AU706762B2 - Interferon tau compositions and methods of use - Google Patents

Description

1A INTERFERON TAU COMPOSITIONS AND METHODS OF USE Field of the Invention The present invention relates to hybrid interferon proteins, to methods for preparing these proteins and methods of using these proteins.

This application is divided from Australian Patent Application No.

54449/94 which in one aspect relates to a novel method of inhibiting tumor cell growth by use of interferon-tau and the claims and description of Australian Application No 54449/94, as filed, is incorporated herein by reference.

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Background of the Invention 15 Conceptus membranes, or trophectoderm, of various mammals produce biochemical signals that allow for the establishment and maintenance of pregnancy (Bazer, et al., 1983). One such protein, ovine trophoblast protein- .**one (oTP-1), was identified as a low molecular weight protein secreted by sheep conceptuses between days 10 and 21 of pregnancy (Wilson, et al., 1979; Bazer, et al., 1986). The protein oTP-1 was shown to inhibit uterine secretion S: of prostaglandin F 2 -alpha, which causes the corpus luteum on the ovary to undergo physiological and endocrinological demise in nonpregnant sheep (Bazer, et al., 1986). Accordingly, oTP-1 has antiluteolytic biological activity.

The primary role of oTP-1 was assumed to be associated with the establishment of pregnancy.

oTP-1 was subsequently found to exhibit limited homology (50-70%) with interferon alphas (IFNa) of various species (Imakawa, et al., 1987), and (ii) bind to a Type I interferon receptor (Stewart, et al., 1987). Despite some similarities with IFNa, oTP-1 has several features that distinguish it from IFNa including the following: oTP-1's role in reproductive biochemistry (other interferons are not known to have any role in the biochemical regulation of reproductive cycles), oTP-1's cellular source trophoblast cells (IFNa is E:\KRH\6894S0SP.DOC

I

derived from lymphocytes cells), oTP-1's size 172 amino acids (IFNc is typically about 166 amino acids), and oTP-1 is weakly inducible by viruses (IFNa is highly inducible by viruses). The International Interferon Society recognizes oTP-1 as belonging to an entirely new class of interferons which have been named interferon-tau (IFN-c). The Greek letter stands for trophoblast.

The interferons have been classified into two distinct groups: type I interferons, including IFNa, IFNp, and IFNeo (also known as IFNall); and type II interferons, represented by IFNy (reviewed by DeMaeyer, et In humans, it is estimated that there are at least 17 IFNa non-allelic genes, at least about 2 or 3 IFNp non-allelic genes, and a single IFNy gene.

IFNa's has been shown to inhibit various types of cellular proliferation.

IFNa's are especially useful against hematologic malignancies such as hairyo* cell leukemia (Quesada, et al., 1984). Further, these proteins have also shown activity against multiple myeloma, chronic lymphocytic leukemia, low-grade lymphoma, Kaposi's sarcoma, chronic myelogenous leukemia, renal-cell carcinoma, urinary bladder tumors and ovarian cancers (Bonnem, et al., 1984; Oldham, 1985). The role of interferons and interferon receptors in the pathogenesis of certain autoimmune and inflammatory diseases has also been 20 investigated (Benoit, et al., 1993).

IFNa's are also useful against various types of viral infections (Finter, et al., 1991). Alpha interferons have shown activity against human papillomavirus infection, Hepatitis B, and Hepatitis C infections (Finter, et al., 1991; Kashima, et al., 1988; Dusheiko, et 1986; Davis, et al., 1989). Significantly, however, the usefulness of IFNa's has been limited by their toxicity: use of interferons in the treatment of cancer and viral disease has resulted in serious side effects, such as fever, chills, anorexia, weight loss, and fatigue (Pontzer, et al., 1991; Oldham, 1985). These side effects often require the interferon dosage to be reduced to levels that limit the effectiveness of treatment, or (ii) the removal of the patient from treatment. Such toxicity has reduced the usefulness of these potent antiviral and antiproliferative proteins in the treatment of debilitating E:<KRH689450SP.DOC 6 human and animal diseases.

Summary of the Invention In one aspect the present invention relates to novel isolated hybrid interferon proteins which are useful for inhibiting tumor cell growth. Two embodiments include the hybrid interferon proteins presented as either SEQ ID NO:2 or SEQ ID NO:4. A number of tumor cells can be targeted for growth inhibition by the hybrid interferon proteins of this application, these include but are not limited to the following: human carcinoma cells and steroid-affected tumor cells mammary tumor cells).

The present invention also includes a method of inhibiting viral replication in a cell. The replication of a number of viruses can be inhibited in cells, these viruses include RNA feline leukemia virus, human immunodeficiency virus, or Hepatitis C Virus) and DNA Hepatitis B Virus) viruses.

The isolated hybrid interferon proteins of the present invention can also be used in a method of enhancing fertility in a female mammal. In this method an amount of the proteins effective to enhance fertility of a female mammal is administered, typically in a pharmaceutically acceptable carrier. Exemplary of such molecules are the protein sequences presented as SEQ ID NO:4 and SEQ ID NO:12.

The present invention also includes an isolated nucleic acid which encodes the hybrid interferon of the present invention. Further, the invention includes expression vectors. Typically the expression vector includes a nucleic acid which encodes the hybrid interferon of the invention; and (b) regulatory sequences effective to express said in a host cell.

The invention includes a method of recombinantly producing the hybrid interferon proteins of the invention. In the method, a recombinant expression system containing an open reading frame (ORF) having a polynucleotide sequence which encodes the hybrid interferon, where the vector is designed to express the ORF in the host, is introduced into suitable host cells. The host is then cultured under conditions resulting in the expression of the ORF sequence.

E: (RHX689450SP.DOC 7 These and other objects and features of the invention will be more fully appreciated when the following detailed description of the invention is read in conjunction with the accompanying drawings.

Brief Description of the Figures Figure 1 presents the nucleic acid coding sequence of a synthetic gene of OvIFNT designed to include 19 unique restriction enzyme sites spaced evenly throughout the coding sequence.

Figure 2 shows the cloning strategy used for making a synthetic gene encoding OvIFNr.

Figure 3 shows a comparison of the predicted protein sequences of a human interferon-t gene and an ovine interferon-T gene. Divergent amino acids are indicated by presentation of the alternative amino acid on the line below the nucleic acid sequences.

Figure 4 presents data demonstrating that both OvIFNT and IFNa were able to drastically reduce growth of HL-60 cells.

Figure 5 presents data demonstrating that rHulFNa is cytotoxic and OvIFNt is not. In the figure, results of one of three replicate experiments are presented as mean viability SD.

Figure 6 presents the sequences of polypeptides derived from the IFNt 20 sequence.

Figure 7 presents the complete nucleic acid and amino acid sequence of an OvIFNr sequence.

Figure 8 presents data supporting the lack of cytotoxicity, relative to IFNa, when IFNT is used to treat peripheral blood mononuclear cells.

Figure 9 shows the results of treatment of a human cutaneous T cell lymphoma line, HUT 78, with IFNT.

Figure 10 shows the results of treatment of a human T cell lymphoma line, H9, with IFN-c.

Figure 11A presents data for the peptide inhibition, relative to FIV (feline immunodeficiency virus) replication, of polypeptides derived from OvlFNt with whole OvlFN-. Figure 11B presents data for the peptide inhibition, relative to E:KRH\89450SP.DOC 8 HIV (human immunodeficiency virus) replication, of polypeptides derived from OvIFN- with whole OvIFNr.

Figure 12 presents data demonstrating the inhibition of the antiviral activity of IFNT by IFNt-derived peptides.

Figure 13 presents data demonstrating the inhibition by IFNr-derived peptides of OvlFNt antiviral activity.

Figure 14 presents data demonstrating the inhibition by IFNt-derived peptides of bovine IFNa antiviral activity.

Figure 15 presents data demonstrating the inhibition by IFNt-derived peptides of human IFNa antiviral activity.

Figure 16 presents data evaluating the lack of inhibition by IFNT-derived peptides of bovine IFNy antiviral activity.

Figure 17 presents data demonstrating the anti-IFNr-derived peptide antisera inhibition of the antiviral activity of IFN-c.

15 Figure 18 presents data demonstrating the anti-IFNT-derived peptide antisera inhibition of the binding of radiolabeled IFNT to cells.

Brief Description of the Sequences SEQ ID NO:1 is the nucleotide sequence of a synthetic gene encoding ovine interferon-r (OvlFN-). Also shown is the encoded amino acid sequence.

20 SEQ ID NO:2 is an amino acid sequence of a mature OvlFN- protein.

SEQ ID NO:3 is a synthetic nucleotide sequence encoding a mature human interferon-- (HulFNr) protein.

SEQ ID NO:4 is an amino acid sequence for a mature HulFNT protein.

SEQ ID NO:5 is the amino acid sequence of fragment 1-37 of SEQ ID NO:2.

SEQ ID NO:6 is the amino acid sequence of fragment 34-64 of SEQ ID NO:2.

SEQ ID NO:7 is the amino acid sequence of fragment 62-92 of SEQ ID NO:2.

SEQ ID NO:8 is the amino acid sequence of fragment 90-122 of SEQ ID NO:2.

E:)KRH\689450SP.DOC I 9 SEQ ID NO:9 is the amino acid sequence of fragment 119-150 of SEQ ID NO:2.

SEQ ID NO:10 is the amino acid sequence of fragment 139-172 of SEQ ID NO:2.

SEQ ID NO:11 is the nucleotide sequence of a natural HulFNT gene with a leader sequence.

SEQ ID NO:12 is the predicted amino acid coding sequence of the SEQ ID NO:11.

SEQ ID NO:13 is a 25-mer synthetic oligonucleotide according to the subject invention.

SEQ ID NO:14 is a 25-mer synthetic oligonucleotide according the subject invention.

SEQ ID NO:15 is the amino acid sequence of fragment 1-37 of SEQ ID NO:4.

15 SEQ ID NO:16 is the amino acid sequence of fragment 34-64 of SEQ ID NO:4.

SEQ ID NO:17 is the amino acid sequence of fragment 62-92 of SEQ ID NO:4.

SEQ ID NO:18 is the amino acid sequence of fragment 90-122 of SEQ ID NO:4.

SEQ ID NO:19 is the amino acid sequence of fragment 119-150 of SEQ ID NO:4.

SEQ ID NO:20 is the amino acid sequence of fragment 139-172 of SEQ ID NO:4.

Interferons and greater than 70% amino acid homology to the sequence presented as SEQ ID NO:2. Interferon- can be isolated from a number of E:\KRH\689450SP.DOC mammalian sources as described below.

An interferon-- polypeptide is a polypeptide having between about and 172 amino acids derived from an interferon-T amino acid coding sequence, where said 15 to 172 amino acids are contiguous in native interferon-T. Such- 15-172 amino acid regions can also be assembled into polypeptides where two or more such interferon-T regions are joined that are normally discontinuous in the native protein.

II. Isolation and Characterization of Interferon-T.

A. Ovine and Bovine Interferon-T.

1. Interferon-T Coding Sequences.

Ovine interferon-- (OvIFNr) is a major conceptus secretory protein produced by the embryonic trophectoderm during the critical period of maternal recognition in sheep. One isolate of mature OvIFN- is 172 amino acids in length (SEQ ID NO:2). The cDNA coding sequence contains an additional 23 15 amino acids at the amino-terminal end of the mature protein (Imakawa, et al., 1987). The coding sequence of this OvIFNr isolate is presented as Figure 7.

For the isolation of OvlFN-r protein, conceptuses were collected from pregnant sheep and cultured in vitro in a modified Minimum Essential Medium as described previously (Godkin, et al., 1982). Conceptuses were collected on 20 various days of pregnancy with the first day of mating being described as Day .i 0. IFNr was purified from conceptus culture medium essentially as described by Vallet, et al., (1987) and Godkin, et al. (1982).

The homogeneity of IFNr was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Maniatis, et al.; Ausubel, et Determination of protein concentration in purified IFN- samples was performed using the bicinchoninic (BCA) assay (Pierce Chemical Co., Rockford, IL; Smith, et al., 1985).

A homologous protein to OvIFNr was isolated from cows (blFNr; Helmer, et al., 1987; Imakawa, et al., 1989). OvlFN- and BolFNt have similar functions in maternal recognition of pregnancy, and (ii) share a high degree of amino acid and nucleotide sequence homology between mature proteins. The E:KRH\689450SP.DOC 11 nucleic acid sequence homology between OvlFNT and BolFNT is 76.3% for the non-coding region, 89.7% for the coding region, and 91.9% for the 3' noncoding region. The amino acid sequence homology is 80.4%.

Example 1 describes the reproductive functions of OvIFNt. OvIFNr and recombinant human a-2-lnterferon (rHulFNCa 2 were infused into uterine lumen of ewes at a variety of concentrations. The life span of the corpus luteum was assessed by examination of interestrous intervals, maintenance of progesterone secretion, and inhibition of prostaglandin secretion (Davis, et al., 1992). Comparison of the data resulting from these examinations demonstrated a considerable lengthening of the interestrous interval when IFNt is administered at 100g/day and no meaningful effect when rHulFNa is administered. These data support the conclusion that IFNT significantly influences the biochemical events of the estrous cycle.

The antiviral properties of interferon-T at various stages of the 15 reproductive cycle were also examined (Example Conceptus cultures were established using conceptus obtained from sheep at days 12 through 16 of the estrus cycle. Antiviral activity of supernatant from each conceptus culture was assessed. Culture supernatants had increasing antiviral activity associated with advancing development of the conceptus up to Day 16 post estrus.

2. Recombinant Production of IFNT.

Recombinant IFNT was produced using bacterial and yeast cells. The amino acid coding sequence for OvIFNT was used to generate a corresponding DNA coding sequence with codon usage optimized for expression in E. coli (Example The DNA coding sequence was synthetically constructed by sequential addition of oligonucleotides. Cloned oligonucleotides were fused into a single polynucleotide using the restriction digestions and ligations outlined in Figure 2. The polynucleotide coding sequence had the sequence presented as SEQ ID NO:1.

For expression of recombinant IFN, this synthetic coding sequence can be placed in a number of bacterial expression vectors: for example, lambda gt11 (Promega, Madison WI); pGEX (Smith, et pNH vectors (Stratagene, E:AKRH689450SP.DOC 12 La Jolla CA). Cloning of the IFNr synthetic polynucleotide into a modified pIN III omp-A expression vector is described in Example 3. Production of the IFNprotein was induced by the addition of IPTG. Soluble recombinant IFN- was liberated from the cells by sonication or osmotic fractionation.

The protein can be further purified by standard methods, including size fractionation (column chromatography or preoperative gel electrophoresis) or affinity chromatography (using, for example, anti-IFNt antibodies (solid support available from Pharmacia, Piscataway NJ). Protein preparations can also be concentrated by, for example, filtration (Amicon, Danvers, Mass.).

The synthetic IFNr gene was also cloned into the yeast cloning vector oo pBS24Ub (Example 4; Sabin, et al.; Ecker, et Synthetic linkers were constructed to permit in-frame fusion of the IFN coding sequences with the 0 ubiquitin coding sequences in the vector. The resulting junction allowed in vivo *0 1 cleavage of the ubiquitin sequences from the IFNT sequences.

The recombinant plasmid pBS24Ub-IFNT was transformed into the yeast S. cerevisiae. Transformed yeast cells were cultured, lysed and the recombinant IFNT (r-IFN) protein isolated from the cell lysates.

The amount of r-IFNT was quantified by radioimmunoassay.

0 Microsequencing of the purified r-IFNT was carried out. The results 20 demonstrated identity with native IFNT through the first 15 amino acids. The results also confirmed that the ubiquitin/IFNT fusion protein was correctly processed in vivo.

Recombinant IFNT obtained by this method exhibited antiviral activity similar to the antiviral activity of IFNT purified from conceptus-conditioned culture medium.

Other yeast vectors can be used in the practice of the present invention including, but are not limited to, vectors with regulatable expression (Hitzeman, et al.; Rutter, et al.; Oeda, et The yeast transformation host is typically Saccharomyces cerevisiae, however, other yeast suitable for transformation can be used as well Schizosaccharomyces pombe).

The DNA encoding the IFNT polypeptide can be cloned into any number E:\KRH\689450SP.DOC 13 of commercially available vectors to generate expression of the polypeptide in the appropriate host system. These systems include the above described bacterial and yeast expression systems as well as the following: baculovirus expression (Reilly, et al.; Beames, et al.; Clontech, Palo Alto CA); and expression in mammalian cells (Clontech, Palo Alto CA; Gibco-BRL, Gaithersburg MD). These recombinant polypeptides can be expressed as fusion proteins or as native proteins. A number of features can be engineered into the expression vectors, such as leader sequences which promote the secretion of the expressed sequences into culture medium. The recombinantly produced polypeptides are typically isolated from lysed cells or culture media.

Purification can be carried out by methods known in the art including salt fractionation, ion exchange chromatography, and affinity chromatography.

Immunoaffinity chromatography can be employed, as described above, using antibodies generated based on the IFN- polypeptides.

B. Human Interferon-T.

1. Identification and Cloning of Human Genomic Sequences Encoding an Interferon- Protein.

DNA was screened for sequences homologous to interferon-r (Example Several sequences that hybridized with the OvlFNt cDNA probe were identified. Several clones containing partial sequences of human interferonwere then isolated (Example Two synthetic 25-mer oligonucleotides, corresponding to sequences from the OvIFNr cDNA (Imakawa, et al., 1987) were synthesized. These primers were employed in amplification reactions using DNA derived from the following two cDNA libraries: human term placenta and human term cytotrophoblast. The resulting amplified DNA fragments were electrophoretically separated and a band containing an IFNt amplification product was isolated. The product was subcloned and the inserted amplification product sequenced using the dideoxy termination method.

Comparison of sequences from three of the clones revealed a high degree of sequence homology between the isolates, but the sequences were not identical. This result suggests the existence of multiple variants of human interferon-T genes.

E:\KRH589450SP.DOC 14 Example 7 describes the isolation of a full-length human IFNT gene.

High molecular weight DNA was isolated from peripheral blood mononuclear cells (PBMCs) and size-fractionated. Fractions were tested for the presence of IFNT sequences using polymerase chain reaction: DNA molecules from fractions that tested amplification positive were used to generate a subgenomic library in Xgt11.

This subgenomic library was plated and hybridized with an OVIFNT cDNA probe (Example 7A). Approximately 20 clones were identified that hybridized to the probe. Plaques corresponding to the positive clones were passaged, DNA isolated and analyzed by amplification reactions using OvIFN- primers. Of ::these twenty plaques, six plaques generated positive PCR signals. The phage from these six clones were purified and the inserts sequenced. One of the inserts from one of these six clones was used as a hybridization probe in the following screening.

15 Recombinant phage from the 2gtll subgenomic library were screened using the hybridization probe just described (Example 7B). Three clones giving positive hybridization signals were isolated and the inserts sequenced. The resulting nucleic acid sequence information is presented as SEQ ID NO:11 and the predicted protein coding sequence is presented as SEQ ID NO:12. The predicted mature protein coding sequence is presented as SEQ ID NO:4.

Comparison of the predicted protein sequences (Figure 3) of the human interferon-T gene (SEQ ID NO:4) and the ovine interferon- gene demonstrates the levels of sequence homology and divergence at the amino acid level.

The human IFNT sequences presented as SEQ ID NO:12 and SEQ ID NO:11, and primers and probes derived therefrom, can be used as specific probes to detect isolates of further human IFNt coding sequences and/or pseudogenes. Further, there may be more than one isoform of the IFN' protein and more than one coding sequence per species. The specific nucleic acid probes used in the practice of the present invention and antibodies reactive with the IFNt polypeptides of the present invention may be useful to isolate unidentified variants of interferon-- in mammals, according to the methods of E:AKRH\689450SP.DOC the invention disclosed herein.

2. Characterization of the Expression of Interferon-T in Human Tissues.

Human placental cDNA libraries and an ovine cDNA library were analyzed by hybridization to the OvIFN- cDNA probe (Example This DNA hybridization analysis suggested that the IFNt-signals from human cDNA libraries were approximately 1/100 of the signal obtained using the ovine cDNA library. OvlFNt cDNAs constitute around 0.4% of the ovine cDNA library.

Accordingly, the abundance of human cDNAs responding to the OvIFN' probe 10 appears to be low, at least in the term placenta from which the cDNA libraries were generated.

The presence of HulFNt mRNA in human term placenta and amniocytes were also analyzed. The results suggest the presence of human IFN mRNA in the feto-placental annex. The aminocytes also expressed the messages correi15 sponding to OvlFNt primers and human probe, suggesting that the expression of IFNr mRNA is not limited to the term placenta.

In addition, a RT-PCR analysis for the presence of HulFNr was applied to the total cellular RNA isolated from human adult lymphocytes: the results suggest that IFNT mRNA exists in lymphocytes.

20 The expression of interferon-r in human tissue was also examined using in situ hybridization (Example Sections from four healthy, different term and first trimester human placentas were examined. This analysis employed a cDNA probe derived from the OvIFNt cDNA sequences (Example 9B). In situ hybridization was performed using an anti-sense RNA probe. In three separate experiments, specific hybridization was observed in all term and first trimester placental tissues.

First trimester placental villi (composed of an outer layer of syncytiotrophoblast, an underlying layer of cytotrophoblast, and a central stromal region with various types of mesenchymal cells) displayed the highest transcript level of IFNT in the cytotrophoblast cells. Less intense but detectable levels were present in both the syncytiotrophoblast and stromal cells. A similar E:AKRH\689450SP.DOC 16 pattern of transcript expression was demonstrated in the placental villi of term tissue but the level of signal detection was low. First trimester extravillous trophoblasts displayed the highest amount of message and stained positive when present in the maternal blood spaces.

Howatson, et al., (1988) noted IFNa production in the syncytiotrophoblast of chorionic villi in both first trimester and term tissues.

Also, Paulesu, et al. (1991) observed IFNa in extravillous trophoblast as well as syncytiotrophoblasts, noting more intense and abundant reactivity in first trimester placental tissue when compared to those taken at term. These investigators employed antibodies raised against human IFNa subtypes, and none observed any IFNa in the villous cytotrophoblasts.

The present results demonstrate that the human IFNr gene is highly expressed in early placental tissues by migrating extravillous trophoblasts, but is also expressed in villous syncytiotrophoblasts, cytotrophoblasts, and various stromal cells. These results demonstrate the detection of IFNt transcripts in human pregnancy tissues, and IFN- expression in the villous cytotrophoblasts as well as the extravillous trophoblast of first trimester placenta.

C. Antiviral Properties of Interferon-r.

The antiviral activity of IFNT has been evaluated against a number of 20 viruses, including both RNA and DNA viruses. The relative specific activity of *OvIFNr, purified to homogeneity, was evaluated in antiviral assays (Example IFNT had a higher specific antiviral activity than either rBolFNa or rBolFNy (Example 10, Table 3).

One advantage of the present invention is that IFNT has potent antiviral activity with limited cytotoxic effects. Highly purified OvIFNt was tested for antiretroviral and cytotoxic effects on peripheral blood lymphocytes exposed to feline AIDS and human AIDS retroviruses (Bazer, et al., (1989)). This feline AIDS lentivirus produces a chronic AIDS-like syndrome in cats and is a model for human AIDS (Pederson, et al., 1987). Replication of either virus in peripheral blood lymphocytes (PBL) was monitored by reverse transcriptase (RT) activity in culture supernatants over time.

E:KRH689450SP.DOC To determine IFNT antiviral activity against FIV and HIV, RNA-dependent DNA polymerase RT activity was assayed in FIV- and HIV-infected feline and human PBL cultures treated with IFNt (Example 11). Replication of FIV was reduced to about one-third of control values when cells were cultured in the presence of IFNr. Addition of OvIFN- produced a rapid, dose-dependent decrease in reverse transcriptase (RT) activity (Example 11, Table While concentrations as low as 0.62 ng/ml of IFNT inhibited viral replication, much higher concentrations (40 ng/ml) having greater effects on RT-activity were without toxic effects on the cells. The results suggest that replication of the S 10 feline immunodeficiency virus was reduced significantly compared to control values when cells were cultured in the presence of OvIFNr.

IFNT appeared to exert no cytotoxic effect on the cells hosting the S. retrovirus. This was true even when IFNT was present at 40 ng per ml of culture medium. This concentration of IFNT is equivalent to about 8,000 anti- 15 viral units of alpha interferon, when IFNr is assayed for its ability to protect Madin-Darby bovine kidney cells from lysis by vesicular stomatitis virus as described by Pontzer, et al. (1988).

IFNT was also tested for activity against HIV replication in human cells.

Human peripheral lymphocytes, which had been infected with HIV were treated 20 with varying concentrations of IFN- (Example 12). Replication of HIV in peripheral blood lymphocytes was monitored by reverse transcriptase activity in culture supernatants over time. Over a range of concentrations of IFN- produced significant anti-HIV effects (Example 12, Table A concentration of only 10 ng/ml resulted in over a 50% reduction in RT activity after only six days.

A concentration of 500 ng/ml resulted in a 90% reduction in RT activity within days. Further, there was no evidence of any cytotoxic effects attributable to the administration of IFNT (Example 12, Table Further, the antiviral effects of IFNT against HIV were evaluated by treating human PBMC cells with various amounts of either recombinant IFN- or recombinant human IFNc 2 at the time of infection with HIV (Example 18). The data from these experiments (Example 18, Table 12) support the conclusion E:\KRH689450SP.DOC 18 that, at similar concentrations, IFNc and IFN- are effective in reducing the replication of HIV in human lymphocytes. However, treatment of cells with IFNa2 resulted in cytoxicity, whereas.no such cytotoxity was observed with treatment using IFNT, even when IFNT was used at much higher concentrations. No cytotoxicity was observed using IFNT, even when IFNT was used at 200 times the dosage of interferon-alpha II.

Both FIV and HIV reverse transcriptase themselves were unaffected by IFNT in the absence of PBL. Therefore, the antiviral activity is not due to a direct effect on the viral RT.

Interferon-T has also been shown to inhibit Hepatitis B Virus DNA replication in hepatocytes (Example 18). A human cell derived from liver cells transfected with Hepatitis B Virus (HBV) was used to test the antiviral effects of l: IFNT. The cells were treated with both the IFNo and IFNT over a range of concentrations. Both IFNa and IFNT reduced DNA production by approximately 15 two-fold compared to the no interferon control.

To demonstrate that the effect of the interferons was specific to the infecting virus and not the result of an effect on general cell metabolism, the hepatocyte was examined for the effects of IFNa and IFNT on hepatospecific mRNA production (Example 18). Two hepatocyte specific proteins, Apo E and Apo A1, were detected by hybridization analysis. There was no apparent reduction of mRNA production for either hepatospecific mRNA at concentrations up to 40,000 units/ml of either IFNa or IFNr. Further, no evidence for hepatotoxicity with IFNr was seen in this assay.

These results suggest that IFNr is an effective antiviral agent against a wide variety of viruses, including both RNA and DNA viruses. One advantage of IFN- over other interferons, such as IFNa, is that treatment with IFNT does not appear to be associated with any cytotoxicity.

D. Antiproliferative Properties of IFNT.

The effects of IFNT on cellular growth have also been examined. In one analysis, anti-cellular growth activity was examined using a colony inhibition assay (Example 13). Human amnion (WISH) or MDBK cells were plated at low E:\KRH\689450SP.DOC 19 cell densities to form colonies originating from single cells. Dilutions of interferons were added to triplicate wells and the plates were incubated to allow colony formation. IFNT inhibited both colony size and number in these assays.

IFNT was more effective at inhibiting cell proliferation of the human cell line (WISH) than human IFNa. The antiproliferative activity of IFNr was dosedependent. High concentrations of IFNr stopped proliferation, while cell viability was not impaired.

Based on cell cycle analysis, using flow cytometry, IFNt appears to inhibit progress of cells through S phase. These results demonstrate the antiproliferative effect of IFN-, and underscore its low cytotoxicity.

The antiproliferative effects of IFNr were also studied for rat and bovine cell lines (Example 14). The rate of 3 H-thymidine incorporation was used to assess the rate of cellular proliferation. The data obtained demonstrate that IFNl drastically reduced the rate of cellular proliferation (Example 14, Table 7) 15 for each tested cell line.

The antiproliferative activity and lack of toxicity of IFN- was further examined using a series of human tumor cell lines (Example 15). A variety of human tumor cell lines were selected from the standard lines used in NIH screening procedure for antineoplastic agents (Pontzer, et al., (1991)). At least one cell line from each major neoplastic category was examined.

The following cell lines were obtained from American Type Culture Collection (12301 Parklawn Dr., Rockville MD 20852): NCI-H460 human lung large cell carcinoma; DLD-1 human colon adenocarcinoma; SK-MEL-28 human malignant melanoma; ACHN human renal adenocarcinoma; human promyelocytic leukemia; H9 human T cell lymphoma; HUT 78 human cutaneous T cell lymphoma; and MCF7 human breast adenocarcinoma.

As above, the antiproliferative activity was evaluated by measuring the E:\KRH689450SP.DOC rate of 3 H-thymidine incorporation into cells which have been treated with IFN-.

Significant differences between treatments were assessed by an analysis of variance followed by Scheffe's F-test. Cell cycle analysis was performed by flow cytometry.

Examination of IFNt inhibition of MCF7 (breast adenocarcinoma) proliferation demonstrated that IFNr reduced MCF7 proliferation in a dosedependent manner. A 50% reduction in 3 H-thymidine was observed with 10,000 units/ml of IFNt (Example 15, Table This cell line had previously been found to be unresponsive to anti-estrogen treatment.

A comparison of the antiproliferative effects of IFNt and IFNa was conducted using HL-60 (human promyelocytic leukemia) cells. Results with the promyelocytic leukemia HL-60 are typical of those obtained comparing IFNt Swith human IFNa (Example 15). Concentrations as low as 100 units/ml of both IFNs produced significant 60%) growth reduction. Increasing amounts of 15 IFNs further decreased tumor cell proliferation (Figure At high doses of IFNa, but not IFNr, was cytotoxic (Figure Cell viability was reduced by approximately 80% by IFN. By contrast, nearly 100% of the IFNr-treated cells 4* remained viable when IFNt was applied at 10,000 units/ml. Thus, while both interferons inhibit proliferation, only IFNt is without cytotoxicity. This lack of 20 toxicity provides an advantage of IFNT for use in vivo therapies.

The human cutaneous T cell lymphoma, HUT 78, responded similarly to when treated with IFNT (Example 15, Figure Both OvIFNr and rHulFNa reduce HUT 78 cell growth, but IFNa demonstrated adverse effects on cell viability.

The T cell lymphoma H9 was less sensitive to the antiproliferative effects of IFN than the tumor cell lines described above. While IFNa was not toxic to the H9 cells, it failed to inhibit cell division significantly at any of the concentrations examined (Example 15, Figure 10). In contrast, IFNT was observed to reduce H9 growth by approximately 60%. Thus, only OvlFNT is an effective growth inhibitor of this T cell lymphoma.

In three additional tumor cell lines (NCI-H460, DLD-1 and SK-MEL-28) E.\KRH\689450SP.DOC IFN and IFNa were equally efficacious antitumor agents. In the melanoma, SK-MEL-28, inhibition of proliferation by IFNa was accomplished by a 13% drop in viability, while IFNr was not cytotoxic. In the majority of tumors examined, IFNr is equal or preferable to IFNa as an antineoplastic agent against human tumors.

IFNr exhibits antiproliferative activity against human tumor cells without toxicity and is as potent or more potent than human IFNa. Clinical trials of the IFNa2s have shown them to be effective antitumor agents (Dianzani, 1992; Krown, 1987). One therapeutic advantage of IFNr as a therapeutic is the 0 elimination of toxic effects seen with high doses IFNas.

An additional application of the IFNr is against tumors like Kaposi's •sarcoma (associated with HIV infection) where the antineoplastic effects of IFNt S are coupled with IFNr ability to inhibit retroviral growth.

The in vivo efficacy of interferon-t treatment was examined in a mouse 15 system (Example 16). B16-F10 is a syngeneic mouse transplantable tumor selected because of its high incidence of pulmonary metastases (Poste, et al., 1981). Interferon treatment was initiated 3 days after the introduction of the tumor cells. The in vivo administration of IFNT dramatically reduced B16-F10 pulmonary tumors. Thus, IFNr appears to be an efficacious antineoplastic 20 agent in vivo as well as in vitro.

III. Interferon-T Polypeptide Fragments, Protein Modeling and Protein Modifications.

The variety of IFN- activities, its potency and lack of cytotoxicity, as taught by the present specification, suggest the importance of structure/function analysis for this novel interferon. The structural basis for OvIFNt function has been examined using six overlapping synthetic peptides corresponding to the entire OvlFN- sequence (Figure The corresponding polypeptides derived from the ovine IFNt sequence are presented as SEQ ID NO:15 to SEQ ID Three peptides representing amino acids 1-37, 62-92 and 139-172 have been shown to inhibit IFNc antiviral activity (Example 17). The peptides were effective competitors at concentrations of 300 p M and above.

E:XKRH1689450SP.DOC The C-terminal peptide of IFNT, OvIFN- (139-172), and the internal peptide OvlFNT (62-92), inhibited IFNT and rBolFNal antiviral activity to the same extent, while the N-terminal peptide OvIFNT (1-37) was more effective in inhibiting OvlFNt antiviral activity. Dose-response data indicated that IFNr (62- 92) and IFN (139-172) inhibited IFNT antiviral activity to similar extents. The same peptides that blocked IFNu antiviral activity also blocked the antiviral activity of recombinant bovine IFNa (rBolFNa); recombinant bovine IFNy was unaffected by the peptides. These two IFNr peptides may represent common receptor binding regions for IFNr and various IFNas.

S 10 The two synthetic peptides OvlFN-(1-37) and OvlFNt(139-172) also blocked OvIFN- anti-FIV and anti-HIV activity (Example 17; Figures 11A and 11B). While both peptides blocked FIV RT activity, only the C-terminal peptide, OvlFNr(139-172), appeared to be an efficient inhibitor of vesicular stomatitis virus activity on the feline cell line, Fc9.

15 The above data taken together suggest that the C-terminal regions of type I interferons may bind to common site on the type I interferon receptor, while the N-terminal region may be involved in the elicitation of unique functions. These results suggest that portions of the IFNT interferon molecule may be used to substitute regions of interferon alpha molecules. For example, 20 the region of an interferon alpha molecule that is responsible for increased a.o cytotoxicity, relative to IFNT treatment, can be identified by substituting polypeptide regions derived from IFN- for regions of an interferon alpha molecule. Such substitutions can be carried out by manipulation of synthetic genes (see below) encoding the selected IFNt and interferon alpha molecules, coupled to the functional assays described herein (such as, antiviral, antiproliferative and cytoxicity assays).

Polyclonal anti-peptide antisera against the IFNt peptides yielded similar results as the polypeptide inhibition studies, described above. Antibodies directed against the same three regions (OvIFNt IFNt (62-92) and IFNr (139-172)) blocked OvIFN- function, confirming the importance of these three domains in antiviral activity (Example 17). These peptides, although apparently E:\KRH\689450SP.DOC 23 binding to the interferon receptor, did not in and of themselves elicit interferonlike effects in the cells.

The antiproliferative activity of IFNr (Example 17, Table 11) involved a further region of the molecule, since IFN-(119-150) was the most effective inhibitor of OvIFNT-induced reduction of cell proliferation. This results suggests that the region of the molecule primarily responsible for inhibition of cell growth is the IFNT(119-150) region. This region of the IFN- molecule may be useful alone or fused to other proteins (such as serum albumin, an antibody or an interferon alpha polypeptide) as an antineoplastic agent. A conjugated protein between an N-terminal peptide derived from human interferon-a and serum albumin was shown to have anticellular proliferation activity (Ruegg, et al., 1990).

Finally, binding of 1251-OvlFN to its receptor on MDBK cells could be blocked by antisera to 4 of the 6 peptides; the 4 polypeptides representing 15 amino acids 1-37, 62-92, 119-150 and 139-172 of OvlFNc. This reflects the multiple binding domains as well as the functional significance of these regions.

Since different regions of IFNT are involved in elicitation of different functions, modification of selected amino acids could potentially result in IFNT-like interferons with selective biological activity.

20 The above data demonstrate the identification of synthetic peptides having four discontinuous sites on the OvIFNT protein that are involved in receptor interaction and biological activity. In order to elucidate the structural relationship of these regions, modeling of the three dimensional structure of IFNT was undertaken. A three dimensional model would be useful in interpretation of existing data and the design of future structure/function studies.

Combining circular dichroism (CD) of both the full length recombinant OvIFNt to IFNp (a protein of known three dimensional structure (Senda, et al., 1992)), a model of OvIFNT has been constructed. The most striking feature of this model is that IFNr falls into a class of proteins with a four-helix bundle motif. The CD spectra of IFNt was taken on an AVIV 60 S spectropolarimeter.

E:\KRH689450SP.DOC Two different methods were employed for secondary structure estimations, the algorithm of Perczel, et al., (1991) and variable selection by W.C. Johnson, Jr.

(1992).

Secondary structure estimations of the spectra indicate 70-75% alpha helix (characterized by minima at 222 and 208 nm and maximum at 190 nm).

The variable selection algorithm estimates the remainder of the molecule to be beta sheet and 10% turn. The Chang method estimates the remainder to be 30% random coil. Alignment of IFNr and IFNp sequences revealed homology between the two molecules, specifically in the regions of known helical structure in IFNp. Sequence analysis of IFN- also showed that proposed helical regions possess an apolar periodicity indicative of a four-helix bundle motif.

The final modeling step was to apply the IFNp x-ray crystallographic coordinates of the IFNp carbon backbone to the IFNT sequence. The 15 functionally active domains of IFNt, identified above, were localized to one side of the molecule and found to be in close spatial proximity. This is consistent with multiple binding sites on IFNr interacting simultaneously with the type I IFN receptor.

The three dimensional modeling data coupled with the function data 20 described above, provides the ability to introduce sequence variations into specific regions of IFNr to generate enhancement of selected functions antiviral or anticellular proliferation) or the ability to substitute a region(s) of selected function into other interferon molecules antiviral, antineoplastic, or reduced cytotoxicity).

The construction of a synthetic gene for OvlFNt is described in Example 3. Briefly, a consensus amino sequence was back-translated using optimal codon usage for E. coli. The sequence was edited to include 20, unique, restriction sites spaced throughout the length of the construct. This 540 base pair synthetic gene sequence was divided into 11 oligonucleotide fragments.

Individual fragments were synthesized and cloned, either single or double stranded, into either pTZ 19R, pTZ 18R or pBluescript, amplified and fused.

E:KRH\689450SP.DOC The synthetic OvIFNr construct was then cloned into a modified plN-Ill-ompA expression vector for expression in bacteria and also cloned into a yeast expression plasmid. A similarly constructed human IFNr synthetic gene (SEQ ID NO:3) has been designed, constructed and expressed in yeast cells.

Expression of the OvIFNt synthetic gene in yeast (Example 4) allowed over production of recombinant IFNT in S. cerevisiae: large quantities (5-20 mg/1) of recombinant IFNr can be purified from soluble yeast extract using sequential ion exchange and molecular sieve chromatography. Recombinant IFNT purified in this fashion exhibited potent antiviral activity (2 to 3 X 108 B 10 units/mg) similar to native OvIFNr.

The synthetic gene construct facilitates introduction of mutations for possible enhancement of antitumor (anticellular proliferative) and antiviral activities. Further, the disparate regions of the molecule responsible for Bo different functions allows for separate manipulation of different functions. For i. 15 example, two deletion mutants, OvlFNT(1-155) and OvlFNt(1-166), have been constructed to examine the role of carboxy terminal sequences in IFN' molecules.

C ~Additional mutant IFNT molecules have been constructed to identify residues critical for antiproliferative activity. For example, one particular 20 residue, Tyr 123 has been implicated in the anticellular proliferative activity of O* IFNa (Mclnnes, et al., 1989). The equivalent of Tyr 123 in IFNT is contained within peptide OvlFNT(119-150): this polypeptide inhibits OvIFNT and human IFN antiproliferative activity. Mutations converting Tyr 123 to conservative (Trp) and nonconservative (Asp) substitutions have been generated, as well as mutant sequences having deletion of this residue. The codon for Tyr 123 is located within an Sspl site; elimination of this site has been used for screening.

The antiproliferative activity of these mutant IFNt is evaluated as described herein.

Synthetic peptides can be generated which correspond to the IFNt polypeptides of the present invention. Synthetic peptides can be commercially synthesized or prepared using standard methods and apparatus in the art E:\KRH\689450SP.DOC 1 (Applied Biosystems, Foster City CA).

Alternatively, oligonucleotide sequences encoding peptides can be either synthesized directly by standard methods of oligonucleotide synthesis, or, in the case of large coding sequences, synthesized by a series of cloning steps involving a tandem array of multiple oligonucleotide fragments corresponding to the coding sequence (Crea; Yoshio et al.; Eaton et Oligonucleotide coding sequences can be expressed by standard recombinant procedures (Maniatis et al.; Ausubel et al.).

The biological activities of the interferon-T polypeptides described above can be exploited using either the interferon-T polypeptides alone or conjugated with other proteins (see below).

IV. Production of Fusion Proteins.

Interferon- or interferon-r-derived polypeptides may be covalently attached to a second polypeptide to form a fused, or hybrid, protein. The interferon-- sequences making up fused proteins can be recombinantly produced interferon-T or a bioactive portion thereof, as described above.

For example, where interferon-T is used to inhibit viral expression, the polypeptides presented as SEQ ID NO:10 and SEQ ID NO:20 may be advantageously fused with a soluble peptide, such as, serum albumin, an antibody specific against an virus-specific cell surface antigen), or an interferon alpha polypeptide. Other examples of fusion proteins include (i) replacing toxicity-associated regions of interferon-a with the interferon-T regions SEQ ID NO:5 and SEQ ID NO:15, and (ii) fusion proteins containing the interferon-T regions SEQ ID NO:9 and SEQ ID NO:19 as anticellular proliferation agents.

The fused proteins may be formed by chemical conjugation or by recombinant techniques. In the former method, the interferon-T and second selected polypeptide are modified by conventional coupling agents for covalent attachment. In one exemplary method for coupling soluble serum albumin to an interferon-T polypeptide, serum albumin is derivatized with N-succinimidyl-Sacetyl thioacetate (Duncan), yielding thiolated serum albumin. The activated E:\KRH\~89450SP.DOC serum albumin polypeptide is then reacted with interferon- derivatized with Nsuccinimidyl 3-(2-pyridyldithio) propionate (Cumber), to produce the fused protein joined through a disulfide linkage.

As an alternative method, recombinant interferon-c may be prepared with a cysteine residue to allow disulfide coupling of the interferon-- to an activated ligand, thus simplifying the coupling reaction. An interferon-c expression vector, used for production of recombinant interferon-z, can be modified for insertion of an internal or a terminal cysteine codon according to standard methods of sitedirected mutagenesis (Ausubel, et al.).

In one method, a fused protein is prepared recombinantly using an expression vector in which the coding sequence of a second selected polypeptide is joined to the interferon-- coding sequence. For example, human serum albumin coding sequences can be fused in-frame to the coding sequence of an interferon- polypeptide, such as, SEQ ID NO:9. The fused protein is then expressed using a suitable host cell. The fusion protein may be .purified by molecular-sieve and ion-exchange chromatography methods, with additional purification by polyacrylamide gel electrophoretic separation and/or HPLC chromatography, if necessary.

It will be appreciated from the above how interferon-T-containing fusion 20 proteins may be prepared. One variation on the above fusion is to exchange positions of the interferon-c and selected second protein molecules in the fusion protein carboxy terminal versus amino terminal fusions). Further, internal portions of a native interferon-- polypeptide (for example, amino acid regions of between 15 and 172 amino acids) can be assembled into polypeptides where two or more such interferon- portions are contiguous that are normally discontinuous in the native protein.

E:\KRH\689450SP.DOC V. Antibodies Reactive with Interferon--.

Fusion proteins containing the polypeptide antigens fused with the glutathione-S-transferase (Sj26) protein can be expressed using the pGEX-GLI vector system in E. coli JM101 cells. The fused Sj26 protein can be isolated readily by glutathione substrate affinity chromatography (Smith). Expression and partial purification of IFNt proteins is described in (Example 20), and is applicable to any of the other soluble, induced polypeptides coded by sequences described by the present invention.

Insoluble GST (sj26) fusion proteins can be purified by preparative gel electrophoresis.

Alternatively, IFNt-p-galactosidase fusion proteins can be isolated as described in Example 19.

Also included in the invention is an expression vector, such as the lambda gt11 or pGEX vectors described above, containing IFNt coding sequences and expression control elements which allow expression of the coding regions in a suitable host. The control elements generally include a promoter, translation initiation codon, and translation and transcription termination sequences, and an insertion site for introducing the insert into the vector.

20 The DNA encoding the desired polypeptide can be cloned into any .0" number of vectors (discussed above) to generate expression of the polypeptide in the appropriate host system. These recombinant polypeptides can be expressed as fusion proteins or as native proteins. A number of features can be engineered into the expression vectors, such as leader sequences which promote the secretion of the expressed sequences into culture medium.

Recombinantly produced IFNr, and polypeptides derived therefrom, are typically isolated from lysed cells or culture media. Purification can be carried out by methods known in the art including salt fractionation, ion exchange chromatography, and affinity chromatography. Immunoaffinity chromatography can be employed using antibodies generated against selected IFNt antigens.

Specific antibodies directed against the polypeptides described above E:\KRH\689450SP.DOC can also be prepared. Typically, to prepare antibodies, a host animal, such as a rabbit, is immunized with the purified antigen or fused protein antigen.

Hybrid, or fused, proteins may be generated using a variety of coding sequences derived from other proteins, such as P-galactosidase or glutathione- S-transferase. The host serum or plasma is collected following an appropriate time interval, and this serum is tested for antibodies specific against the antigen. Example 20 describes the production of rabbit serum antibodies which are specific against the IFNt antigens in a Sj26/IFNT hybrid protein. These techniques can be applied to the all of the IFNr molecules and polypeptides derived therefrom.

The gamma globulin fraction or the IgG antibodies of immunized animals can be obtained, for example, by use of saturated ammonium sulfate or DEAE Sephadex, or other techniques known to those skilled in the art for producing i* polyclonal antibodies.

Alternatively, purified protein or fused protein may be used for producing monoclonal antibodies. Here the spleen or lymphocytes from a animal immunized with the selected polypeptide antigen are removed and immortalized or used to prepare hybridomas by methods known to those skilled in the art (Harlow, et Lymphocytes can be isolated from a peripheral blood sample. Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes or a fusion partner can be used to produce hybridomas.

Antibodies secreted by the immortalized cells are screened to determine the clones that secrete antibodies of the desired specificity, for example, by using the ELISA or Western blot method (Ausubel et Experiments performed in support of the present invention have yielded four hybridomas producing monoclonal antibodies specific for ovine IFNt have been isolated.

Antigenic regions of polypeptides are generally relatively small, typically 7 to 10 amino acids in length. Smaller fragments have been identified as antigenic regions. Interferon-- polypeptide antigens are identified as described above. The resulting DNA coding regions can be expressed recombinantly E:V(RH\68945SPDOC either as fusion proteins or isolated polypeptides.

In addition, some amino acid sequences can be conveniently chemically synthesized (Applied Biosystems, Foster City CA). Antigens obtained by any of these methods may be directly used for the generation of antibodies or they may be coupled to appropriate carrier molecules. Many such carriers are known in the art and are commercially available Pierce, Rockford IL).

Antibodies reactive with IFNt are useful, for example, in the analysis of structure/function relationships.

VI. Utility A. Reproductive.

Although IFNt bears some similarity to the IFNa family based on structure and its potent antiviral properties, the IFNas do not possess the reproductive properties associated with IFNt. Also, recombinant bovine IFNa has little or no effect on interestrous interval compared to IFN- (Davis, et al., 1992).

Therefore, although IFNt has some structural similarities to other interferons, it has very distinctive properties of its own: for example, the capability of significantly influencing the biochemical events of the estrous cycle.

The human IFNT of the present invention can be used in methods of

S.

enhancing fertility and prolonging the life span of the corpus luteum in female o.

mammals as generally described in Hansen, et al., herein incorporated by reference. Further, the human interferon-r of the present invention could be used to regulate growth and development of uterine and/or fetal-placental tissues. The human IFNt is particularly useful for treatment of humans, since potential antigenic responses are less likely using such a same-species protein.

B. Antiviral Properties.

The antiviral activity of IFNt has broad therapeutic applications without the toxic effects that are usually associated with IFNas. Although the presence of IFNt in culture medium inhibited reverse transcriptase activity of the feline immunodeficiency virus (Example 11), this is not due to a direct effect of IFN- E:\KRH\689450SP.DOC on the FIV. Rather, IFNT appears to induce the host cell to produce a factor(s) which is inhibitory to the reverse transcriptase of the virus.

IFN, was found to exert its antiviral activity without adverse effects on the cells: no evidence of cytotoxic effects attributable to the administration of IFNt was observed. It is the lack of cytotoxicity of IFNT which makes it extremely valuable as an in vivo therapeutic agent. This lack of cytotoxicity sets IFNr apart from most other known antiviral agents and all other known interferons.

Formulations comprising the IFNt compounds of the present invention can be used to inhibit viral replication.

The human IFN- of the present invention can be employed in methods for affecting the immune relationship between fetus and mother, for example, in preventing transmission of maternal viruses HIV) to the developing fetus.

The human interferon-r is particularly useful for treatment of humans, since potential antigenic responses are less likely using a homologous protein.

C. Anticellular Proliferation Properties.

IFNr exhibits potent anticellular activity. IFN- can also be used to inhibit cellular growth without the negative side effects associated with other interferons which are currently known. Formulations comprising the IFNcompounds of the subject invention can be used to inhibit, prevent, or slow tumor growth.

The development of certain tumors is mediated by estrogen.

Experiments performed in support of the present invention indicate that IFN can suppress estrogen receptor numbers. Therefore, IFN- can be used in the treatment or prevention of estrogen-dependent tumors.

D. Interfering with the Binding of Interferons to Receptors.

IFNr appears to interact with the Type I IFN receptor via several epitopes on the molecule, and these regions either separately or in combination may affect distinct functions of IFNr differently.

The polypeptides of the present invention are useful for the selective inhibition of binding of interferons to the interferon receptor. Specifically, as E:\KRH\689450SP.DOC described herein, certain of the disclosed peptides selectively inhibit the antiviral activity of IFNT while others inhibit the antiproliferative activity.

Combinations of these peptides could be used to inhibit both activities.

Advantageously, despite binding to the interferon receptor and blocking IFNt activity, these peptides do not, themselves, elicit the antiviral or antiproliferative activity.

Therefore, such polypeptides can be used as immunoregulatory molecules when it is desired to prevent immune responses triggered by interferon molecules. These peptides could be used as immunosuppressants to prevent, for example, interferon-mediated immune responses to tissue transplants. Other types of interferon mediated responses may also be blocked, such as the cytotoxic effects of alpha interferon.

E. Pharmaceutical Compositions.

IFNT proteins can be formulated according to known methods for preparing pharmaceutically useful compositions. Formulations comprising :interferons or interferon-like compounds have been previously described (for example, Martin, 1976). In general, the compositions of the subject invention will be formulated such that an effective amount of the IFNT is combined with a suitable carrier in order to facilitate effective administration of the composition.

The compositions used in these therapies may also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, a such as tablets, pills, powders, liquid solutions or suspensions, liposomes, suppositories, injectable, and infusible solutions. The preferred form depends on the intended mode of administration and therapeutic application. The compositions also preferably include conventional pharmaceutically acceptable carriers and adjuvants which are known to those of skill in the art. Preferably, the compositions of the invention are in the form of a unit dose and will usually be administered to the patient one or more times a day.

IFNT, or related polypeptides, may be administered to a patient in any pharmaceutically acceptable dosage form, including intravenous, intramuscular, intralesional, or subcutaneous injection. Specifically, compositions and E:AKRH\689450SP.DOC methods used for other interferon compounds can be used for the delivery of these compounds.

One primary advantage of the compounds of the subject invention, however, is the extremely low cytotoxicity of the IFNr proteins. Because of this low cytotoxicity, it is possible to administer the IFNT in concentrations which are greater than those which can generally be utilized for other interferon IFNa) compounds. Thus, IFN- can be administered at rates from about 5 x 10 4 to 20 x 106 units/day to about 500 x 106 units/day or more. In a preferred embodiment, the dosage is about 10 6 units/day. High doses are preferred for systemic administration. It should, of course, be understood that the compositions and methods of this invention may be used in combination with other therapies.

~Once improvement of a patient's condition has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment should cease. Patients may, .9 however, require intermittent treatment on a long-term basis upon any 9999 recurrence of disease symptoms.

The compositions of the subject invention can be administered through standard procedures to treat a variety of cancers and viral diseases including those for which other interferons have previously shown activity. See, for example, Finter, et aL. (1991); Dianzani, et al. (1992); Francis, et al. (1992) and U.S. Patent Nos. 4,885,166 and 4,975,276. However, as discussed above, the compositions of the subject invention have unique features and advantages, including their ability to treat these conditions without toxicity.

F. Treatment of Skin Disorders.

Disorders of the skin can be treated intralesionally using IFNT, wherein formulation and dose will depend on the method of administration and on the size and severity of the lesion to be treated. Preferred methods include intradermal and subcutaneous injection. Multiple injections into large lesions E:'1KRH689450SP.DOC may be possible, and several lesions on the skin of a single patient may be treated at one time. The schedule for administration can be determined by a person skilled in the art. Formulations designed for sustained release can reduce the frequency of administration.

G. Systemic Treatment.

Systemic treatment is essentially equivalent for all applications. Multiple intravenous or subcutaneous doses are possible, and in the case of implantable methods for treatment, formulations designed for sustained release are particularly useful. Patients may also be treated using implantable subcutaneous portals, reservoirs, or pumps.

H. Regional Treatment.

Regional treatment with the IFN- polypeptides of the present invention is useful for treatment of cancers in specific organs. Treatment can be accomplished by intraarterial infusion. A catheter can be surgically or angiographically implanted to direct treatment to the affected organ. A subcutaneous portal, connected to the catheter, can be used for chronic treatment, or an implantable, refillable pump may also be employed.

The following examples illustrate, but in no way are intended to limit the present invention.

Materials and Methods Restriction endonucleases, T4 DNA ligase, T4 polynucleotide kinase, Taq DNA polymerase, and calf intestinal phosphatase were purchased from New England Biolabs (Beverly, MA) or Promega Biotech (Madison, WI): these reagents were used according to the manufacturer's instruction. For sequencing reactions, a "SEQUENASE DNA II" sequencing kit was used (United States Biochemical Corporation, Cleveland OH). Immunoblotting and other reagents were from Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific (Needham, MA). Nitrocellulose filters are obtained from Schleicher and Schuell (Keene, NH).

Synthetic oligonucleotide linkers and primers are prepared using commercially available automated oligonucleotide synthesizers an ABI model 380B-02 DNA synthesizer (Applied Biosystems, Foster City, CA)).

E:KRHA689450SP.DOC Alternatively, custom designed synthetic oligonucleotides may be purchased, for example, from Synthetic Genetics (San Diego, CA). cDNA synthesis kit and random priming labeling kits are obtained from Boehringer-Mannheim Biochemical (BMB, Indianapolis, IN).

Oligonucleotide sequences encoding polypeptides can be either synthesized directly by standard methods of oligonucleotide synthesis, or, in the case of large coding sequences, synthesized by a series of cloning steps involving a tandem array of multiple oligonucleotide fragments corresponding to the coding sequence (Crea; Yoshio et al.; Eaton et Oligonucleotide coding sequences can be expressed by standard recombinant procedures (Maniatis et al.; Ausubel et al.).

~Alternatively, peptides can be synthesized directly by standard in vitro techniques (Applied Biosystems, Foster City CA).

Common manipulations involved in polyclonal and monoclonal antibody work, including antibody purification from sera, are performed by standard procedures (Harlow et Pierce (Rockford, IL) is a source of many antibody reagents.

Recombinant human IFNa (rHulFNa) and rBolFNy was obtained from Genentech Inc. (South San Francisco, CA). The reference preparation of recombinant human IFNo (rHulFNa) was obtained from the National Institutes of Health: rHulFNa is commercially available from Lee Biomolecular (San Diego, CA).

All tissue culture media, sera and IFNs used in this study were negative for endotoxin, as determined by assay with Limulus amebocyte lysate (Associates of Cape Cod, Woods Hole, MA) at a sensitivity level of 0.07 ng/ml.

General ELISA Protocol for Detection of Antibodies.

Polystyrene 96 well plates Immulon II (PGC) were coated with 5 pg/mL (100 pL per well) antigen in 0.1 M carb/bicarbonate buffer, pH 9.5. Plates were sealed with parafilm and stored at 4 0 C overnight.

Plates were aspirated and blocked with 300 uL 10% NGS and incubated at 37 0 C for 1 hr.

E:\KRHk689450SP.DOC Plates were washed 5 times with PBS 0.5% Antisera were diluted in 0.1 M PBS, pH 7.2. The desired dilution(s) of antisera (0.1 mL) were added to each well and the plate incubated 1 hours at 37 0 C. The plates was then washed 5 times with PBS 0.5% Horseradish peroxidase (HRP) conjugated goat anti-human antiserum (Cappel) was diluted 1/5,000 in PBS. 0.1 mL of this solution was added to each well. The plate was incubated 30 min at 37 0 C, then washed 5 times with

PBS.

Sigma ABTS (substrate) was prepared just prior to addition to the plate.

The reagent consists of 50 mL 0.05 M citric acid, pH 4.2, 0.078 mL hydrogen peroxide solution and 15 mg ABTS. 0.1 mL of the substrate was added to each well, then incubated for 30 min at room temperature. The reaction was stopped with the addition of 0.050 mL 5% SDS The relative absorbance is determined at 410 nm.

EXAMPLE 1 Reproductive Functions of IFNt The effect of interferon-c on the lifespan of the corpus lutem was examined.

IFN- was infused into uterine lumen of ewes at the concentrations given 20 in Table 1. Recombinant human IFNa (rHulFNa) was infused at similar concentrations. In addition, control animals, which received control proteins, were also used. The life span of the corpus luteum was assessed by examination of interestrous intervals, maintenance of progesterone secretion, and inhibition of prostaglandin secretion (Davis, et al., 1992).

E:'KRH689450SP.DOC Table 1 Effect of Interferons on Reproductive Physiology Interferon Treatment Interestrous Interval (days) Control 17.3 100 pg/day 16.0 rHulFNa 200 pg/day 16.0 2000 pg/day 19.0 OvIFNT 100 pg/day 27.2 Comparison of the interestrous intervals for the control animals and for animals receiving OvlFNt demonstrate a considerable lengthening of the interval, when IFN- is administered at 100pg/day. On the other hand, comparison of the interestrous interval for the control animal and for animals 10 receiving recombinant human IFNa, demonstrated that rHulFN( had no meaningful effect.

These results demonstrate that interferon-r has the capability of significantly influencing the biochemical events of the reproductive cycle.

EXAMPLE 2 15 Antiviral Properties of Interferon- at Various Stages of the Reproductive Cycle Conceptus cultures were established using conceptus obtained from sheep at days 12 through 16 of the estrous cycle. Antiviral activity of supernatant from each conceptus culture was assessed using a cytopathic effect assay (Familetti, et al., 1981). Briefly, dilutions of IFNt or other IFNs were incubated with Madin-Darby bovine kidney (MDBK) cells for 16-18 hours at 370C. Following incubation, inhibition of viral replication was determined in a cytopathic effect assay using vesicular stomatitis virus (VSV) as the challenge virus.

One antiviral unit caused a 50% reduction in destruction of the E:URH\89450SP.DOC monolayer, relative to untreated MDBK cells infected with VSV (control plates).

Specific activities were further evaluated using normal ovine fibroblasts (Shnf) in a plaque inhibition assay (Langford, et 1981). A minimum of three samples were examined at each time point, and each sample was assayed in triplicate. The results presented in Table 2 are expressed as mean units/ml.

Table 2 IFNt Antiviral Activity of Conceptus Cultures and Allantoic and Amniotic Fluids

S

*S.

S

S

S.

SS*

S

55 S S S.

S

5*5 Day ISamples JUnits/ml 10 9 <3 Conceptus Cultures 12 5 34 13 6 4.5 x 10 3 14 3 7.7 x 10 3 16 12 2.0 x 10 6 60 3 1.4 x 10 3 Allantoic Fluid 100 4 11 140 3 <3 60 3 22 mniotic Fluid 100 <3 Culture supernatants had increasing antiviral activity associated with advancing development of the conceptus (Table 2).

EXAMPLE 3 Expression of IFNr in Bacteria The amino acid coding sequence for OvlFNt (Imakawa, et al., 1987) was used to generate a corresponding DNA coding sequence with codon usage optimized for expression in E. coli. Linker sequences were added to the 5' and 3' ends to facilitate cloning in bacterial expression vectors. The nucleotide E:\KRH89450SP.DOC sequence was designed to include 19 unique restriction enzyme sites spaced evenly throughout the coding sequence (Figure 1).

The nucleotide sequence was divided into eleven oligonucleotide fragments ranging in sizes of 33 to 75 bases. Each of the eleven oligonucleotides were synthesized on a 380-B 2-column DNA synthesizer (Applied Biosytems) and cloned single- or double-stranded into one of the following vectors: "pBLUESCRIPT (Stratagene, LaJolla, CA), pTZ18R (Pharmacia, Piscataway, NJ), or pTZ19R (Pharmacia, Piscataway, NJ) cloning vectors.

The vectors were transformed into E. coli K. strain "XL1-BLUE" (recA1 endA1 gyrA96 thi hsdR17 supE44 relA1 (lac), proAB, lacqZAM15, Tnl0(tetR}) which is commercially available from Stratagene (LaJolla, CA). Transformed cells were grown in L broth supplemented with ampicillin (50 pg/ml). Oligonucleotide cloning and fusion was performed using standard recombinant DNA techniques.

Cloning vectors were cut with the appropriate restriction enzymes to insert the synthetic oligonucleotides. The vectors were treated with calf intestine alkaline phosphatase (CIP) to remove terminal phosphate groups.

Oligonucleotides were phosphorylated and cloned, as either single- or doublestranded molecules, into the appropriate vector using T4 DNA ligase. When single-strands were introduced into cloning vectors, the second strand was completed by the bacterial host following transfection.

For double-stranded cloning, oligonucleotides were first annealed with their synthetic complementary strand then ligated into the cloning vector. E.

coli K12 strains SB221 or NM522 were then transformed with the ligation. E.

coli strain GM119 was used for cloning when the methylation-sensitive Stul and Clal restriction sites were involved. Restriction analyses were performed on isolated DNA at each stage of the cloning procedure.

Cloned oligonucleotides were fused into a single polynucleotide using the restriction digestions and ligations outlined in Figure 2. Oligonucleotidecontaining-DNA fragments were typically isolated after electrophoretic size fractionation on low-melting point agarose gels (Maniatis, et al.; Sambrook, et E:KRHk689450SP.DOC al.; Ausubel, et The resulting IFNt polynucleotide coding sequence spans position 16 through 531: a coding sequence of 172 amino acids.

The nucleotide sequence of the final polynucleotide was confirmed by DNA sequencing using the dideoxy chain termination method.

The full length StullSstl fragment (540 bp; Figure 2) was cloned into a modified pIN III omp-A expression vector and transformed into a competent SB221 strain of E. coli. For expression of the IFN protein, cells carrying the expression vector were grown in L-broth containing ampicillin to an OD (550 nm) of 0.1-1, induced with IPTG for 3 hours and harvested by centrifugation.

Soluble recombinant IFNr was liberated from the cells by sonication or osmotic fractionation.

EXAMPLE 4 Expression of IFNr in Yeast S. The synthetic IFNr gene, synthesized in Example 3, was flanked at the 15 5' end by an Stul restriction site and at the 3' end by a Sac/ restriction site.

A. Isolation of the Synthetic IFNr Gene.

Two oligonucleotide primers (SEQ ID NO:13 and SEQ ID NO:14) were used to attach linkers to the synthetic IFNT gene using polymerase chain reaction. The linker at the 5' end allowed the placement of the synthetic IFN- 20 gene in correct reading with the ubiquitin coding sequence present in the yeast cloning vector pBS24Ub (Chiron Corp., Emeryville, CA). The linker also constructed a ubiquitin-IFNr junction region that allowed in vivo cleavage of the ubiquitin sequences from the IFN- sequences. The 5' oligonucleotide also encoded a Sacll restriction endonuclease cleavage site. The 3' oligonucleotide contained a Stul cleavage site.

The vector carrying the synthetic IFN- gene (Example 3) was isolated from E. coli strain "XLI-BLUE" by the alkaline lysis method. Isolated vector was diluted 500-fold in 10 mM Tris, pH 8.0/1 mM EDTA/10 mM NaCI. The PCR reaction was performed in a 100 pl volume using Taq DNA polymerase and primers SEQ ID NO:13/SEQ ID NO:14. The amplified fragments were digested with Stul and Sacll. These digested fragments were ligated into the Sacll and E:\KRH\689450SP.DOC 41 Smal sites of "pBLUESCRIPT+(KS)." The resulting plasmid was named pBSY-IFN-. The DNA sequence was verified using double stranded DNA as the template.

B. Construction of the Expression Plasmid.

Plasmid pBSY-IFN was digested with Sacll and EcoRV and the fragment containing the synthetic IFNt gene was isolated. The yeast expression vector pBS24Ub (Sabin, et al.; Ecker, et al.) was digested with Sail.

Blunt ends were generated using T4 DNA polymerase. The vector DNA was extracted with phenol and ethanol precipitated (Sambrook, et al., 1989). The recovered linearized plasmid was digested with Sacll, purified by agarose gel electrophoresis, and ligated to the Sacll-EcoRV fragment isolated from pBSY- IFN-. The resulting recombinant plasmid was designated pBS24Ub-IFNT.

The recombinant plasmid pBS24Ub-IFNT was transformed into E. coli.

Recombinant clones containing the IFNT insert were isolated and identified by 15 restriction enzyme analysis. Plasmid DNA from clones containing IFN coding sequences was used for transformation of S. cerevisiae (Rothstein, 1986).

Transformation mixtures were plated on uracil omission medium and incubated for 3-5 days at 30 0 C. Colonies were then streaked and maintained on uracil and leucine omission medium (Rothstein, 1986).

20 C. Expression Experiments.

For small-scale expression, a single colony of S. cerevisiae AB116 containing pBS24Ub-IFNT was picked from a leucine and uracil omission plate and grown at 30 0 C in YEP medium yeast extract, 2% peptone) containing 1% glucose for inducing conditions or 8% glucose for noninducing conditions.

Cell lysates were recovered and subjected to SDS-PAGE in 15% acrylamide, 0.4% bisacrylamide (Sambrook, et al., 1989). The fractionated proteins were visualized by Coomassie blue staining.

Recombinant IFNT was visualized specifically by immunoblotting with monoclonal antibody or polyclonal antiserum against ovine IFNT upon electrotransfer of the fractionated cell extract to "NYTRAN" paper (Rothstein, 1986).

E:\KRH\689450SP.DOC For large-scale expression, pBS24-IFNT was grown for 24 hours at in 5 x uracil and leucine omission medium containing 8% glucose. This culture was then diluted 20-fold in YEP medium containing 1% glucose and further incubated for another 24-36 hours.

Cells were harvested by centrifugation, washed in 50 mM Tris, pH 7.6,/1 mM EDTA and resuspended in wash buffer containing 1 mM PMSF. The cells were lysed using a Bead-beater apparatus (Biospec Products, Bartlesville, OK).

The lysate was spun at 43,000 x g for 20 minutes. The supernatant fraction was recovered and subjected to the purification protocol described below.

D. Purification of r-IFNt from Yeast Cell Lysate.

The supernatant was loaded on a 1 x 10 cm DEAE column and washed with 10 mM Tris, pH 8.0. Retained proteins were eluted with a 300 ml, 0 to M NaCI gradient in 10 mM Tris, pH 8.0. Three-milliliter fractions were collected.

Ten-microliter samples of fractions 17-26 containing the recombinant (r-IFNr) 15 were electrophorectically separated on 15% SDS-polyacrylamide gels. The gels were stained with Coomassie blue.

Fractions 18, 19, and 20 contained largest amount of r-IFN-c. These fractions were loaded individually on a 1.5 x 90 cm Sephadex S-200 column and proteins were resolved in two peaks. Aliquots of each protein peak (25 pl) were electrophoretically separated on 15% SDS-polyacrylamide gels and the proteins visualized with Coomassie staining.

.0 Purified r-IFN-r-containing fractions were combined and the amount of r- IFNt quantified by radioimmunoassay (Vallet, et al., 1988). Total protein concentration was determined by using the Lowry protein assay (Lowry, et al., 1951).

Microsequencing of purified r-IFNT demonstrated identity with native IFNT through the first 15 amino acids, confirming that the ubiquitin/r-IFNr fusion protein was correctly processed in vivo.

Purified r-IFN- exhibited 2 to 3 x 108 units of antiviral activity per milligram of protein (n 3 replicate plates) which is similar to the antiviral activity of IFNT purified from conceptus-conditioned culture medium (2 x 108 E:\KRH689450SP.DOC 43 U/mg).

EXAMPLE Southern Blot Analysis of Human High Molecular Weight DNA Human venous blood samples from healthy donors were collected in heparinized tubes and peripheral blood lymphocytes were isolated by densitygradient centrifugation using a Ficoll-lsopaque gradient (1.077 g/ml) (Sigma Chemical High molecular weight (HMW) DNA was isolated from these cells (Sambrook, et al., 1989).

Two 10 pg samples of HMW DNA were digested with the restriction endonucleases Hindlll or Pstl (Promega) for 2 hours at 370C, and the DNA fragments electrophoretically separated in a 0.8% agarose gel (Bio-Rad, Richmond, CA) at 75 volts for 8 hours. The DNA fragments were transferred onto a nylon membrane (IBI-lnternational Biotechnologies, Inc., New Haven, 15 CT). The membrane was baked at 800C for 2 hours and incubated at 42 0 C for 4 hours in the following prehybridization solution: 5 x SSC (1 x SSC is 0.15 M "NaCI and 0.15 M sodium citrate), 50% vol/vol formamide, 0.6% (wt/vol) SDS, 0.5% (wt/vol) nonfat dry milk, 20 mM Tris-HCI (pH 4 mM EDTA, and mg/ml single stranded herring sperm DNA (Promega).

20 The filter was then incubated in a hybridization solution (5 x SSC, vol/vol formamide, 0.6% (wt/vol) SDS, 0.5% (wt/vol) nonfat dry milk, 20 mM Tris-HCI (pH 4 mM EDTA, and 2 x 10 cpm/ml 32P-labelled OvlFNt cDNA (Imakawa, et al., 1987)) for 18 hours at 420C. The filter was washed at 420C for 15 minutes with 2 x SSC and 0.1% (wt/vol) SDS and exposed to X-ray film (XAR, Eastman Kodak, Rochester, NY) at -80°C for 48 hours in the presence of an intensifying screen.

Autoradiography detected a hybridization signal at approximately 3.4 kb in DNA digested with Pstl and a slightly smaller 3.0 kb) fragment in the Hindlll digested DNA. These results indicate the presence of human DNA sequences complementary to the OvlFNt cDNA probe.

E:\KRH\689450SP.DOC 44 EXAMPLE 6 Isolation of Partial Sequence of Human IFN cDNA by PCR Two synthetic oligonucleotides (each 25-mer), corresponding to the sequence 231 to 255 (contained in SEQ ID NO:13) and 566 to 590 (contained in SEQ ID NO:14) of OvIFNt cDNA (numbering relative to the cap site, Imakawa, et al., 1987) were synthesized. These primers contained, respectively, cleavage sites for the restriction endonucleases Pstl and EcoRI.

SEQ ID NO:13 was modified to contain the EcoRI site, which begins at position 569.

DNA was isolated from approximately 1 x 10 5 plaque forming units (pfu) of the following two cDNA libraries: human term placenta (Clontech, Inc., Palo Alto, CA) and human term cytotrophoblast (Dr. J.F. Strauss, University of Pennsylvania, Philadelphia PA). The DNA was employed in polymerase chain 15 reaction (PCR) amplifications (Mullis; Mullis, et al.; Pekin Elmer Cetus Corp.

Norwalk CT). Amplification reactions were carried out for 30 cycles (45 0 C, 1m; 72 0 C, 2m; 94 0 C, 1m) (thermal cycler and reagents, Perkin Elmer Cetus) using primers SEQ ID NO:13/SEQ ID NO:14.

Amplification products were electrophoretically separated (100 volts in a 1.5% agarose gel (Bio-Rad)) and transferred onto a nylon membrane (IBI). The membrane was baked at 80 0 C for 2 hours and prehybridized and hybridized with 32P-labelled OvIFNT cDNA as described above. The membrane was Swashed in 5 x SSC/0.1% (wt/vol) SDS for 5 minutes at 42 0 C and in 2 x SSC/0.1% (wt/vol) SDS for 2 minutes at 42 0 C. It was then exposed at -80 0 C to "XAR" (Eastman Kodak) X-ray film for 24 hours in the presence of an intensifying screen. An amplification product that hybridized with the labelled probe DNA was detected.

PCR was performed again as directed above. Amplified products were digested with the restriction endonucleases EcoRI and Pstl (Promega) for minutes at 37 0 C. The resulting DNA fragments were electrophoretically separated as described above and the band containing the IFNr amplification E:'KRH\689450SP.DOC

I

product was excised from the gel. DNA fragments were recovered by electroelution, subcloned into EcoRIIPstl digested-dephosphorylated plasmid pUC19 and transformed into E. coli strain JM101 (Promega) by calcium chloride method (Sambrook, et al., 1989). The plasmids were isolated and the inserted amplification product sequenced using the dideoxy termination method (Sanger, et al., 1977; "SEQUENASE" reactions, United States Biochemical, Cleveland, OH). Nucleotide sequences were determined, and comparison of these as well as the deduced amino acid sequences to other IFN sequences were performed using DNA Star Software (Madison, WI).

Comparison of the sequences of these clones revealed three different clones: from the human placental library, Clone 15 (306 bp) and Clone 21 (315 bp), which exhibit 95% identity in their nucleotide sequences; from the cytotrophoblast library clone CTB 35 (301 basepairs), which shares 95% and 9 98% identity with Clone 15 and Clone 21, respectively.

EXAMPLE 7 Isolation of Full-Length Human IFNt Gene Ten micrograms PBMC HMW DNA was digested with restriction endonuclease EcoRI and subjected to electrophoretic analysis in a 0.8% agarose gel. A series of samples containing ranges of DNA fragments sized 20 1.5 to 10 kb 1.5 to 2.5 kb, 2.5 kb to 3 kb) were excised from the gel. The DNAs were electroeluted and purified. Each DNA sample was amplified as described above using the OvIFNr primers. The DNA molecules of any sample that yielded a positive PCR signal were cloned into ,gt11 (the subgenomic ?gtl 1 library).

A. PCR Identification of Clones Containing Sequences Complementary to OvIFNc.

The Xgt11 phage were then plated for plaques and plaque-lift hybridization performed using the 32 P-labelled OvlFN cDNA probe.

Approximately 20 clones were identified that hybridized to the probe.

Plaques that hybridized to the probe were further analyzed by PCR using the OvIFNt primers described above. Six plaques which generated positive E:\KRH\689450SP.DOC 46 PCR signals were purified. The phage DNA from these clones was isolated and digested with EcoRI restriction endonuclease. The DNA inserts were subcloned into pUC19 vectors and their nucleotide sequences determined by dideoxy nucleotide sequencings.

B. Hybridization Identification of Clones Containing Sequences Complementary to PCR-Positive Phage.

Recombinant phage from the Xgt 1 subgenomic library were propagated in E. coli Y1080 and plated with E. coli Y1090 at a density of about 20,000 plaques/150 mm plate. The plates were overlaid with duplicate nitrocellulose filters, which were hybridized with a 32 P-labelled probe from one of the six human IFN- cDNA clones isolated above. Three clones giving positive hybridization signals were further screened and purified. The phage DNAs were isolated, digested with EcoRI, subcloned into pUC19 vector and S. sequenced. The three clones yielded sequence information for over 800 bases 15 relative to cap site (clones were sequenced in both orientations). The nucleic acid sequence information is presented as SEQ ID NO:11 and the predicted protein coding sequence is presented as SEQ ID NO:12. Comparison of the predicted mature protein sequence (SEQ ID NO:12) of this gene to the predicted protein sequence of OvlFNr is shown in Figure 3.

EXAMPLE8 Analysis of the Presence of HulFNt mRNA by RT-PCR a.

Human placental cDNA libraries and an ovine cDNA library, constructed from day 15-16 conceptuses, were analyzed by hybridization to the OvIFNt cDNA probe, described above. cDNAs were size-fractionated on agarose gels and transferred to filters (Maniatis, et al.; Sambrook, et Southern blot analysis with OvlFNT probe showed that the autoradiographic signals from human cDNA libraries were approximately 1/100 of the signal obtained using the OvIFNT cDNA library.

The presence of HulFNt mRNA in human term placenta and amniocytes (26 weeks, 2 million cells) was analyzed by using reverse transcriptase-PCR E:\KRH\689450SP.DOC (RT-PCR) method (Clontech Laboratories, Palo Alto CA).

Total cellular RNA (tcRNA) isolated from human placenta, amniocytes and ovine conceptuses were reverse transcribed using the primer SEQ ID NO:14. The primer SEQ ID NO:13 was then added to the reaction and polymerase chain reaction carried out for 40 cycles. The PCR products were size fractionated on agarose gels and transferred to filters. The DNA on the filters was hybridized with 32P-labelled OvIFNr and HulFNr cDNAs. The results of these analyses demonstrate the presence of human IFNr mRNA in the fetoplacental annex. The aminocytes also expressed the messages corresponding to OvIFNt primers and human probe.

In addition, a RT-PCR analysis for the presence of HulFN- was applied to the tcRNA isolated from human adult lymphocytes. A densitometric analysis revealed that IFNr mRNA exists in lymphocytes.

EXAMPLE 9 15 In Situ Hybridization A. Tissue Slides of semiserial 5-p paraffin embedded sections from four healthy, Sdifferent term and first trimester human placentas were examined.

S: B. cRNA Probe Preparation From the cDNA clone isolated from OvlFNt amplified library a fragment corresponding to the OvIFNr cDNA bases #77-736 (base #1 is cap site; open reading frame of OvlFNr cDNA is base #81-665; Figure 7) was subcloned into the transcription vector, pBS (New England Biolabs). Several pBS clones were isolated, subcloned, and their nucleotides sequenced. From this clone a 3' fragment (bases #425-736) was excised using the restriction endonucleases NIalV and EcoRI and subcloned into the transcription vector pBS. This vector was designated pBS/OvlFNr.

After linearization of the pBS/OvlFNt plasmid, an antisense cRNA probe was synthesized by in vitro transcription (Sambrook, et al., 1989) using T 7

RNA

polymerase (Stratagene). A trace amount of 3 H-CTP (NEN-DuPont, Cambridge, MA) was used in the transcription reaction. dUTP labeled with E:KRH1689450SP.DOC 48 digoxigenin (Boehringer-Mannheim, Indianapolis, IN) was incorporated into the cRNA and yield was estimated through TCA precipitation and scintillation counting.

C. Hybridization In situ hybridization was performed using the anti-sense RNA probe, as described by Lawrence, et al. (1985) with the following modifications.

Deparaffinized and hydrated sections were prehybridized for 10 minutes at room temperature in phosphate buffered saline (PBS) containing 5 mM MgCI 2 Nucleic acids in the sections were denatured for 10 minutes at 65 0 C in formamide/2 x SSC. Sections were incubated overnight at 37°C with a hybridization cocktail (30 pl/slide) containing 0.3 pg/ml digoxigenin-labelled :i cRNA probe and then washed for 30 minutes each at 37°C in 50 formamide/1 x SSC. Final washes were performed for 30 minutes each at room temperature in 1 x SSC and 0.1 x SSC. The sections were blocked for 30 minutes with 9 15 0.5% Triton X-100 (Sigma) and 0.5% non-fat dry milk.

Hybridization signal was detected using purified sheep antidioxigenin "Fab fragments conjugated to alkaline phosphatase (Boehringer-Mannheim).

I After unbound antibody was removed, nitroblue tetrazolium/5-bromo-4-chloro-3indolyl-phosphate substrate (Promega) and levamisole (Bector Laboratories, 20 Burlingame, CA) were added for signal detection via colorimetric substrate generation. The tissues were counterstained in methyl green (Sigma), dehydrated, and mounted.

As a control, some tissue sections were pretreated with 100 pg/ml of pancreatic RNaseA (Sigma) for 30 minutes at 370C. The RNase was inactivated on the slide with 400 units of RNase inhibitor (Promega). The slides were then washed twice in 250 ml of PBS/5 mM MgCI 2 In other control experiments, tRNA (Sigma) was substituted for the digoxigenin probes.

Specific hybridization was observed in all term and first trimester placental tissues in three separate experiments with various OvIFN'c cRNA probe concentrations and blocking reagents.

First trimester placental villi composed of an outer layer of E:\KRH1689450SP.DOC 49 syncytiotrophoblast, an underlying layer of cytotrophoblast, and a central stromal region with various types of mesenchymal cells, displayed the highest transcript level of IFNt in the cytotrophoblast cells. Less intense but detectable levels were present in both the syncytiotrophoblast and stromal cells. A similar pattern of transcript expression was demonstrated in the placental villi of term tissue but the level of signal detection was low. First trimester extravillous trophoblast displayed the highest amount of message and stained positive when present in the maternal blood spaces.

EXAMPLE Antiviral Activity of IFN- The relative specific activity of OvlFNr, purified to homogeneity, was evaluated in antiviral assays. The antiviral assays were performed essentially as described above in Example 2. Specific activities are expressed in antiviral units/mg protein obtained from antiviral assays using either Madin-Darby bovine 15 kidney (MDBK) cells or sheep normal fibroblasts (Shnf). All samples were assayed simultaneously to eliminate interassay variability. The results, *presented in Table 3, are the means of four determinations where the standard deviation was less than 10% of the mean.

Table 3 20 Antiviral Activity of IFNt and Known IFNs 595 OvIFN-u 2 x 108 3 x 108 rBolFNa 6 x 10 7 1 x 10 7 rBolFNy 4.5 x 106 3 x 10 6 NIH rHulFNa 2.2 x 108 2.2 x 108 rHulFNa 2.9 x 10 5 4.3 x 10 IFNt had a higher specific activity than either rBolFNa or rBolFNy (Table The NIH standard preparation of rHulFNa had a similar specific activity, while a commercial preparation of rHulFNa exhibited low specific antiviral E:\KRH\689450SP.DOC a a. a a a.

a activity. Comparable relative antiviral activity was demonstrated using either bovine or ovine cells.

EXAMPLE 11 Anti-Retroviral Activity and Cytotoxic Effects of IFNt Highly purified OvIFNT was tested for anti-retroviral and cytotoxic effects on feline peripheral blood lymphocytes exposed to the feline immunodeficiency retrovirus. This lentivirus produces a chronic AIDS-like syndrome in cats and is a model for human AIDS (Pederson, et aL, 1987). Replication of the virus in peripheral blood lymphocytes is monitored by reverse transcriptase activity in culture supernatants over time. The data from these assays are presented in Table 4.

Table 4 Effect of OvIFNr on FIV Replication Harvest Days Experiment I Day 2 Day5 I Day8 Day 12 Day 0.00 93,908 363,042 289,874 171,185 125,400 0.62 77,243 179,842 172,100 218,281 73,039 1.25 94,587 101,873 122,216 71,916 50,038 2.50 63,676 72,320 140,783 75,001 36,105 5.00 69,348 82,928 90,737 49,546 36,299 Harvest Days Experiment 2 Day 2 Day 5 Day 8 Day 13 Day 17 0.0 210,569 305,048 279,556 500,634 611,542 121,082 106,815 108,882 201,676 195,356 223,975 185,579 108,114 175,196 173,881 10.0 167,425 113,631 125,131 131,649 129,364 20.0 204,879 80,399 59,458 78,277 72,179 40.0 133,768 54,905 31,606 72,580 53,493 a.

a a.

4 a a a .a E:AKRH\689450SP.DOC Addition of OvIFNT produced a rapid, dose-dependent decrease in reverse transcriptase (RT) activity (Table While concentrations as low as 0.62 ng/ml of IFN- inhibited viral replication, much higher concentrations ng/ml) having greater effects on RT-activity were without toxic effects on the cells. The results suggest that replication of the feline immunodeficiency virus was reduced significantly compared to control values when cells were cultured in the presence of OvlFNr.

IFNT appeared to exert no cytotoxic effect on the cells hosting the retrovirus. This was true even when IFNT was present at 40 ng per ml of culture medium.

EXAMPLE 12 Effects of IFNc on HIV Infected Human Peripheral Lymphocytes 6: IFNr was also tested for activity against HIV infection in human cells.

Human peripheral blood lymphocytes, which had been infected with HIV (Crowe, et were treated with varying concentrations of OvIFNr. Replication of HIV in peripheral blood lymphocytes was monitored by reverse transcriptase activity in culture supernatants over time. Reverse transcriptase activity was measured essentially by the method of Hoffman, et al. The data from these assays are presented in Table Table Effect of OvlFNT on HIV Replication in Human Peripheral LymDhocvtes 4,214 25,994 2,046 51 9,883 62 1,794 57 4,962 81 11 100 E:N(RH89450SP.DOC 1,770 3,012 ~RT Ativit ANT4 Concetration (ngm9 Day 6 Day 500 1,686 60 2,670 1000 1,499 64 2,971 89 As shown in Table 5, concentrations of OvIFNr produced significant antiviral effects. A concentration of only 10 ng/ml resulted in over a reduction in RT activity after only six days. A concentration of 500 ng/ml resulted in a 90% reduction in RT activity within 10 days.

The viability of human peripheral blood lymphocytes after treatment with IFNt, over a range of concentrations for 3-13 days, was evaluated by trypan blue exclusion. The results of this viability analysis are presented in Table 6.

Table 6 0 Effect of OvlFNT on Viability of HIV Infected

S.

S S

*SSS..

S

S S

S

S S

*S

S. S S 5555

S

S.

S S S. S S S

S.

Human Peripheral Lymphocvtes IFNi VibeClsllx1* (ng/mI l K 0 16.0 7.5 5.3 13.0 7.5 13.0 11.5 100 15.0 8.5 500 16.5 12.0 11.0 1000 21.9 9.5 The data presented in Table 6 show no evidence of cytotoxic effects attributable to the administration of IFNt.

E:\KRH\689450SP.DOC EXAMPLE 13 Inhibition of Cellular Growth The effects of IFNT on cellular growth were also examined. Anti-cellular growth activity was examined using a colony inhibition assay. Human amnion (WISH) or MDBK cells were plated at low cell densities to form colonies originating from single cells. Cells were cultured at 200 or 400 cells/well in 24 well plates in HMEM supplemented with 2% fetal bovine serum (FBS) and essential and nonessential amino acids. Various dilutions of interferons were added to triplicate wells, and the plates were incubated for 8 days to allow l0 colony formation. Colonies were visualized after staining with crystal violet, and counted. Cell cycle analysis was performed with HMEM containing "spent" media for an additional 7 days. WISH cells were used without being synchronized.

For examination of IFNt activity, cells were replated at 2.5 x 10 15 cells/well in HMEM with 10% FBS in 6 well plates. Various dilutions of IFNalone or in combination with peptides were added to achieve a final volume of 1 ml. Plates were incubated at 37 0 C in 5% Co 2 for 12, 15, 18, 24, or 48 hours.

Cells were treated with trypsin, collected by low speed centrifugation and washed. The cell pellet was blotted dry and 250 pi of nuclear staining solution (5 mg propidium iodide, 0.3 ml NP40 and 0.1 gm sodium citrate i 100 ml distilled H 2 0) was added to each tube. The tubes were incubated at room temperature. After 10 minutes, 250 pl of RNase (500 units/ml in 1.12% sodium citrate) was added per tube and incubated an additional 20 minutes. Nuclei were filtered through 44 pm mesh, and analyzed on a FACStar (Becton Dickinson, Mountain View, CA) using the DNA Star 2.0 software.

In the cellular growth assay using colony formation of both the bovine epithelial line, MDBK, and the human amniotic line, WISH, OvIFN- inhibited both colony size and number. Ovine IFNT was more effective than human IFNo on the human cell line; thus, it is very potent in cross-species activity. Its activity was dose-dependent, and inhibition of proliferation could be observed at concentrations as low as 1 unit/mI. Concentrations as high as 50,000 units/mI E:M(RH689450SP.DOC (units of antiviral activity/ml) stopped proliferation, while cell viability was not impaired.

Cell cycle analysis by flow cytometry with propidium iodide-stained WISH cells revealed an increased proportion of cells in G2/M after 48 hours of OvIFNtreatment. IFNr, therefore, appears to inhibit progress of cells through S phase. Ovine IFNt antiproliferative effects can be observed as early as 12 hours after the initiation of culture and are maintained through 6 days.

The results presented above demonstrate both the antiproliferative effect of IFNc as well as its low cytotoxicity.

EXAMPLE 14 Further Antiproliferative Effects of IFNt The antiproliferative effects of OvIFNt were studied for a rat cell line and a bovine cell line. The rate of 3H-thymidine incorporation was used to assess the rate of cellular proliferation.

15 Rat (MtBr7 .c5) or bovine kidney (MDBK) cells were seeded in phenol red-free DME-F12 medium supplemented with 3% dextran-coated charcoal stripped Controlled Process Serum Replacement 2 (CPSR 2, Sigma) and dextran-coated charcoal stripped fetal bovine serum (FBS). After attaching for approximately 15-18 hours, the cells were washed once with serum-free DME- F12 medium. The medium was replaced with phenol red-free DME-F12 medium supplemented with 3% stripped CPSR2, 1% stripped FBS medium) or 3/1 medium containing OvIFNt at various units of antiviral activity as determined in the vesicular stomatitis virus challenge assay for interferons (Example Media containing a similar dilution of buffer (undiluted buffer mM Tris, 330 mM NaCI, in which the OvIFN- was dissolved was used for controls.

Cells were pulse labeled with 3 H-thymidine for 2 hours at approximately 48 hours post-treatment. The trichloroacetic acid (TCA) precipitable incorporated counts were determined by scintillation counting. Three replicates were included per treatment. Mean values for OvlFNt treatments were compared to samples containing comparable dilutions of carrier TS buffer.

E:\KRH\689450SP.DOC 45 4 4 0**4

S

4S

S

S

S.

S.

Results of these experiments are shown in Table 7.

Table 7 3 H-Thymidine Incorporation Experiment 1: MtBr7 .c5 (Rat) 3/1 u OvlFNT/ml 0(+12) 1:5000 TS 104 u OvFNT/ml 24 1:500 TS 10 u OvlFNr/ml 87 Experiment 2: MDBK 3/1 10 3 u OvIFNT/ml 74 1:5000 TS 104 u OvlFNT/ml 83 1:500 TS 5 u OvlFNT/ml 83 As can be seen from Table 7, OvIFNr drastically reduced the rate of cellular proliferation (based on thymidine incorporation) for each of the cell lines tested.

EXAMPLE Anti roliferative Effects of IFNr on Human Tumor Cell Lines The antiproliferative activity of OvlFNT on human tumor cell lines was evaluated by measuring the rate of 3 H-thymidine incorporation into cells which have been treated with OvlFNT.

For experiments on tumor lines that grow in suspension, 1 ml of cells were plated at from 2.5 5 X 10 5 cells/well in 24-well plates. Triplicate wells received either the appropriate media, 100, 1,000 or 10,000 units/mi of OvIFNT E:AKRH689450SP.DOC or equivalent antiviral concentrations of rHulFNa2A (Lee Biomolecular). After 48 hours of incubation, cells were counted and viability assessed by trypan blue exclusion.

Adherent tumor lines were plated at 2.5 X 10 5 cells/well in 1 ml in 6-well plates. They received interferon treatments as just described, but were trypsinized prior to counting.

Significant differences between treatments were assessed by an analysis of variance followed by Scheffe's F-test. Cell cycle analysis was performed by flow cytometry using propidium iodide.

A. Breast Adenocarcinoma Cells.

Human MCF7 breast adenocarcinoma cells were seeded from logarithmically growing cultures in phenol red-free DME-F12 medium supplemented with 3% dextran-coated charcoal stripped CPSR and dextran-coated FBS. After attaching for approximately 15-18 hours, the cells 15 were washed once with serum-free DME-F12 medium. The medium was replaced with phenol red-free DME-F12 medium supplemented with 3% stripped CPSR2, 1% stripped FBS medium) or 3/1 medium containing OvIFNT at the indicated number of units of antiviral activity as determined in the vesicular stomatitis virus challenge assay for interferons. Media containing a 20 similar dilution of buffer (undiluted buffer 10 mM Tris, 330 mM NaCI was used for controls. Cells were pulse labeled with 3 H-thymidine for 2 hours at approximately 48 hours post-treatment.

The trichloroacetic acid (TCA) precipitable incorporated counts were determined by scintillation counting. Three replicates were included per treatment. Mean values for OvIFNt treatments were compared to samples containing comparable dilutions of carrier TS buffer. The results of these analyses are shown in Table 8.

E:\KRH\689450SP.DOC ___________111111111II 57 Table 8 3 H-Thymidine Incorporation reatment Reduction 3 H-Thymidin Incorporation MCF7 Human 3/1 3 u OvlFNT/ml 1:5000 TS 04 u OvlFNT/ml 53 1:500 TS 5 u OvFNT/ml

S*

a a. a *5*a S. a a a.

As can be seen from the results shown in Table 8, OvIFN- was able to substantially reduce the rate of 3H-thymidine incorporation in the human carcinoma cell line. This demonstrates the efficacy of OvlFN' in inhibiting tumor cell proliferation, in particular, mammary tumor cell proliferation.

B. Human Promyelocytic Leukemia.

10 A comparison of the antiproliferative effects of OvlFNt and IFNa was conducted using HL-60 (human leukemia) cells (Foa, et al.; Todd, et a.) essentially as described above for MDBK cells. Both OvIFNT and rHulFNa inhibit HL-60 cell proliferation. Results of one of three replicate experiments are presented as mean growth reduction SD in Figure 4. Figure 4 shows that both OvlFNr and IFNa were able to drastically reduce growth of HL-60 cells.

The growth reduction for each compound exceeded 60% for each concentration tested. At 10,000 units/ml, OvlFNr caused an approximately reduction in growth while IFNa caused a 100% reduction in growth.

However, the data presented in Figure 4 reveal, that a substantial factor in the ability of IFNa to reduce growth was its toxic effect on the cells. At 10,000 units/ml, the toxicity of IFNa resulted in less than 25% of the cells remaining viable. By contrast, nearly 100% of the cells remained viable when OvlFNt was applied at 10,000 units/ml.

E:KRH\689450SP.DOC Figure 5 presents data demonstrating that rHulFNo is cytotoxic. In the figure, results of one of three replicate experiments are presented as mean viability SD.

C. Human Cutaneous T Cell Lymphoma.

The cutaneous T cell lymphoma, HUT 78, responded similarly to when treated with IFNt (Figure Both OvIFNt and rHulFNa reduce HUT 78 cell growth, but 10,000 units/ml of rHulFNa decreased the cell number below that originally plated (5X10 5 This is indicative of a reduction in cell viability to approximately Cell cycle analysis (performed by cell flow cytometry) revealed an increased proportion of cells in G2/M phase of the cell cycle upon 48 hours of treatment with both interferons (Table 10). In Table 10 the results from one of three replicate experiments are presented as the percentage of cells in each phase of the cell cycle. 10,000 events were analyzed per sample.

15 This result is likely due to the slower progress of cells through the cell cycle. In the sample treated with 10,000 units/ml of rHulFNa, a large percentage of events with low forward and high side scatter, identifying dead cells, were present. This is consistent with the data obtained from proliferation experiments, where only OvIFNT inhibited HUT 78 proliferation without toxicity.

Table 10. HUT 78 Cell Cycle Analysis.

Treatment (units/mi) GOIG1 S G2/M Media 44.43 49.95 5.61 100 OvlFNr 44.35 47.45 8.20 100 rHulFNa 40.01 57.53 2.45 1,000 OvIFNt 41.29 50.50 8.21 1,000 rHulFNa 41.73 44.91 13.36 10,000 OvIFNT 42.79 42.61 14.60 10,000 rHulFNa 18.01 71.31 10.67 (cell death) E:KRH\689450SP.DOC D. Human T Cell Lymphoma.

The T cell lymphoma cell line H9 was slightly less sensitive to the antiproliferative effects of the IFNs than the tumor cell lines described above.

Results of one of three replicate experiments are presented in Figure 10 as mean growth reduction SD. While rHulFNa was not toxic to the H9 cells, it failed to inhibit cell division significantly at any of the concentrations examined.

In contrast, OvIFNr was observed to reduce H9 growth by approximately (Figure 10). Thus, only OvIFN- is an effective growth inhibitor of this T cell lymphoma.

The results presented above demonstrate both the antiproliferative effect of IFN- as well as its low cytotoxicity.

EXAMPLE 16 Preliminary In Vivo Treatment with OvIFNT 15 Three groups of 4 C57BI/6 mice per group were given 2.5 X 10 4 B16-F10 cells via the tail vein: B16-F10 is a syngeneic mouse transplantable tumor selected because of its high incidence of pulmonary metastases (Poste, et al., 1981). Interferon treatment was initiated 3 days after the introduction of the tumor cells. Each mouse received 100 p1 of either PBS alone, PBS containing 1 X 10 5 units of OvlFNr, or PBS containing 1 X 10 5 units of recombinant murine IFN (MulFNa), i.v. per day for 3 consecutive days.

Mice were sacrificed at 21 days and the lungs were preserved in buffered formalin. The frequency of pulmonary metastases were compared between control mice (PBS), OvlFNT-treated mice, and MulFNa-treated mice.

The results of these in vivo administrations demonstrated that OvlFNr dramatically reduced B16-F10 pulmonary tumors. These results support the use of IFN- as an efficacious antineoplastic agent in vivo.

E:\KRH689450SP.DOC EXAMPLE 17 Competitive Binding of IFNT Peptide Fragments A. The Ability of IFN--Based Peptides to Block IFN- and IFN-a Antiviral Activity.

Overlapping synthetic peptides were synthesized corresponding to the entire IFNT sequence (Figure Average hydropathicity values were calculated by taking the sum of the hydropathy values for each amino acid divided by the total number of amino acids in each sequence. Hydropathy values were taken from Kyte, et a. (1982).

These peptides were of approximately the same molecular weight but differed slightly in overall hydrophilicity. Despite this difference, all peptides were antigenic as demonstrated by the production of rabbit antisera with titers greater than 1:3,000 as assessed by ELISA (Harlow, et al.).

The peptides were used to inhibit the antiviral activity (Example 2) of OvlFNt and rBolFNa. The results of this analysis are presented in Figure 12: 1 mM N- and C-terminal peptides both effectively blocked the antiviral activity of OvIFNt using MDBK cells. A third peptide, representing amino acids 62-92, also reduced IFNT antiviral activity (70% inhibition). The peptide OvlFN- (119- 150) showed minimal inhibitory activity. The OvIFNT (34-64) and (90-122) peptides had no apparent inhibitory activity.

Peptide inhibition of OvIFNT antiviral activity was also examined as follows. Monolayers of Madin Darby bovine kidney cells were incubated with units/mi OvIFNT in the presence or absence of various concentrations of OvIFNt peptides (see Figure 13). Results in Figure 13 are expressed as the percent of control antiviral activity: that is, in the absence of any competing peptide. Data presented are the means of 6 replicate experiments. The data demonstrate that inhibition by OvlFNr (62-92), (119-150), and (139-172) were significantly different than OvIFNT (34-64) and (90-122) at 10 3 M and 3 x 10-3 M. OvlFNt (139-172) was significantly different than all other peptides at 10 3 M. Significance was assessed by analysis of variance followed by Scheffe's F test at p 0.05. Thus, OvlFNt (1-37) (62-92), (119-150), and (139- E:\KRH\689450SP.DOC 61 172), in particular (139-172), may represent receptor binding regions for IFN.

The ability of the OvlFNt peptides to inhibit bovine IFNa (BolFNa) antiviral activity was examined as follows. Monolayers of Madin Darby bovine kidney cells were incubated with 40 units/ml bovine IFNa in the presence or absence of various concentrations of OvIFNt peptides. The results are presented in Figure 14 and are expressed as the percent of control antiviral activity in the absence of OvIFN- peptides. The data presented are the means of 4 replicate experiments. The results indicate that inhibition by OvIFN- (62- 92), (119-150), and (139-172) were significantly different from OvlFNt (1-37), (34-64) and (90-122) at 10- 3 M. OvIFNT (139-172) was significantly different than OvlFNt (34-64) and (90-122) at 3 x 10- 3 M. Significance was assessed by analysis of variance followed by Scheffe's F test at p 0.05.

Thus, OvlFNt (62-92), (119-150), and (139-172), in particular (139-172), may Srepresent common receptor binding regions for IFNt and bovine IFN.

Peptide inhibition by OvlFN- peptides of human IFNa antiviral activity was also examined. Monolayers of Madin Darby bovine kidney cells were incubated with 40 units/ml human IFNa in the presence or absence of various concentrations of OvlFNt peptides. The results are expressed as the percent *of control antiviral activity in the absence of OvIFNT peptides. The data are presented in Figure 15 and are the means of 3 replicate experiments. OvIFN- (139-172) was significantly different from all other peptides at 10- 3

M.

"Significance was assessed by analysis of variance followed by Scheffe's F test at p 0.05. Thus, OvIFNt (139-172) may represent a common receptor binding region for IFNr and various IFNa(s).

The OvIFNt peptides described above appear to have no effect on the antiviral activity of IFNy. Peptide inhibition of bovine IFNy antiviral activity was evaluated as follows. Monolayers of Madin Darby bovine kidney cells were incubated with 40 units/ml bovine IFN gamma in the presence or absence of various concentrations of OvlFNT peptides. Results are expressed as the percent of control antiviral activity in the absence of OvIFNT peptides. The data are presented in Figure 16 and are the means of 3 replicate experiments.

E:\KRH\689450SP.DOC There were no significant differences among peptides as assessed by analysis of variance followed by Scheffe's F test at p 0.05.

The two synthetic peptides OvlFN-(1-37) and OvlFNt(139-172) also blocked OvIFNt anti-FIV and anti-HIV activity. Reverse transcriptase (RT) activity (Examples 12 and 13) was monitored over a 14 day period in FIVinfected FET-1 cells (1 X 10 6 /ml) and HIV-infected HPBL (1 X 10 6 Control cultures received no OvIFNt. OvlFNt was used at 100 ng/ml, and peptides were used at 200 pM. Data from a representative experiment are expressed as cpm/ml culture supernatant and are presented for FIV infected cells, Figure 11A, and HIV infected cells, Figure 11B. Both the N- and C-terminus of OvlFNt appear to be involved in its anti-retroviral activity. While both peptides blocked 0 FIV RT activity, only the C-terminal peptide, OvlFN-r(139-172), was an efficient inhibitor of vesicular stomatitis virus activity on the feline cell line, Fc9. Thus the C-terminal regions of type I IFNs may bind to common site on the type I IFN 15 receptor, while the N-terminal region may be involved in the elicitation of unique functions.

B. Anti-Peptide Sera.

The ability of anti-peptide antisera to inhibit OVIFNT antiviral activity was also determined. Antipeptide antisera inhibition of OvIFN-r antiviral activity was evaluated as follows. Monolayers of MDBK cells were incubated with units/mi of OvlFNt in the presence a 1:30 dilution of either preimmune sera or antisera to each of the OvIFNr peptides described above. In Figure 17 the data from duplicate experiments are presented as the mean percent inhibition of OvIFNt antiviral activity produced by antipeptide antisera relative to the appropriate preimmune sera standard error. Significant differences were assessed by analysis of variance followed by Scheffe's F test at p 0.05. Consistent with peptide inhibition of antiviral activities, sera containing antibodies immunoreactive to OvIFNr OvlFNr (62-92), and OvlFNT (139-172) were also the most effective inhibitors of OvlFNr antiviral activity, with antibodies directed against the N-terminal and C-terminal peptides being the most efficacious.

E:\KRH\689450SP.DOC 63 The same sera were also used to examine their effect on the binding of IFN, to its receptor.

The IFNT binding assay was carried out as follows. Five pg of IFN- was iodinated for 2 minutes with 500 pCi of Na 1 25 1 (15 mCi/pg; Amersham Corporation, Arlington Heights, IL) in 25 pl of 0.5 M potassium phosphate buffer, pH 7.4, and 10 pl of chloramine-T (5 mg/ml) (Griggs, et al., 1992). The specific activity of the iodinated protein was 137 pCi/pg. For binding assays, monolayers of MDBK cells were fixed with paraformaldehyde and blocked with nonfat dry milk. Cells were incubated with 5 nM 1 25 1-IFNT in phosphate So buffered saline with 1% BSA for 2 hours at 4 0 C in the presence or absence of a 1:30 dilution of sera containing antibodies raised against IFNT peptides or the appropriate preimmune sera. Specific binding was assessed by incubation with a 100-fold molar excess of unlabeled IFNc. Specific binding of 36% was determined by competition with 500 nM unlabeled IFNT. For example, total counts bound were 6850 133, and a 100-fold molar excess of OvIFNproduced 4398 158 counts per minute. After incubation, the monolayers were washed three times, solubilized with 1% sodium dodecyl sulfate, and the radioactivity counted. Data from three replicate experiments are presented in Figure 18 as the mean percent reduction of OvIFNr specific binding produced by antipeptide antisera relative to the appropriate preimmune sera standard S: deviation. Significant differences were assessed by analysis of variance followed by Scheffe's F test.

The same sera (containing antibodies immunoreactive to OvlFNT (1-37), OvlFNt (62-92), and OvlFNT (139-172)) were the most effective inhibitors of 1251-IFNt binding to its receptor on MDBK cells. The lack of effect of sera immunoreactive with other IFNt-derived peptides was not a function of titer against OvIFNT, since each sera had equal or greater titer to their respective peptide relative to the three inhibiting sera: similar results were obtained when sera reactivity against the whole OvlFNt molecule was assessed by ELISA for each sera.

These peptides, although apparently binding to the interferon receptor, E:\KRH\689450SP.DOC 64 did not in and of themselves elicit interferon-like effects in the cells.

C. Anti-Proliferative Activity.

Functionally important sites for the antiproliferative activity of IFN- were also examined using synthetic peptides (Table 11). Cellular proliferation was assayed as described above using MDBK cells. MDBK cells were cultured at x 105 cells/well in experiments 1 and 2 or 10 x 10 5 cells in experiment 3 and treated with medium alone, IFNr at a concentration of 300 units/ml and peptides at 1 mM for 48 hours. Duplicate wells were counted in each of three replicate experiments. For statistical analysis, data were normalized based on medium alone and assessed by analysis of variance followed by Least Significant Difference multiplate comparison test (p 0.05).

Table 11 Peptide Inhibition of IFNt Antiproliferative Activity Experiment I Experiment 2 Experiment 3 Cell Via- Cell Via- Cell Via- Count bility Count bility Count bility Medium alone 9.8x10" 99% 13.0x105 96% 27.3x10 '.00 a a. 0 0 IFNT 5.0x10 5 98% 5.6x10 5 97% 8.3x10 5 97% IFNT+IFNT(1-37) 6.3x10 5 100% 10.6x10 5 98% 13.4x10 100% IFNT+IFNT(34-64) 5.3x10 5 96% 6.9x10 5 95% 16.0x10 5 98% IFNT+IFNt(62-92) 6.5x10 5 97% 9.2x10 5 93% 8.9x10 5 96% IFNT+IFNT(90-122) 5.9x10 5 100% 11.0x10 5 97% 19.6x10 5 98% IFNT+IFNT(119-150) 8.4x10 5 100% 13.2x10 5 96% 31.8x10 5 IFNt+IFNT(139-172) 5.1x10 s 100% 12.7x10 5 98% 18.9x10 5 98% When proliferation of MDBK cells was monitored over a two-day period, cell number increased roughly 2-fold with greater than 95% viability. Addition of 300 units/ml of OvlFNt entirely eliminated cell proliferation without a decrease in cell viability. Ovine IFNr (119-150) was the most effective inhibitor of IFNt antiproliferative activity.

Antisera to IFN- (119-150), which inhibited binding of OvlFNT to receptor, also reversed the OvlFNt antiproliferative effect. Several other E:\KRH\689450SP.DOC peptides, notably IFNr (139-172), reversed the OvlFNT antiproliferative effect, but to a lesser extent.

EXAMPLE 18 Further Analysis of the Cellular and Anti-Viral Effects of IFNT A. HIV Anti-Viral Effects.

The antiviral effects of IFN-r against HIV were evaluated by treating human PBMC cells with various amounts of either recombinant ovine IFN- (r- OvlFNT) or recombinant human IFNUo 2 at the time of infection with HIV. Drug was present throughout the experiment. At day 7 and day 14, p24 production was determined (by ELISA (Wang, et al., 1988, 1989) and compared to a zero drug control. The results of this analysis are presented in Table 12.

Table 12 Inhibition Inhibition Day 7 Day 14 IFNx IFNT 58%, 48% 91%, 91% 26 48%, 45% 88%, 59% 100 68%, 74% 94%, 91% 260 58%, 51% 82%, 1,000 89%, 86% 97%, 93% 2,600 65%, 68% 87%, 79% 10,000 90%, 86% 99%, 99% 26,000 77%, 85% 77%, 96% 260,000 85%, 84% 96%, 86% The data from these experiments support the conclusion that, at relatively low concentrations, IFNa and IFNr are effective in reducing the replication of HIV in human lymphocytes.

E:.KRH\689450SP.DOC 66 B. In vitro Cytotoxicity Test in PBMC's Human PBMC's were seeded at 5 x 10 5 cells/mi. Cells were stimulated at day 0 with 3 gg/ml PHA. Cells were treated with recombinant human IFN2A (at concentrations of 10, 100, 1,000 and 10,000 units/ml) and IFNr (at concentrations of 2.6, 26, 260, 2,600, 26,000, 260,000, and 2,600,000 units/mi) in 200 gl/wells (4 replicates of each concentration using 96 well flat bottom plates). Control cultures were given no interferons. After 4 days of incubation, cells were pulsed for 9 hours using 3H-thymidine at 1 uCi/well. The cells were harvested and the incorporation of labeled thymidine into DNA was determined (Figure 8).

No cytotoxicity was observed by measuring the uptake of thymidine at any concentration of IFNr. However, rHulFNa2 was toxic to cells at 1,000 units/ml.

In a second experiment, the same human PBMC's were treated with either IFNt or human IFNa2A at concentrations of 100 units/ml or 10,000 units/ml. After 3 days or 8 days of incubation, viable cells were counted by flow cytometry. The results of this analysis are presented in Table 13.

TABLE 13 Day 3 Day 8 No Treatment 735 840 INFt 100 units/ml 745 860 IFNr 10,000 units/ml 695 910 IFNa 100 units/ml 635 750 IFN 10,000 units/ml 680 495 No cytotoxicity was observed in the cells treated with IFNt. However, there was 10% cell death in IFNa treated cells at Day 3 and 49% cell death at Day 8.

C. Inhibition of Hepatitis B Virus DNA Replication in Hepatocytes The cell line used, HepG2-T14, is a human cell that was derived from E:\KRH\689450SP.DOC 67 liver cells transfected with Hepatitis B Virus (HBV). The cell line semi-stably produces HBV virus: over time the cell line's production of HBV intracellular DNA and secreted virus decreases. In order to maximize production of HBV DNA and virus, the cells are pre-treated with deAZA-C (5-azacytidine; Miyoshi, et al.) to induce production of the virus. Treatment was for 2-3 days and the amount of induction was about a factor of two.

The cells were then treated with either the IFNa and IFNT at levels of 0, 5,000, 10,000, 20,000 and 40,000 units per ml.

All levels of either IFNa or IFN- reduced DNA production by about a factor of 2 compared to the no drug control.

D. Inhibition of Hepatospecific Messenger RNA Production in S Hepatocytes The hepatocyte cell line HepG2-T14 (described above) was examined for the effects of IFNa and IFNr on hepatospecific mRNA production. Cells were incubated in concentrations of IFNa or IFNt at 0, 5,000, 10,000, 20,000, and 40,000 units per ml. The messenger RNAs for the hepatocyte specific proteins Apo E and Apo Al were detected by hybridization analysis (Sambrook, et al.; Maniatis, et al.) using probes specific for these two mRNA's (Shoulders, et al., and Wallis, et al.).

No reduction of mRNA production was seen for Apo E or Apo Al mRNA production with up to 40,000 units of either IFNa or IFNc. This result suggests that the reduction of viral DNA replication in previous experiments was not due to the effects of IFNs on cellular house-keeping activities; rather the reduction was likely due to specific inhibition of viral replication in the host cells.

EXAMPLE 19 Isolation of Interferon-T Fusion Protein Sepharose 4B beads conjugated with anti-beta galactosidase is purchased from Promega. The beads are packed in 2 ml column and washed successively with phosphate-buffered saline with 0.02% sodium azide and ml TX buffer (10 mM Tris buffer, pH 7.4, 1% aprotinin).

The IFNr coding sequence Figure 7) is cloned into the polylinker E:~KRH\689450SP.DOC site of lambda gt11. The IFN coding sequence is placed in-frame with the amino terminal p-galactosidase coding sequences in lambda gt11. Lysogens infected with gt11/IFNT are used to inoculate 500 ml of NZYDT broth. The culture is incubated at 32°C with aeration to an O.D. of about 0.2 to 0.4, then brought to 43°C quickly in a 43°C water bath for 15 minutes to induce gt11 peptide synthesis, and incubated further at 37°C for 1 hour. The cells are pelleted by centrifugation, suspended in 10 ml of lysis buffer (10 mM Tris, pH 7.4 containing 2% "TRITON X-100" and 1% aprotinin added just before use.

The resuspended cells are frozen in liquid nitrogen then thawed, resulting in substantially complete cell lysis. The lysate is treated with DNasel to digest bacterial and phage DNA, as evidenced by a gradual loss of viscosity in the lysate. Non-solubilized material is removed by centrifugation.

The clarified lysate material is loaded on the Sepharose column, the ends of the column closed, and the column placed on a rotary shaker for 2 hrs.

at room temperature and 16 hours at 4°C. After the column settles, it is washed with 10 ml of TX buffer. The fused protein is eluted with 0.1 M a a carbonate/bicarbonate buffer, pH10. Typically, 14 ml of the elution buffer is passed through the column, and the fusion protein is eluted in the first 4-6 ml of eluate.

The eluate containing the fusion protein is concentrated in cartridges (Amicon, Danvers, Mass.). The final protein concentrate is resuspended in, for example, 400 pl PBS buffer. Protein purity is analyzed by SDS-PAGE.

For polyclonal antibodies, the purified fused protein is injected subcutaneously in Freund's adjuvant in a rabbit. Approximately 1 mg of fused protein is injected at days 0 and 21, and rabbit serum is typically collected at 6 and 8 weeks.

EXAMPLE Preparation of Anti-IFNT Antibody A. Expression of Glutathione-S-Transferase Fusion Proteins.

The IFNr coding sequence Figure 7) is cloned into the pGEX E:\KRH\689450SP.DOC 69 vector (Boyer, et Frangioni, et al.; Guan, et al.; Hakes, et al.; Smith, et al., 1988). The pGEX vector (Smith, et al.) was modified by insertion of a thrombin cleavage sequence in-frame with the glutathione-S-transferase protein (GST sj26 coding sequence). This vector is designated pGEXthr. The IFNr coding sequence is placed in-frame with the sj26-thrombin coding sequences (Guan, et al.; Hakes, et The IFNt coding sequence insert can be generated by the polymerase chain reaction using PCR primers specific for the insert.

The IFNr fragment is ligated to the linearized pGEXthr vector. The ligation mixture is transformed into E. coli and ampicillin resistant colonies are selected. Plasmids are isolated from the ampicillin resistant colonies and :i analyzed by restriction enzyme digestion to identify clones containing the IFNT insert (vector designated pGEXthr-IFN).

E. coli strain XL-I Blue is transformed with pGEXthr-IFNr and is grown at 37 0 C overnight. DNA is prepared from randomly-picked colonies. The presence of the insert coding sequence is typically confirmed by restriction digest mapping, (ii) hybridization screening using labelled IFNr probes Southern analysis), or (iii) direct DNA sequence analysis.

B. Partial Purification of Fusion Proteins.

A pGEXthr-IFNt clone is grown overnight. The overnight culture is 20 diluted 1:10 with LB medium containing ampicillin and grown for one hour at 370C. Alternatively, the overnight culture is diluted 1:100 and grown to OD of 0.5-1.0 before addition of IPTG (isopropylthio-p-galactoside). IPTG (GIBCO- BRL, Gaithersburg MD) is added to a final concentration of 0.2-0.5 mM for the induction of protein expression and the incubation is typically continued for hours, preferably 3.5 hours.

Bacterial cells are harvested by centrifugation and resuspended in 1/100 culture volume of MTPBS (150 mM NaCI, 16 mM Na 2

HPO

4 4 mM NaH 2

PO

4 Cells are lysed by lysozyme, sonication or French press, and lysates cleared of cellular debris by centrifugation.

An aliquot of the supernatant obtained from IPTG-induced cultures of pGEXthr-IFN-r-containing cells and an aliquot of the supernatant obtained from E:\KRH\689450SPDOC IPTG-induced cultures of pGEXthr-vector alone are analyzed by SDSpolyacrylamide gel electrophoresis followed by Western blotting, as described below.

If necessary, the extracts can be concentrated by ultrafiltration using, for example, a "CENTRICON 10" filter.

Alternatively, the fusion proteins are partially purified over a glutathione agarose affinity column as described in detail by Smith, et al. In this method, 100 ml cultures are grown overnight. The cultures are diluted to 1 liter, and the cells grown another hour at 37 0 C. Expression of the fusion proteins is induced using IPTG. The induced cultures are grown at 37°C for 3.5 hours. Cells are harvested and a sonicator used to lyse the cells. Cellular debris is pelleted and the clear lysate loaded onto a glutathione "SEPHAROSE" column. The column is washed with several column volumes. The fusion protein is eluted from the affinity column with reduced glutathione and dialyzed. The IFN- can be liberated from the hybrid protein by treatment with thrombin. The sj26 and IFNr fragments of the hybrid protein can then be separated by size fractionation over columns or on gels.

Alternatively, the IFNt portion of the hybrid protein is released from the column by treatment with thrombin (Guan, et al.; Hakes, et al.).

C. Antibodies Against the Fusion Protein.

The purified Sj26/IFNt fused protein is injected subcutaneously in Freund's adjuvant in a rabbit. Approximately 1 mg of fused protein is injected at days 0 and 21, and rabbit serum is typically collected at 6 and 8 weeks. A second rabbit is similarly immunized with purified Sj26 protein obtained from control bacterial lysate.

Minilysates from the following bacterial cultures are prepared: (1) KM392 cells infected with pGEXthr and pGEXthr containing the IFN insert; and cells infected with lambda gt11 containing the IFNr insert. The minilysates and a commercial source P-galactosidase are fractionated by SDS-PAGE, and the bands transferred to nitrocellulose filters for Western blotting (Sambrook, et al.; Ausubel, et al.).

E:\KRH\689450SP.DOC 71 Summarizing the expected results, serum from control (Sj26) rabbits is immunoreactive with each of the Sj26 and Sj26 fused protein antigens. Serum from the animal immunized with Sj26/IFN- fused protein is reactive with all Sj- 26 and beta-gal fusion proteins containing IFNt coding sequences, indicating the presence of specific immunoreaction with the IFNT antigen. None of the sera are expected to be immunoreactive with beta-galactosidase.

Anti-IFNt antibody present in the sera from the animal immunized with the Sj26/IFNT is purified by affinity chromatography (using immobilized recombinantly produced IFN- as ligand, essentially as described above in Example 12 for the anti-beta-galactosidase antibody).

W" hile the invention has been described with reference to specific methods and embodiments, it will be appreciated that various modifications and changes may be made without departing from the invention.

E:\KRH\689450SP.DOC SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: University of Florida The Women's Research Institute (ii) TITLE OF INVENTION: Interferon Tau Compositions and Methods of Use .(iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Law Offices of Peter J. Dehlinger 15 STREET: 350 Cambridge Ave., Suite 300 CITY: Palo Alto STATE: CA S.I. COUNTRY: USA ZIP: 94306 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS 25 SOFTWARE: PatentIn Release Version #1.25 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 07/969,890 FILING DATE: 30-OCT-1992 (viii) ATTORNEY/AGENT INFORMATION: NAME: Fabian, Gary R.

REGISTRATION NUMBER: 33,875 REFERENCE/DOCKET NUMBER: 5600-0001.41 (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: 415-324-0880 73 TELEFAX: 415-324-0960 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 518 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Ovis aries S(B) STRAIN: Domestic 20 DEVELOPMENTAL STAGE: Blastula (blastocyst) TISSUE TYPE: Trophectoderm CELL TYPE: Mononuclear trophectoderm cells (vii) IMMEDIATE SOURCE: CLONE: oTP-la (viii) POSITION IN GENOME: UNITS: bp (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..518 PUBLICATION INFORMATION: AUTHORS: Ott, Troy L Van Heeke, Gino Johnson, Howard M Bazer, Fuller W TITLE: Cloning and Expression in Saccharomyces cerevisiae of a Synthetic Gene for the Type I Trophoblast Interferon Ovine Trophoblast Protein-1:Purification and Antiviral Activity JOURNAL: J. Interferon Res.

VOLUME: 11 PAGES: 357-364 DATE: 1991 RELEVANT RESIDUES IN SEQ ID NO:1: FROM I TO 518 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: TGC TAC CTG TCG CGA AAA CTG ATG CTG GAC OCT CGA 9999 99 9 9 999.

999.99 9 9* *t 9 9 9 999.

9 *9 9 9* *9*9 99 9* 9 99 9 9 9 9.9.

99 99 9 999 9 .9 9 9* Cys Tyr Leu Ser Arg Lys Leu Met Leu Asp Ala A-rg 10 GAA AAT TTA AAA Glu Asn Leu Lys is 15 CTG CTG GAC Leu Leu Asp COG AAA GAC 20 Arg Lye Asp CGT ATG Arg Met 2D AAT CGA TTG Asn Arg Leu TCT CCG CAC Ser Pro His 25 AGC TGC Ser Cys

CTG

Leu CAA GAC Gin Asp TTC GOT CG CCC Phe Gly Leu Pro

CAG

Gin 40 GAA ATG OTT GAA GOT GAG CAA CTG Glu Met Val Giu Gly Asp Gin Leu CAA AAA Gin Lys 50 GAC CAA OCT TTC Asp Gin Ala Phe

CCG

Pro 55 OTA CTO TAT OAA Val Leu Tyr Glu

ATO

Met CTG GAG CAC TCT Leu Gin Gin Ser

TTC

Phe AAG CTG TTC TAC Asn Leu Phe Tyr GAA CAT TCT TCG Glu His Ser Ser

GCC

Ala OCT TG-G GAG ACT Ala Trp Asp Thr CTT CTA GAA CAA CTO TG ACT OCT CTG Leu Leu Giu Gin Leu Gys Thr Oly Leu CAP CAA CTO GAG Gin Gin Leu Asp CAT GTG His Leu 288 GAG ACT TG Asp Thr Gys

COT

Arg 100 C CGAG OTT ATG OGT OAA Gly Gin Val Met Gly Olu 105 GAA GAG TOT 01u Asp Ser GAA CTO GOT Glu Leu Gly 110 AAC ATO OAT Asn Met Asp 115 CCG ATC OTT ACT OTT AAA AAA TAT TTC GAG GGT ATC TAG Pro Ile Val Thr Val Lye Lye Tyr Phe Gin Gly Ile Tyr GAC TAC CTG CAG GAA A.AA GOT TAC Asp Tyr Leu Gin Olu Lys Gly Tyr 130 135 CGC OTT OAA ATG ATG COG 0CC CTG Arg Val Giu Met Met Arg Ala Leu 145 150 CGG TTA ACT AAA ATG GOT GGT GAC Arg Leu Thr Lys Met Gly Gly Asp 165 INFORMATION FOR SEQ ID NO:2: TCT GAC Ser Asp TG OCT TOO GAA ATC OTA Cys Ala Trp Olu Ile Val ACT OTO TCG Thr Val Ser 155 ACT ACT CTO CAA Thr Thr Leu Gin

AAA

Lys 160 CTO AAT TCT CCO Leu Asn Ser Pro 170 518 boom too, **o boo* so 9 *t S of 9 0.

SEQUENCE CHARACTERISTICS: LENGTH: 172 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Cys Tyr Leu Ser Arg Lye Leu Met Leu Asp Ala Arg Giu Asn Leu Lye Leu Leu Asp Arg Met Aen Arq Leu Ser Pro 25 His Ser Cys Arg Lye Asp Phe Gly Leu Pro Gin Giu Met Vai Giu Gly 40 Leu Gin Asp Asp Gin Leu Gin Gin Ser Gin Lys Asp Gin Ala Phe Pro Vai Leu Tyr Giu Met Leu 55 Phe Aen Leu Phe Tyr Thr Giu His Ser Ser Ala Ala Trp Asp Thr Thr 70 75 Leu Leu Giu Gin Leu Cys Asp Thr Cys Arg Gly Gin 100 Thr Gly Leu Gin Gin Leu Asp His Leu Val Met Gly 105 Lys Giu Giu Asp Ser Asn Met Asp Pro Ile Vai 115 Asp Tyr Leu Gin Giu Lys 130 Arg Val Giu Met Met Arg 145 150 Arg Leu Thr Lys Met Gly 165 Thr Gly 135 Lys Tyr Phe Gin 125 Trp Glu Leu Gly 110 Giy Ile Tyr Glu Ile Val Ser Asp Cys Aia 140 a a 0 C. a a.

ta a a a.

a.

Ala Leu Thr Val Gly Asp Leu Asn 170 Ser Thr 155 Ser Pro Thr Leu Gin Ly s 160 20 INFORMATION FOR SEQ ID NO:]: SEQUENCE CHARACTERISTICS: LENGTH: 516 base pairs TYPE: nucleic acid 25 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TGTGACTTGT CTCAAAACCA CGTTTTGGTT GGTAGAAAGA ACTTAAGACT ACTAGACGAA ATGAGACGTC TATCTCCACA CTTCTGTCTA CAAGACAGAA AGGACTTCGC TTTGCCTCAG GAAATGGTTG AAGGTGGCCA ACTACAAGAA GCTCAAGCGA TATCTGTTTT GCACGAAATG TTGCAACAAA GCTTCAACTT GTTCCACACC GAACACTCTT CGGCCGCTTG GGACACCACC TTGTTGGAAC CATGTAGAAC CGGTTTGCAC CAACAATTGG ACAACTTGGA TGCATGTTTG 120 180 240 300 GGTCAAGTTA TGGGTGAAGA AGACTCTCCT CTCGGGAGAA CCGGTCCAAC GCTAGCTTTG AAGAGATACT TCCAAGGTAT CCACGTTTAC TTGA' AGGAAA AGGGTTACTC TCACTGTGCT TGGGAAACCG TGCGTCTAGA AATCATGCGT AGCTTCTCTT CTTTGATCAG CTTGCAAAA AGATTACGTA TGATGGACGG TGACTTGTCG AGCCCA INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 172 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein 360 420 480 516 4* 4* *4 4.*9 44 *4 4 U 0* 4 44 4* (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Cys Asp Leu Ser Gin Asn His Val Leu Val 1 5 10 Leu Leu Asp Giu Met Arg Arg Leu Ser Pro 20 25 Arg Lys Asp Phe Ala Leu Pro Gin Giu Met 40 Gin Glu Ala Gin Ala Ile Ser Val Leu His 55 Phe Asn Leu Phe His Thr Giu His Ser Ser 70 Leu Leu Giu Pro Cys Arg Thr Gly Leu His 90 Gly Arg Lys Asn Leu Arg His Phe Cys Val Giu Gly Leu Leu Gin Asp Gly Gin Leu Gin Gin Ser Glu Met Ala 75 Gin Ala Trp Asp Thr Thr Gin Leu Asp Asn Leu 78 Asp Ala Cys Leu Gly Gin Val Met Gly Glu Glu Asp Ser Ala Leu Gly 100 105 110 Arg Thr Gly Pro Thr Leu Ala Leu Lys Arg Tyr Phe Gin Gly Ile His 115 120 125 Val Tyr Leu Lys Glu Lys Gly Tyr Ser Asp Cys Ala Trp Glu Thr Val 130 135 140 Arg Leu Glu Ile Met Arg Ser Phe Ser Ser Leu Ile Ser Leu Gin Glu 145 150 155 160 Arg Leu Arg Met Met Asp Gly Asp Leu Ser Ser Pro 165 170 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: **20 LENGTH: 37 amino acids TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Cys Tyr Leu Ser Arg Lye Leu Met Leu Asp Ala Arg Glu Asn Leu Lys 10 Leu Leu Asp Arg Met Asn Arg Leu Ser Pro His Ser Cys Leu Gin Asp 25 Arg Lys Asp Phe Gly INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 31 amino acids 79 TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Lys Asp Phe Gly Leu Pro Gin Glu Met Val Glu Gly Asp Gin Leu Gin 10 Lys Asp Gin Ala Phe Pro Val Leu Tyr Glu Met Leu Gin Gin Ser 25 99* INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 31 amino acids TYPE: amino acid 20 TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Gin Gin Ser Phe Asn Leu Phe Tyr Thr Glu His Ser Ser Ala Ala Trp 5 10 Asp Thr Thr Leu Leu Glu Gin Leu Cys Thr Gly Leu Gin Gin Gin 20 25 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 33 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Gin Gin Gin Leu Asp His Leu Asp Thr Cys Arg Gly Gin Val Met Gly 10 Glu Glu Asp Ser Glu Leu Gly Asn Met Asp Pro Ile Val Thr Val Lys 25 Lys INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: 15 LENGTH: 32 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Thr Val Lys Lys Tyr Phe Gin Gly Ile Tyr Asp Tyr Leu Gln Glu Lys 10 Gly Tyr Ser Asp Cys Ala Trp Glu Ile Val Arg Val Glu Met Met Arg 20 25

S.

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 34 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Cys Ala Trp Glu Ile Val Arg Val Glu Ser Thr Thr Leu Gin Lys Arg Leu Thr 25 Met Met Arg Ala Leu Thr Val 10 Lys Met Gly Gly Asp Leu Asn Ser Pro a a.

p 9 p. p p p

C*~

S.

a app INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 588 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 20 (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: 25 INDIVIDUAL ISOLATE: Human Interferon Tau Coding Sequences (ix) FEATURE: NAME/KEY: CDS LOCATION: 585 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: ATG GCC TTC GTG CTC TCT CTA CTC ATG GCC CTG GTG CTG GTC Met Ala Phe Val Leu Ser Leu Leu Met Ala Leu Val Leu Val AGC TAC Ser Tyr GGC CCA GGA GGA TCC CTG GGT TGT GAC CTG TCT CAG AAC Gly Pro Gly Gly Ser Leu Gly Cys Asp Leu Ser Gin Asn CAC GTG CTG His Val Leu GTT GGC Val Gly AGG AAG Axg Lys AAC CTC AGG CTC CTG Asn Leu Arg Leu Leu 40 GAC GAA ATG AGG Asp Giu Met Arg AGA CTC TCC Arg Leu Ser CCT CAC Pro His TTT TGT CTG CAG Phe Cys Leu Gin

GAC

Asp 55 AGA AAA GAC TTC Arg Lys Asp Phe

GCT

Ala TTA CCC CAG GAA Leu Pro Gin Giu

ATG

Met GTG GAG GGC GGC Val Glu Gly Gly

CAG

Gin 70 CTC CAG GAG GCC Leu Gin Giu Ala

CAG

Gin 75 GCC ATC TCT GTG Ala Ile Ser Val

CTC

Leu ta..

a .a a a a.

CAT GAG ATG CTC CAG CAG AGC TTC AAC His Giu Met Leu Gin Gin Ser Phe Asn 85

CTC

Leu 90 TTC CAC ACA GAG Phe His Thr Glu CAC TCC His Ser 288 336 TCT GCT GCC Ser Ala Ala CAT CAG CAG His Gin Gin 115

TGG

Trp 100 GAG ACC ACC CTC Asp Thr Thr Leu

CTG

Leu 105 GAG CCA TGC CC Giu Pro Cys Arg ACT GGA CTC Thr Gly Leu 110 GTG ATG GGA Val Met Gly CTG GAC AAC CTG Leu Asp Asn Leu

GAT

Asp 120 GCC TGC CTG GGG Ala Cys Leu Gly

CAG

Gin 125 GAG GAA Giu Giu 130 GAC TCT GCC CTG Asp Ser Ala Leu AGG ACO GGC CCC Arg Thr Gly Pro CTG GCT CTG AAG Leu Ala Leu Lys

AGG

Arg 145 TAC TTC CAG GCC Tyr Phe Gin Cly

ATC

Ile 150 CAT GTC TAC CTG His Vai Tyr Leu*

AAA

Lys 155 GAG AAG GGA TAG Ciu Lys Gly Tyr GAC TGC GCC TGG Asp Cys Ala Trp

GAA

Giu 165 ACC GTC AGA CTG Thr Val Arg Leu ATC ATG AGA TC Ile Met Arg Ser TTC TCT Phe Ser 175 TCA TTA ATC AGC TTG CAA GAA AGG TTA AGA ATG ATG GAT GGA GAC CTG Ser Leu Ile Ser Leu Gin Giu Arg Leu Arg Met Met Asp Gly Asp Leu 180 185 190 AGC TCA CCT TGA Ser Ser Pro 195 INFORMATION FOR SEQ ID NO:i2: SEQUENCE CHARACTERISTICS: LENGTH: 195 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECUL~E TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: S.

S

*5 5 9 *5 *55t

S

a.

S.

S a *SS9

S

*5 a

S

S. S S *5 Met Ala Phe Val Leu Ser Leu Leu Met Ala Leu Val Leu Val Ser Tyr 1 20 Gly 5 Pro Gly Gly Ser 20 Leu Gly Cys 10 Asp Leu 25 Leu Asp Val Leu Ser Gin Asn His Val Giy Arg Pro His Phe 50 Lys Asn Leu Arg Giu Met A-rg Leu Ser Cys Leu Gin Asp Lys Asp Phe Leu Pro Gin diii Met 65 Val Glu Gly Gly Gin Leu Gin Giu Ala Ile Ser Val His Glu Met Leu Gin Ser Ala Ala Trp Asp 100 His Gin Gin Leu Asp 115 Gin Ser Phe Asn Leu Phe His Thr 90 Thr Thr Leu Leu Giu Pro Cys Arg 105 Asn Leu Asp Ala Cys Leu Gly Gin 120 125 dlu His Ser Thr Gly Leu 110 Val Met Gly Giu Glu 130 Asp Ser Ala Leu Gly 235 Ile His 150 A-rg Thr Gly Pro Leu Ala Leu Lys Arg 145 Tyr Phe Gin Gly Val Tyr Leu Giu Lys Gly Tyr Ser 160 Ser Asp Cys Ala Trp Glu 165 Thr Val Arg Leu Ile Met A-rg Ser Ser Leu Ile Ser Leu Gin Giu Arg Leu Arg Met Met Asp Gly Asp Leu 280 285 190 S. .5

S

S

*SS.

S.

5 9 5* *5 S

S

55.5

S

S.

S

5 *5 S 5* Ser Ser Pro 195 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 25 bases TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: iinear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i3: CCTGTCTGCA GGACAGAAAA GACTT INFORMATION FOR SEQ ID NO:i4: SEQUENCE CHARACTERISTICS: LENGTH: 25 bases TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: TCTGAATTCT GACGATTTCC CAGGC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 37 amino acids TYPE: amino acid TOPOLOGY: linear S(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: INDIVIDUAL ISOLATE: Amino acid sequence of fragment 15 1-37, Human Tau-IFN

**S

*a (xi) SEQUENCE DESCRIPTION: SEQ ID Cys Asp Leu Ser Gin Asn His Val Leu Val Gly Arg Lys Asn Leu Arg 1 5 10 Leu Leu Asp Glu Met Arg Arg Leu Ser Pro His Phe Cys Leu Gin Asp 20 25 Arg Lys Asp Phe Ala INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 31 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: INDIVIDUAL ISOLATE: Amino acid sequence of fragment 34-64, Human Tau-IFN (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Lys Asp Phe Ala Leu Pro Gin Glu Met Val Glu Gly Gly Gin Leu Gin 1 5 10 Glu Ala Gin Ala Ile Ser Val Leu His Glu Met Leu Gin Gin Ser 20 25 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: 15 LENGTH: 31 amino acids TYPE: amino acid TOPOLOGY: linear S(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: INDIVIDUAL ISOLATE: Amino acid sequence of fragment 62-92, Human Tau-IFN (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Gin Gin Ser Phe Asn Leu Phe His Thr Glu His Ser Ser Ala Ala Trp 1 5 10 Asp Thr Thr Leu Leu Glu Pro Cys Arg Thr Gly Leu His Gin Gin 25 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 33 amino acids TYPE: amino acid TOPOLOGY: linear 87 (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: INDIVIDUAL ISOLATE: Amino acid sequence of fragment 90-122, Human Tau-IFN (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: His Gin Gin Leu Asp Asn Leu Asp Ala Cys Leu Gly Gin Val Met Gly 1 5 10 15 Glu Glu Asp Ser Ala Leu Gly Arg Thr Gly Pro Thr Leu Ala Leu Lys 25 Arg INFORMATION FOR SEQ ID NO:19: S(i) SEQUENCE CHARACTERISTICS: LENGTH: 32 amino acids 25 TYPE: amino acid TOPOLOGY: linear

S

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: INDIVIDUAL ISOLATE: Amino acid sequence of fragment 119-150, Human Tau-IFN (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Ala Leu Lys Arg Tyr Phe Gin Gly Ile His Val Tyr Leu Lys Glu Lys 1 5 10 88 Gly Tyr Ser Asp Cys Ala Trp Giu Thr Val. Arg Leu Glu Ile Met A~rg 25 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 34 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: INDIVIDUAL ISOLATE: Amino acid sequence of fragment 139-172, Human Tau-IFN (xi) SEQUENCE DESCRIPTION: SEQ ID Cys Ala Trp Glu Thr Val. Arg Leu Glu Ile Met Arg Ser Phe Ser Ser a..1 5 10 Leu Ile Ser Leu Gin Giu Arg Leu Arg Met Met Asp Gly Asp Leu Ser 25 Ser Pro 89 THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. An isolated hybrid interferon protein, wherein a portion of a non-tau type I interferon consisting of the first 15 to 37 N-terminal amino acids of the mature sequence of the non-tau interferon, is replaced with the analogous first 15 to 37 N-terminal amino acids of the mature sequence of an interferon-tau, and said hybrid interferon has reduced cytotoxicity compared to that of the non-tau type I interferon.

2. The hybrid interferon of claim 1, wherein said non-tau type I interferon is selected from the group consisting of interferon-alpha and interferon-beta.

3. The hybrid interferon of claim 1 or claim 2, wherein said interferon-tau is selected from the group consisting of ovine interferon-tau and human interferon-tau.

4. The hybrid interferon of any one of claims 1 to 3, wherein the mature 15 amino acid sequence of said interferon-tau is substantially identical to SEQ ID NO:2 or SEQ ID NO:4.

S' 5. The hybrid interferon of claim 1 substantially as hereinbefore described with reference to any one of the examples.

6. An isolated nucleic acid which encodes the hybrid interferon of any one 20 of claims 1 to 7. An expression vector including the nucleic acid of claim 6; and regulatory sequences effective to express said nucleic acid in a host cell.

8. A method of recombinantly producing the hybrid interferon of any one of claims 1 to 5 including; introducing into suitable host cells, a recombinant expression system containing an open reading frame (ORF) having a polynucleotide sequence which encodes the hybrid interferon, where the vector is designed to express the ORF in said host, and culturing said host under conditions resulting in the expression of the ORF sequence.

E:\KRH689450SP.DOC

Claims (1)

1. 9. A method of inhibiting tumor cell growth, including contacting the cells with the hybrid interferon of any one of claims 1 to 5 at a concentration effective to inhibit growth of the tumor cells. A method of inhibiting viral replication in cells, including contacting cells infected with a virus with the hybrid interferon of any one of claims 1 to 5 at a concentration effective to inhibit viral replication within said cells. DATED: 24 June 1998 PHILLIPS ORMONDE FITZPATRICK Attorneys for: UNIVERSITY OF FLORIDA, THE WOMEN'S RESEARCH INSTITUTE 0 ss* *OO S. •O E:\KRH689450SP.DOC ABSTRACT The present invention relates to novel isolated hybrid interferon proteins which are useful for inhibiting tumor cell growth and to methods of preparing these proteins. 00 000* E'J(RH\689450SP.DOC