Multiclass Hybrid Interferons

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 crossspecies 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 G0-G1 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.

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 antiviral activity.

FIG. 1 shows the amino acid sequence for several different interferons indicated as .beta.1, .alpha.A through .alpha.H and .alpha.61A with regions of sequence homology being enclosed by dark lines. The one letter abbreviations recommended by the IUPAC-IUB Commission on Biochemical Nomenclature are used; A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; and Y, tyrosine.

FIG. 2 illustrates the structure of plasmid pGW5 used in the methodology of the invention.

FIG. 3 illustrates the nucleotide sequence between the HindIII site and the EcoRI site of pGW5, as well as the amino acid sequence of HuIFN-.alpha.1 which the plasmid expresses.

FIG. 4 illustrates the structure of a plasmid pDM101/trp/.beta.1 used in the methodology of the invention.

FIG. 5 illustrates the nucleotide sequence between the HindIII site and the BglII sites of the plasmid pDM101/trp/.beta.1 as well as the amino acid sequence of the expressed HuIFN-.beta.1.

FIG. 6 illustrates the amino acid sequences of HuIFN-.alpha.1 and HuIFN-.beta.1 at around amino acid 70 of both proteins.

FIG. 7 illustrates the 217 base pair (bp) HindIII-HinfI fragment and the 285 bp HinfI-BglIII fragment of the HuIFN-.beta.1 gene, as generated in the methodology of the invention.

FIG. 8 illustrates the 213 base pair HindIII-HinfI fragment and the 65 base pair HinfI-PvuII fragment of the HuIFN-.alpha.1 gene, as generated in the methodology of the invention.

FIG. 9 illustrates the structure of the plasmid coding for the hybrid protein of Example I infra.

FIG. 10 is the structure of the coding region of the hybrid gene incorporated in the plasmid of FIG. 9.

FIG. 11 illustrates the nucleotide sequence of the region coding for the hybrid protein of Example I, as well as showing the amino acid sequence of the hybrid protein.

FIG. 12 illustrates the structure of the plasmid coding for the hybrid protein of Example II, infra.

FIG. 13 illustrates the structure of the coding region of the hybrid gene incorporated in the plasmid of FIG. 12.

FIG. 14 illustrates the nucleotide sequence of the hybrid gene shown in FIG. 13, as well as showing the corresponding amino acid sequence of the hybrid protein expressed by said gene.

FIG. 15 illustrates the structure of plasmid mid p.alpha.61A used in the methodology of the invention.

FIG. 16 illustrates the nucleotide sequence of the E.coli trp promoter as well as the nucleotide sequence of the HuIFN-.alpha.61A gene including some of the flanking 3' non coding region of the gene which was inserted between the EcoRI and HindIII sites of the plasmid pBW11. The region coding for the HuIFN-.alpha.61A gene begins with the ATG codon at position 113 and terminates with the TGA codon at position 614. The corresponding amino acid sequence of the HuIFN-.alpha.61A protein is also shown.

FIG. 17 illustrates the nucleotide and amino acid sequences of HuIFN-.beta.1 and HuIFN-.alpha.61A at around amino acid 40 of both proteins.

FIG. 18 illustrates the 387 bp EcoRI-PvuII fragment and the 120 bp (Alpha) HindIII-DdeI fragment of the HuIFN-.alpha.61 gene, as generated in the methodology of the invention.

FIG. 19 illustrates the 381 bp (Beta) DdeI-BglII fragment of the HuIFN-.beta.1 gene, as generated in the methodology of the invention.

FIG. 20 illustrates the structure of a plasmid ptrp3 used in the methodology of the invention.

FIG. 21 illustrates the structure of the plasmid coding for the hybrid protein of Example III infra.

FIG. 22 is the structure of the coding region of the hybrid gene incorporated in the plasmid of FIG. 21.

FIG. 23 illustrates the nucleotide sequence of the region coding for the hybrid protein of Example III, as well as showing the amino acid sequence of the hybrid protein.

FIG. 24 depicts a protein gel showing the phosphorylation of the protein kinase in bovine cells.

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 (e.g., 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 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.

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.

The plasmids used in the construction of the HuIFN-.alpha.1.beta.1 Hybrid 1 are plasmids pGW5 and pDM101/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 pDM101/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 pDM101/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 pDM101/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 pDM101/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 pDM101 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).

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 pDM101/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 pDM101/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.

Bacterial extracts made from the E.coli strain carrying pDM101/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)).

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.

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.

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.

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.

As reported in Table II, the hybrid interferon showed substantially less natural killer activity than HuIFN-.beta.1 and HuIFN-.alpha.1.

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.

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.

The plasmids used in the construction of HuIFN-.beta.1.alpha.1 hybrid 1 are plasmids pGW5 and pDM101/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.

The hybrid can be constructed by ligating 0 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 pDM101/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.

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.

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 determinat which governs cell specificity.

Antiviral assays were carried out using the HuIFN-.beta.1.alpha.1 Hybrid 1. Within the realm of sensitivity of the CPE inhbition assay no antiviral activity in the bacterial extracts containing the hybrid interferon was detected.

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.

The plasmids used in the construction of HuIFN-.alpha.61A.beta.1 hybrid are plasmids p.alpha.61A and pDM101/trp/.beta.1 Example I and FIG. 4).

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, Bg1II, 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 32P-labelled 260 probe. This procedure localized the IFN-.alpha.61 gene to a 1.9 kb Bg1II 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 preceoperator, ding 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, pDM101/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 Bg1II and the 381 bp fragment (denoted as Beta) purified (FIG. 19).

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

25 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.

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.

No inhibition of either Daudi or Clone 6 cells was exhibited. Similarly no activation of natural killer cells was detected.

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.

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, "Interferon-Induced Translational Regulation," Texas Rep Biol Med 35:212-219 (1977)). Several cellular inhibitions of mRNA translation have been identified in interferonm-treated 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 interferon-treated 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.

The cell growth regulating activity exhibi

ted 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.