour. The aliquots were then loaded onto a gel and the DNA fragments 
analyzed. The time point that provides the highest yield of the DNA 
fragment needed is chosen for a preparative digestion with the restriction 
enzyme and the appropriate fragment purified from the gel by 
electroelution. The other HindIII-BglII fragment, (.beta.-C in FIG. 9) 
consisting of the plasmid pDM1O1 and trp promoter, is also saved and used 
in the vector for the HuIFN-.alpha.1.beta.1 hybrid. 
In the case of HuIFN-.alpha.1, pGW5 is digested with HindIII and PvuII and 
a 278 bp fragment which contains two HinfI sites is purified from the 
digest. This fragment is then digested partially with HinfI to obtain two 
fragments, a 213 bp HindIII-HinfI fragment (.alpha.-A) and a 65 bp 
HinfI-PvuII fragment (.alpha.-B) (FIG. 8). 
Vector Preparation and Selection 
Assembly of the plasmid for the direct expressions of the 
HuIFN-.alpha.1.beta.1 interferon gene can be constructed by ligating 
fragments .alpha.-A, .beta.-B and .beta.-C together as shown in FIG. 9. 
The ligated DNA was then used to transform competent E. coli cells 
("Advanced Bacterial Genetics" pp 140-141). Transformants were plated onto 
broth plates containing 50 .mu.g per ml of ampicillin and incubated at 
37.degree. C. Ampicillin resistant colonies were grown up in rich medium 
in the presence of 50 .mu.g/ml of ampicillin and plasmid DNA isolated from 
each individual clone ("Advanced Bacterial Genetics", pp 116-125). 
The gene structure of the desired hybrid clone is shown in FIG. 10. The 
correct hybrid clone was identified by digesting the plasmid DNA with the 
restriction enzymes HindIII and BglII and screening for the presence of a 
498 bp restriction fragment on 1.5% agarose gel in Tris-Borate buffer 
("Advanced Bacterial Genetics", p 148). To further characterize the hybrid 
clone, the plasmid DNA was digested with HinfI and screened for the 
presence of the 145 bp and 167 bp restriction fragments. By following this 
scheme, a number of hybrid clones were identified, one of which (denoted 
pDM1O1/trp/hybrid 41) was selected for further characterization and 
culturing to produce the hybrid interferon. 
The nucleotide sequence of the region coding for the hybrid protein is 
shown in FIG. 11. Also shown in FIG. 11 is the amino acid sequence of the 
hybrid protein. This hybrid interferon is denoted HuIFN-.alpha.1.beta.1 
Hybrid 1 herein. The amino terminal portion of this polypeptide starting 
with methionine is composed of the amino acid sequence 1-73 of 
HuIFN-.alpha.1 and the carboxy terminal portion is composed of amino acids 
74-166 of HuIFN-.beta.1. 
The E. coli strain carrying pDM1O1/trp/hybrid 41 was grown in minimal 
medium containing 50 .mu.g/ml of ampicillin to express the hybrid protein. 
The culture was harvested when it reached A600 =1.0, concentrated by 
centrifugation, resuspended in buffer containing 50 mM Tris-HCL pH 8.0, 10 
mM ethylenediaminetetraacetic acid (EDTA), 15% sucrose and 1% sodium 
dodecylsulfate (SDS), and the cells lysed by sonication in a Branson 
Sonicator. The cell free extract was assayed for 
(1) inhibiting the growth of transformed cells, 
(2) activating natural killer cells, and 
(3) antiviral activity. 
Biological Testing of HuIFN-.alpha.1.beta.1 Hybrid 1 
(1) Growth Inhibition Assays 
Bacterial extracts made from the E. coli strain carrying pDM1O1/trp/hybrid 
41, together with various control extracts, were assayed for their ability 
to inhibit the growth of two human tumor cell lines, the Daudi line 
(American Type Culture Collection, Catalog of Cell Strains III, 3rd 
Edition, Rockville, MD (1979)) and the melanoma line HS294T Clone 6 (A. A. 
Creasey et al, PNAS, 77:1471-1475, (1980); A. A. Creasey et al, Exp Cell 
Res, 134:155-160 (1981)). 
(a) Inhibition of Growth of Daudi Cells 
About 2.times.10.sup.4 cells are seeded into each well of a sterile 96-well 
round bottom microtiter plate. Cells are then incubated overnight at 
37.degree. C. Bacterial extracts together with the appropriate controls 
are added to the cells and then allowed to incubate at 37.degree. C. for 
three days. On the third day, cells are pulse labeled with 4.mu.Ci/well of 
.sup.3 H-thymidine for 2-3 hours. The labeling is terminated by addition 
of 5% trichloroacetic acid (TCA) to precipitate the nucleic acids. The 
precipitates are filtered and the filters are counted in the scintillation 
counter. The results for the cells incubated with the bacterial extracts 
are compared to the results for the controls to obtain a percent 
inhibition of growth. The results are reported in Table I below. 
(b) Inhibition of HS294T Clone 6 
About 1.5.times.10.sup.4 cells are seeded into each well of a sterile, 
flexible 48-well flat bottom tissue culture plate. Cells are incubated 
overnight at 37.degree. C. with 10% CO.sub.2. Bacterial extracts together 
with various controls are added to the cells and then incubated for three 
days at 37.degree. C. On the third day, cells are pulse labeled with 
2.mu.Ci/well of .sup.3 H-thymidine for 2-3 hours. The labeling reactions 
is terminated by addition of cold TCA in 0.3% Na.sub.4 P.sub.2 O.sub.7 
(TP). Plates are washed two times with TP solution and three times with 
cold absolute ethanol, and left to dry at room temperature. A sheet of 
adhesive tape is stuck to the bottom of the assay plate, securing all the 
wells in place. The plate is then run through a hot wire cutter. The top 
of the plate is removed and the individual wells are picked off the 
adhesive tape and put into scintillation vials containing 5 ml of 
scintillation fluid and counted in the scintillation counter. Percent 
growth inhibition was obtained as above. The results are also reported in 
Table I below. 
TABLE I 
______________________________________ 
U/ml or Percent Inhibition of 
*dilution of 
Growth Cell Lines 
HS294T 
HuIFN Extract Daudi Clone 6 
______________________________________ 
.alpha..sup.1 
100 70 0 
500 80 9 
.beta..sup.1 
100 68 43 
500 72 80 
Hybrid of *1:2000 46 4 
Example I *1:20,000 24 0 
______________________________________ 
Note: 
Percent inhibition of growth by negative control (pDM101/trp) was include 
in the calculations to obtain the numbers shown above) 
As reported in Table I the hybrid interferon HuIFN-.alpha.1.beta.1 Hybrid 1 
inhibited the growth of Daudi cells but it did not inhibit the HS294T 
Clone 6 cells. Since the HS294T Clone 6 cells are resistant to 
HuIFN-.alpha.1 the hybrid appears to be behaving like HuIFN-.alpha.1 in 
these tests. Therefore, it appears that since the hybrid has the 
HuIFN-.alpha.1 amino terminal sequence as its amino terminus, that portion 
of the protein may carry the determinant which governs cell specificity. 
(2) Stimulation of Natural Killer Cells 
Whole blood is obtained from a donor and kept clot-free by adding EDTA. 
Lymphocytes are separated by centrifugation on a Ficoll/Hypaque gradient. 
The upper band of lymphocytes is harvested and washed. Interferon samples 
and various control samples are diluted into 1 ml of Dulbecco's Modified 
Eagle's Medium (DME) containing 10% fetal calf serum (FCS) and then mixed 
with 1 ml of lymphocytes (10.sup.7 cells) and incubated at 37.degree. C. 
for 18 hours. The treated lymphocytes are then washed and resuspended in 
RPMI 1640 medium containing 10% FCS. 
Two hours before the lymphocytes are harvested, the target cells (Daudi 
line) are labeled with .sup.51 Cr by incubating 2.times.10.sup.6 Daudi 
cells with 100 .mu.Ci of .sup.51 Cr in 1 ml of RPMI 1640. After two hours, 
the target cells are washed four times to remove excess label, 
concentrated by centrifugation and resuspended to 2.times.10.sup.5 cells 
per ml in RPMI 1640. About 2.times.10.sup.4 labeled target cells are added 
to each well of a microtiter plate. Primed lymphocytes together with 
unprimed controls are added to the target cells in triplicate and 
incubated for four hours at 37.degree. C. The plate is then centrifuged 
and 100 .mu.l of media is removed from each well and counted in the gamma 
counter. Percent killing by the activated natural killer cells is 
dependent on the interferon concentration. Thus, small amounts of 
interferon will result in a small percentage of killing and minimal lysis 
of target cells. By determining the amount of label released into the 
medium, the amount of natural killer activity can be quantitated. The 
results of the tests are reported in Table II below. 
TABLE II 
______________________________________ 
ACTIVATION OF NATURAL KILLER CELLS 
U/ml or 
*dilution of 
Percent 
HuIFN extract Killing (%) 
______________________________________ 
.alpha..sup.1 100 39 
10 29 
.beta..sup.1 100 38 
10 2 
Hybrid of *1:1000 13 
Example I 
Controls: 
pDM101/trp/ *1:1000 10 
Cell Control 
(Spontaneous release of label) 
7 
______________________________________ 
As reported in Table II, the hybrid interferon showed substantially less 
natural killer activity than HuIFN-.alpha.1 and HuIFN-.alpha.1. 
(3) Antiviral Assays 
Interferon antiviral activity in bacterial extracts was determined by 
comparison with NIH interferon standards using cytopathic effect (CPE) 
inhibition assays as reviewed previously (W. E. Stewart, "The Interferon 
System" Springer-Verlag, 17-18, (1979)). The assays were performed on two 
different cell lines: the human trisomic 21 line (GM2504), and the bovine 
MDBK line, with vesicular stomatitis virus as the challenge virus within 
the limits of the sensitivity of the CPE inhibition assay (.gtoreq.30 
U/ml) no antiviral activity in the bacterial extracts containing the 
hybrid interferon of Example I was detected. 
EXAMPLE II: Construction of HuIFN-.beta.1.alpha.1 Hybrid 1. 
This example describes the construction of a hybrid interferon containing 
sequences from HuIFN-.alpha.1 and HuIFN-.beta.1. It involves the fusion of 
the amino terminal coding region of the HuIFN-.beta.1 DNA to the DNA 
coding for the carboxy terminal region of HuIFN-.alpha.1 in such a way 
that the translational reading frame of the two genes are preserved and 
the resulting protein being expressed from this hybrid gene will have the 
amino acid sequence of HuIFN-.beta.1 at its amino terminus and the amino 
acid sequence of HuIFN-.alpha.1 at its carboxy terminus. 
Purification and Isolation of HuIFN-.alpha.1 and HuIFN-.beta.1 DNA 
Sequences. 
The plasmids used in the construction of HuIFN-.beta.1.alpha.1 hybrid 1 are 
plasmids pGW5 and pDM1O1/trp/.beta.1 as set forth in Example I. 
As in Example I, the hybrid gene of this example was constructed by taking 
advantage of the homologies between HuIFN-.alpha.1 and HuIFN-.beta.1 at 
around amino acid 70 of both proteins (FIG. 6). The DNA sequence 
5'-proximal to the cutting site of the HuIFN-.beta.1 DNA (the arrow in 
FIG. 6 depicts the cutting site), is ligated to the DNA sequence 
3'-proximal to the cutting site of HuIFN-.alpha.1, to create a fusion of 
the two genes while preserving the translational reading frame of both 
genes. 
Since there are several HinfI sites in the coding regions of both 
HuIFN-.alpha.1 and HuIFN-.beta.1 it is not possible to carry out a 
straightforward exchange of DNA sequences. Thus the procedures of Example 
I were followed for the isolation of the 217 bp fragment (denoted as 
.beta.-A) as shown in FIG. 7. 
In the case of HuIFN-.alpha.1, pGW5 was digested with HindIII and PvuII and 
two fragments were purified. One of the fragments is 278 bp in length (the 
small fragment) and contains two HinfI sites. This fragment is digested 
partially with HinfI to obtain two fragments, a 213 bp HindIII-HinfI 
fragment (.alpha.-A) and a 65 bp HinfI-PvuII fragment (.alpha.-B) (FIG. 
8). The other HindIII-PvuII fragment containing the carboxy terminus 
coding region of HuIFN-.alpha.1 (.alpha.-C fragment) is saved for use as 
vector for cloning the hybrid. 
Vector Preparation and Selection 
The hybrid can be constructed by ligating fragments .beta.-A, .alpha.-B and 
.alpha.-C together as shown in FIG. 12. This ligated DNA was then used to 
transform competent E. coli cells. Transformants were plated onto broth 
plates containing 50 .mu.g/ml of ampicillin and incubated at 37.degree. C. 
Ampicillin resistant colonies were grown up in rich medium in the presence 
of 50 .mu.g/ml of ampicillin and plasmid DNA isolated from each individual 
clone. 
The gene structure of the desired hybrid clone is shown in FIG. 13. 
Therefore, the correct hybrid clone could be identified by digesting the 
plasmid DNA with the restriction enzyme PvuII and screening for the 
presence of the characteristic 141 bp PvuII fragment (FIG. 13) on 5% 
polyacrylamide gel. To further characterize the hybrid clone, the plasmid 
DNA was digested with HinfI and screened for the presence of the 197 bp, 
159 bp, 129 bp, and 39 bp HinfI restriction fragments. By following this 
scheme, a number of hybrid clones were identified, one of which (denoted 
pDM1O1/trp/hybrid 1) was selected for further characterization and 
culturing to produce the hybrid interferon. 
The nucleotide sequence of the region coding for the hybrid protein is 
shown in FIG. 14. Also shown in FIG. 14 is the amino acid sequence of the 
hybrid protein. This hybrid interferon is denoted HuIFN-.beta.1.alpha.1 
Hybrid 1 herein. The amino terminal portion of this polypeptide starting 
with methionine is composed of the amino acid sequence 1-73 of 
HuIFN-.beta.1 and the carboxy terminal portion is composed of amino acids 
74-166 of HuIFN-.alpha.1. 
Biological Testing of HuIFN-.beta.1.alpha.1 Hybrid 1 
The assays used to determine interferon activities were identical to those 
used in Example I. The following Tables III and IV report the results of 
the cell growth regulatory assays and the natural killer cell activity 
assay. 
TABLE III 
______________________________________ 
U/ml or Percent Inhibition of 
*dilution of 
Growth Cell Lines 
HS294T 
HuIFN Extract Daudi Clone 6 
______________________________________ 
.alpha..sup.1 
100 70 0 
500 80 9 
.beta..sup.1 
100 68 43 
500 72 80 
Hybrid of *1:2000 80 16 
Example II 
*1:20,000 23 28 
______________________________________ 
Note: 
Percent inhibition of growth by negative control (pDM101/trp) was include 
in the calculations to obtain the numbers shown above. 
As reported and in contrast to Example I, the hybrid interferon of Example 
II inhibited the growth of both Daudi and HS294T Clone 6 cells, thus 
behaving like HuIFN-.beta.1. Therefore, HuIFN-.beta.1.alpha.1 Hybrid 1 
supports the hypothesis expressed in Example I that the amino terminal 
portion of the interferon carries the determinant which governs cell 
specificity. 
TABLE IV 
______________________________________ 
ACTIVATION OF NATURAL KILLER CELLS 
U/ml or 
*dilution of 
Percent 
HuIFN Extract Killing (%) 
______________________________________ 
.alpha..sup.1 100 39 
10 29 
.beta..sup.1 100 38 
10 2 
Hybrid of *1:000 14 
Example II 
Controls: 
pDM101/trp *1:000 10 
Cell Control 
(Spontaneous release of label) 
7 
______________________________________ 
Antiviral assays were carried out using the HuIFN-.beta.1.alpha.1 Hybrid 1. 
Within the realm of sensitivity of the CPE inhibition assay no antiviral 
activity in the bacterial extracts containing the hybrid interferon was 
detected. 
EXAMPLE III: Construction of HuIFN-.alpha.61A.beta.1 Hybrid 
This example describes the construction of a hybrid interferon containing 
sequences from HuIFN-.alpha.61A and HuIFN-.beta.1. It involves the fusion 
of the amino acid terminal coding region of the HuIFN-.alpha.61A DNA to 
the DNA coding for the carboxy terminal region of HuIFN-.beta.1 in such a 
way that the translational reading frame of the two genes are preserved 
and the resulting protein being expressed from this hybrid gene will have 
the amino acid sequence of HuIFN-.alpha.61A at its amino terminus and the 
amino acid sequence of HuIFN-.beta.1 at its carboxy terminus. 
Purification and Isolation of HuIFN-.alpha.61A and HuIFN-.beta.1 DNA 
Sequences 
The plasmids used in the construction of HuIFN-.alpha.61A.beta.1 hybrid are 
plasmids p.alpha.61A and pDM1O1/trp/.beta.1 (Example I and FIG. 4). 
Preparation of plasmid p.alpha.61A 
In order to assemble the plasmid p.alpha.61A, the Namalwa cell human IFN 
enriched mRNA was used to construct complementary DNA (cDNA) clones in E. 
coli by the G/C tailing method using the PstI site of the cloning vector 
pBR322 (Bolivar, F., et al, Gene, 2:95-113 (1977)). A population of 
transformants containing approximately 50,000 individual cDNA clones was 
grown in one liter of medium overnight and the total plasmid DNA was 
isolated. 
The sequences of two IFN-.alpha. clones (IFN-.alpha.1 and IFN-.alpha.2) 
have been published (Streuli, M., et al, Science, 209:1343-1347 (1980)). 
Examination of the DNA sequences of these two clones revealed that the 
restriction enzyme XhoII would excise a 260 bp fragment from either the 
IFN-.alpha.1 or the IFN-.alpha.2 gene (see FIG. 1). XhoII was prepared in 
accordance with the process described by Gingeras, T. R., and Roberts, R. 
J., J Mol Biol, 118:113-122 (1978). 
One mg of the purified total plasmid DNA preparation was digested with 
XhoII and the DNA fragments were separated on a preparative 6% 
polyacrylamide gel. DNA from the region of the gel corresponding to 260 bp 
was recovered by electroelution and recloned by ligation into the BamHI 
site of the single strand bacteriophage m13:mp7. Thirty-six clones were 
picked at random, the single stranded DNA isolated therefrom, and the DNA 
was sequenced. The DNA sequences of four of these clones were homologous 
to known IFN-.alpha. DNA sequences. Clone mp7:.alpha.-260, with a DNA 
sequence identical to IFN-.alpha.1 DNA (Streuli, M. et al, Science, 
209:1343-1347 (1980)) was chosen as a highly specific hybridization probe 
for identifying additional IFN-.alpha. DNA sequences. This clone is 
hereinafter referred to as the "260 probe." 
In order to isolate other IFN-.alpha. gene sequences, a .sup.32 P-labelled 
260 probe was used to screen a library of human genomic DNA by in situ 
hybridization. The human gene bank, prepared by Lawn, R. M., et al, Cell, 
15:1157-1174 (1978), was generated by partial cleavage of fetal human DNA 
with HaeIII and AluI and cloned into bacteriophage .lambda. Charon 4A with 
synthetic EcoRI linkers. Approximately 800,000 clones were screened, of 
which about 160 hybridized with the 260 probe. Each of the 160 clones was 
further characterized by restriction enzyme mapping and comparison with 
the published restriction maps of 10 chromosomal IFN genes (Nagata, S., et 
al, J Interferon Research, 1:333-336 (1981)). One of the clones, hybrid 
phage .lambda.4A:.alpha.61 containing a 18 kb insert, was characterized as 
follows. A DNA preparation of .lambda.4A:.alpha.61 was cleaved with 
HindIII, BglII, and EcoRI respectively, the fragments separated on an 
agarose gel, transferred to a nitrocellulose filter (Southern, E. M., J 
Mol Biol, 98:503-517 (1977)) and hybridized with .sup.32 P-labelled 260 
probe. This procedure localized the IFN-.alpha.61 gene to a 1.9 kb BglII 
restriction fragment which was then isolated and recloned, in both 
orientations, by ligation of the fragment into BamHI cleaved m13:mp7. The 
two subclones are designated mp7:.alpha.61-1 and mp7:.alpha.61-2. The -1 
designation indicates that the single-stranded bacteriophage contains 
insert DNA complementary to the mRNA (the minus strand) and the -2 
designation indicates that the insert DNA is the same sequence as the mRNA 
(the plus strand). 
The Sanger dideoxy-technique was used to determine the DNA sequence of the 
HuIFN-.alpha.61A gene. The DNA sequence of the IFN-.alpha.61A gene and the 
amino acid sequence predicted therefrom differ substantially from the 
other known IFN-.alpha. DNA and IFN-.alpha. amino acid sequences. In this 
regard Goeddel, D. V., et al Nature (1981) 290: 20-26 discloses the DNA 
sequence of a partial IFN cDNA clone, designated LeIF-G. The sequence of 
the partial clone is similar to the 3'-end of the IFN-.alpha.61A DNA 
sequence, except for a nucleotide change in the codon for amino acid 128. 
As compared to the partial clone the IFN-.alpha.61A gene contains 
additional DNA that codes for the first 33 amino acids of IFN-.alpha.61A. 
Assembly of the p.alpha.61A plasmid involved replacing the DNA fragment 
encoding the 23 amino acid signal polypeptide of preinterferon with a 120 
bp EcoRI/Sau3A promoter fragment E. coli trp promoter, operator, and trp 
leader ribosome binding site preceding an ATG initiation codon) and using 
HindIII site that was inserted, 59 nucleotides 3'- of the TGA 
translational stop codon, to insert the gene into the plasmid pBW11 (a 
derivative of pBR322 having a deletion between the HindIII and PvuII 
sites). The complete DNA sequence of the promoter and gene fragments 
inserted between the EcoRI and HindIII sites of pBW11 is shown in FIG. 16 
which also shows the exact location of relevant cloning sites. Details of 
the construction are described below. 
The coding region for mature IFN-.alpha.61 has three Sau3A sites, one of 
which is between codons for amino acids 2 and 3. A synthetic HindIII site 
was inserted 59 nucleotides 3'- of the coding region and the resulting 
construct was subjected to a HindIII/partial Sau3A digest. A 560 bp 
fragment was isolated from the digest. This fragment and a 120 bp EcoRI to 
Sau3A E. coli promoter fragment were ligated together in a three way 
directed ligation into the EcoRI to HindIII site of pBW11. The promoter 
fragment, contained a synthetic HindIII restriction site, ATG inititation 
codon, the initial cysteine codon (TGT) common to all known IFN-.alpha.s, 
and a Sau3A "sticky end". The ligation mixture was used to transform E. 
coli . The final expression plasmid obtained, p.alpha.61A, is shown in 
FIG. 15. 
As in Examples I and II, the hybrid gene of the example was constructed by 
taking advantage of the homologies between HuIFN-.alpha.61A (the DNA 
sequence of the HuIFN-.alpha.61A gene and the amino acid sequence it 
encodes are shown in FIG. 16) and HuIFN-.beta.1 at around amino acid 40 of 
both proteins (FIG. 17). The DNA sequence 5'-proximal to the DdeI 
restriction enzyme cutting site of the HuIFN-.alpha.61A DNA (the arrow in 
FIG. 17 depicts the cutting site), is ligated to the DNA sequence 
3'-proximal to the cutting site of HuIFN-.beta.1, to create a fusion of 
the two genes while preserving the translational reading frame of both 
genes. 
Since there are several DdeI sites in the coding regions of both 
HuIFN-.alpha.61A and HuIFN-.beta.1, and the DdeI cohesive ends are not 
identical, therefore, it is not possible to carry out a straightforward 
exchange of DNA fragments. Thus variations of the procedures described in 
Examples I and II were used. 
In the case of HuIFN-.alpha.61A, p.alpha.61A was digested with EcoRI and 
PvuII and the 387 bp fragment containing three DdeI sites was purified. 
This fragment was digested partially with DdeI, the cohesive ends repaired 
to a blunt end by the action of DNA Polymerase I Klenow fragment as 
described by Maniatis et al., ("Molecular Cloning" Cold Spring Harbor 
Laboratory, Cold Spring Harbor, N.Y. p. 113-114 (1982)). The repaired DNA 
fragments were then digested with HindIII and the 120 bp fragment (denoted 
as Alpha) purified from an acrylamide gel (FIG. 18). 
In the case of HuIFN-.beta.1, pDM1O1/trp/.beta.1 was digested with EcoRI 
and BamHI and the smaller fragment, containing the interferon gene 
purified (FIG. 4). This fragment was partially digested with DdeI, the 
cohesive ends removed by treatment with S1 nuclease as described by 
Maniatis et al., ("Molecular Cloning", Cold Spring Harbor Laboratory, Cold 
Spring Harbor, N.Y. p. 140 and 237-238 (1982)). The S1 nuclease treated 
DNA was then digested with BglII and the 381 bp fragment (denoted as Beta) 
purified (FIG. 19). 
Vector Preparation 
The plasmid ptrp3 (FIG. 20) is a derivative of pBR322, with the EcoRI-ClaI 
region replaced by the E. coli trp promoter sequence. This plasmid was 
digested with HindIII and BamHI and the large plasmid fragment containing 
the E. coli trp promoter was purified (FIG. 20). 
The hybrid was constructed by ligating this vector fragment to the Alpha 
and Beta fragments as shown in FIG. 21. This ligated DNA was transformed 
into competent E. coli cells and plated on plates containing ampicillin. 
Resistant colonies were grown up individually in rich medium and plasmid 
DNA isolated from them. The plasmid DNA were digested with DdeI and 
screened on acrylamide gels for the presence of the 91 bp and 329 bp DdeI 
fragments characteristic of the hybrid as shown in FIG. 22. A number of 
hybrid clones were identified, one of which (denoted as p.alpha..beta.62) 
was selected for further characterization and culturing to produce the 
hybrid interferon. 
The nucleotide sequence of the region coding for the hybrid protein is 
shown in FIG. 23. Also shown in FIG. 23 is the amino acid sequence of the 
hybrid protein. This hybrid interferon is denoted HuIFN-.alpha.61A.beta.1 
herein. The amino terminal portion of this polypeptide starting with 
methionine is composed of the amino acid sequence 1-41 of HuIFN-.alpha.61A 
and the carboxy terminal portion is composed of amino acids 47-166 of 
HuIFN-.beta.1. 
Biological Testing of HuIFN-.alpha.61A.beta.1 Hybrid 
The assays used to determine interferon activities were identical to those 
used in Examples I and II. However, an additional assay was incorporated, 
the protein kinase phosphorylation assay, to confirm the change we 
observed in host range specificity of the antiviral activity of this 
hybrid as compared to its parents. 
Growth Inhibition and Natural Killer Cell Assays 
No inhibition of either Daudi or Clone 6 cells was exhibited. Similarly no 
activation of natural killer cells was detected. 
Antiviral Assays 
We performed our biological antiviral assays as described for Examples I 
and II on two different cell lines: the human trisomic 21 cell line 
(GM2504), and the bovine MDBK line, with vesicular stomatitis virus as the 
challenge virus. Our results are summarized in Table V. As compared to the 
previous two examples, HuIFN-.alpha.61A.beta.1 had antiviral activity on 
bovine cells (.about.10.sup.3 U/ml), but no detectable antiviral activity 
on human GM2504 cells. 
69K Protein Phosphorylation 
The biological activity of interferons has usually been studied by 
infecting treated cell cultures and measuring the inhibition of virus 
replication. A more direct approach would be to measure, in the cells, 
some interferon-induced biochemical changes associated with the 
establishment of the antiviral state. One of the clearest biochemical 
alterations observed after interferon treatment is an impairment of viral 
protein synthesis (M. Revel, "InterferonInduced Translational Regulation," 
Texas Rep Biol Med 35:212-219 (1977)). Several cellular inhibitions of 
mRNA translation have been identified in interferontreated cells and 
shown, after purification, to be enzymes that act on various components of 
the mRNA translation machinery. One cellular enzyme is a specific protein 
kinase, phosphorylating a 69,000 Mr polypeptide (P.sub.1) and the small 
subunit of eukaryotic initiation factor 2 (eIF-2). (For review, see C. 
Samuel, "Procedures for Measurement of Phosphorylation of Ribosome 
Associated Proteins in Interferon Treated Cells." Methods in Enzymology, 
79:168-178. (1981)). Phosphorylation of protein P.sub.1 is considered one 
of the most sensitive biochemical markers of interferon action and is 
significantly enhanced in interferontreated cells as compared to untreated 
cells. To confirm the change in the host range in the antiviral activity 
of HuIFN-.alpha.61A.beta.1, we used the protein kinase phosphorylation 
assay as has been described by A. Kimchi et al, "Kinetics of the Induction 
of Three Translation-Regulatory Enzymes by Interferon", Proc Natl Acad 
Sci, 76:3208-3212 (1979). We have found that the HuIFN-.alpha.61A.beta.1 
indicated in FIG. 24 as .alpha..beta.62, induced the phosphorylation of 
the kinase in the bovine MDBK cells and not in the human GM2504 cells. The 
+ and - symbols in FIG. 24 indicate the presence or absence of polyIC 
double stranded RNA in the reaction. The arrow points to the bands 
indicating the interferon-induced phosphorylation of the 69K double 
stranded RNA dependent cellular protein (P.sub.1). These results confirm 
the antiviral activity of HuIFN-.alpha.61A.beta.1 on bovine cells. 
TABLE V 
______________________________________ 
Antiviral activity of recombinant parent and hybrid 
interferons on bovine and human cells in culture 
Cell Line 
Human Fibroblasts 
Bovine Fibroblasts 
(GM2504) (MDBK) 
IFN/type IFN Titer (U/ml) 
______________________________________ 
IFN-.alpha.61A 
&gt;10.sup.6 10.sup.6 
IFN-.beta.1 
5 .times. 10.sup.5 
5 .times. 10.sup.3 
IFN-.alpha.61A.beta.1 
&lt;30.sup. 10.sup.3 
trp control 
&lt;30.sup. &lt;30 
______________________________________ 
The cell growth regulating activity exhibited by certain .alpha.-.beta. 
hybrid interferons makes these hybrids potentially useful for treating 
tumors and cancers such as osteogenic sarcoma, multiple myeloma, Hodgkin's 
disease, nodular, poorly differentiated lymphoma, acute lymphocytic 
leukemia, breast carcinoma, melanoma, and nasopharyngeal carcinoma. 
Because of their restricted activity such treatment is not expected to be 
associated with side effects such as immunosuppression that often is 
observed with conventional nonhybrid interferon therapy. Also it is 
expected that the .alpha.-.beta. hybrid interferons exhibiting interferon 
activity restricted to antiviral activity may be used to treat viral 
infections with a potential for interferon therapy such as 
encephalomyocarditis virus infection, influenza and other respiratory 
tract virus infections, rabies and other viral zoonoses and arbovirus 
infections. 
Pharmaceutical compositions that contain a hybrid interferon as an active 
ingredient will normally be formulated with an appropriate solid or liquid 
carrier depending upon the particular mode of administration being used. 
For instance, parenteral formulations are usually injectable fluids that 
use pharmaceutically and physiologically acceptable fluids such as 
physiological saline, balanced salt solutions, or the like as a vehicle. 
Oral formulations, on the other hand, may be solid, eg tablet or capsule, 
or liquid solutions or suspensions. The hybrid interferon will usually be 
formulated as a unit dosage form that contains approximately 100 .mu.g of 
protein per dose. 
The hybrid interferons of the invention may be administered to humans or 
other animals on whose cells they are effective in various manners such as 
orally, intravenously, intramuscularly, intraperitoneally, intranasally, 
intradermally, and subcutaneously. The particular mode of administration 
and dosage regimen will be selected by the attending physician taking into 
account the particulars of the patient, the disease and the disease state 
involved. For instance, viral infections are usually treated by daily or 
twice daily doses over a few days to a few weeks; whereas tumor or cancer 
treatment typically involves daily or multidaily doses over months or 
years. The same dose levels as are used in conventional nonhybrid 
interferon therapy may be used. A hybrid interferon may be combined with 
other treatments and may be combined with or used in association with 
other chemotherapeutic or chemopreventive agents for providing therapy 
against neoplasms or other conditions against which it is effective. 
Modifications of the above described modes for carrying out the invention, 
such as, without limitation, use of alternative vectors, alternative 
expression control systems in the vector, and alternative host 
microorganisms and other therapeutic or related uses of the hybrid 
interferons, that are obvious to those of ordinary skill in the 
biotechnology, pharmaceutical, medical and/or related fields are intended 
to be within the scope of the following claims.