HK1129128B - Recombinant human interferon-like proteins - Google Patents

Description

Recombinant human interferon-like proteins

Technical Field

The present application relates to recombinant proteins having human interferon-like biological activity.

Background

The Interferon (IFN) term published in Nature (1) is used in this application.

Human interferons (huifns), discovered by Isaacs and Lindenmann in 1957, are a well-known family of cytokines that are secreted by various eukaryotic cells following exposure to various stimuli such as viral infection or exposure to mitogens. IFNs can cause a variety of changes in cell behavior, including affecting cell growth and differentiation and modulating the immune system (3-7). HuIFN is divided into 6 subtypes, namely IFN-alpha, IFN-beta, IFN-gamma, IFN-omega, IFN-epsilon and IFN-kappa. HuIFN- α (leukocyte-derived interferon) is produced in human leukocytes, while a small amount of HuIFN- β (fibroblast-derived interferon) is produced in lymphoblastoid cells. Huifns are further divided into two major classes, i.e., class I and class II, based on their chemical and biological characteristics. Type I includes IFN-alpha and IFN-beta subtypes as well as the more recently discovered IFN-omega, IFN-epsilon and IFN-kappa subtypes. Type II has only one member: IFN-gamma (immune interferon).

Different interferon subtypes have different structural and biological characteristics. HuIFN- β is an N-linked glycoprotein (8, 9) that has been purified to homogeneity and characterized. Its size is not uniform, probably due to its sugar moiety. However, only one human IFN- β gene exists, which encodes a protein of 166 amino acids. IFN- β has low homology to IFN- α, sharing about 30-40% identity.

Unlike the uniqueness of the IFN- β gene, HuIFN- α is a subtype composed of a multigene family of roughly 14 genes. Small variants formed by one or two amino acid differences account for the presence of multiple alleles (10). In addition to the pseudogene IFNAP22, there were 13 genes encoding 13 proteins. Each protein consists of 165-166 amino acids. The protein encoded by the IFNA13 gene is the same as the IFNA1 protein. Thus, there are 12 different interferon- α proteins: IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA14, IFNA16, IFNA17, and IFNA 21. The amino acid sequence identity between IFN-alpha subtypes is typically 80-85% (11).

Mature IFN-omega shows 60% nucleotide sequence homology with a family of IFN-alpha species, but has a6 amino acid C-terminus length. IFN- ω is more closely related to interferon- β (sharing about 30% sequence homology). Human IFN-omega is not classified in the IFN-alpha group because it differs antigenically from IFN-alpha and from its interaction with the type I IFN-alpha receptor (12). IFN-omega is secreted by virus-infected leukocytes as a major component of human leukocyte interferon.

Mature human IFN-. epsilon.protein contains 185 amino acids and shares about 33% and 37% sequence homology with IFN-. alpha.2 and IFN-. beta.respectively (13, 14). The functional and biophysical properties of IFN-epsilon have not been effectively characterized in detail; however, it functions like a type I interferon. IFN-epsilon may also play a role in reproductive function (15).

IFN-. kappa.is a 180 amino acid human cytokine, a recently identified type I IFN. The coding sequence for IFN-. kappa.has-30% identity to other type I interferons found in humans. One distinguishing feature of IFN- κ is: detectable constitutive expression of its transcript in non-induced cells, particularly keratinocytes. IFN- κ may play a role in regulating systemic or local immune function by affecting cells of the innate immune system (16). However, IFN-. kappa.exhibits low antiviral activity (17).

Human type I interferons appear to bind 2 receptor subunits, IFNAR-1 and-2, which are widely distributed on the cell surface of various cell types. The ligand participation induced coupled to IFNAR-1 and-2 tyrosine kinase TYK2 and JAK-1 phosphorylation. Once phosphorylated, STAT proteins are released from the receptor and form homodimers and heterodimers (18, 19). Upon release, the dimers of STATA bind to interferon-responsive factor 9(IRF-9), a DNA binding protein, thereby forming a complex called IFN-stimulated gene factor-3 (ISGF-3), which migrates into the nucleus. The ISGF-3 complex then binds to a DNA element present upstream of all IFN-inducible genes. This is the so-called "classical" signal transduction pathway.

New modes of action and biochemical pathways regulated by type I IFNs are continually being discovered. For example, downstream of PI3K in the signal transduction pathway, nuclear factors kappa-B (NF-. kappa.B) and PKC-d were associated with the anti-apoptotic effects observed in neutrophils incubated with IFN- β (20).

More than 300 genes (called interferon-induced genes) respond to IFN treatment. The most studied IFN proteins are those with antiviral properties. For example, enzymes of the 2, 5 oligoadenylate synthetase family (OAS-1 and-2) catalyze the synthesis of short oligoadenylates, which bind to and activate RNAseL, an enzyme that cleaves viral and cellular RNA, thereby inhibiting protein synthesis. DsRNA-activated Protein Kinase (PKR) phosphorylates the translational inhibitor eIF2a and may also lead to inhibition of viral and cellular protein synthesis. Recently, PKR has also been found to be essential for the activation of the transcription factor NF-. kappa.B, a central player in inflammatory cytokine induction, immunomodulation and apoptosis. The Mx (myxovirus resistance) protein inhibits RNA virus replication by preventing the transport of viral particles within cells or transcription of viral RNA. RNA-specific Adenosine Deaminase (ADAR) converts adenosine to inosine, causing hypermutation of the viral RNA genome (21).

Huifns have a broad spectrum of biological activities, including antiviral, antitumor, and immunomodulatory functions. The clinical potential of human interferons has been extensively studied and is summarized below.

In anti-tumor applications, huifns may mediate anti-tumor effects indirectly through modulation of immunomodulatory and anti-angiogenic responses or directly by affecting proliferation or cell differentiation of tumor cells (22). Interferon therapy has been used to treat a variety of leukemias (23), such as hairy cell leukemia (24), acute and chronic myelogenous leukemias (25-27), osteosarcoma (28), basal cell carcinoma (29), glioma (30), renal cell carcinoma (31), multiple myeloma (32), melanoma (33), kaposi's sarcoma (23), and hodgkin's disease (34). Thorough research into the combination therapy of IFN- α with cytarabine (ara-C), 5-FU, hydroxyurea (hydroxyura) and IL-2 has been conducted, and most showed significantly better results than HuIFN- α alone (3). Synergistic treatment of advanced cancer in combination with HuIFN and temozolomide has also been reported (35).

In terms of antiviral applications, huifns have been used clinically in antiviral therapies, for example, in the treatment of AIDS (36), viral hepatitis including chronic hepatitis b, hepatitis c (37, 38), papillomavirus infection (39), herpes virus infection (40), viral encephalitis (41), and in the prevention of rhinitis and respiratory infections (40).

HuIFN has also been used clinically for anti-bacterial therapy (42), e.g., aerosolized HuIFN- γ (43) and HuIFN- α have been used in patients with multidrug resistant tuberculosis (44). HuIFN-gamma has been used to treat multidrug resistant tuberculosis (45).

Huifns have also been used clinically for immunomodulatory therapy, for example, to prevent graft-versus-host rejection, or to slow the progression of autoimmune diseases such as multiple sclerosis (46, 47) and sjogren's syndrome (48). IFN- β has been approved by the FDA for the treatment of multiple sclerosis in the United states. Recently, it has been reported that patients with multiple sclerosis have reduced production of type I interferon and interleukin-2 (49). Furthermore, immunomodulatory treatment with HuIFN- α appears to be an effective therapy in Chronic Myelogenous Leukemia (CML) patients who relapse after bone marrow transplantation (50).

In terms of vaccine adjuvantation, huifns have been used clinically as adjuvants for the treatment of melanoma (51), and may also be used as adjuvants or co-adjuvants in the prophylactic or therapeutic vaccination of many other diseases to enhance or stimulate the immune response (52).

HuIFN- α 2a was the first angiogenesis inhibitor used in clinical trials to be effective in children for the treatment of life-threatening hemangiomas (53, 54). Another clinical indication is giant cell tumor of the bone. Kaban et al reported significant regression of large, rapidly growing recurrent giant cell tumors of the mandible (55).

Although huifns have many important clinical applications, they also exhibit significant side effects and other limitations. Most cytokines, including HuIFN, have a relatively short circulating half-life because they are produced in vivo to act locally and transiently. Since they are usually administered as systemic therapeutics, they need to be administered frequently and at relatively high doses. Frequent parenteral administration is inconvenient and painful. Furthermore, the toxic side effects associated with HuIFN administration are often so severe that some patients cannot tolerate the treatment. These side effects are likely associated with systemic administration of high doses. In addition, it has been found in clinical studies that some patients produce antibodies against rhufn that neutralize the biological activity of rhufn (56).

It is clear that there is an urgent need for the development of new, potency-enhanced interferon proteins for a variety of applications (e.g., anti-cancer therapies, as well as anti-viral therapies, immunotherapy, anti-parasitic therapies, anti-bacterial therapies, or any medical condition or situation where enhanced interferon activity and/or reduced side effects are desired). In conclusion, huifns are likely to play an important role in the next generation of novel anti-tumor and anti-viral therapies (10).

It is well known in the art that the most effective way to improve the pharmacological properties of cytokine drugs is to mutate the cytokine protein itself. Over time, various strategies and techniques for mutating interferon peptides have been developed. In general, there are currently 3 strategies available for generating HuIFN- α mutants.

The first strategy is to prepare IFN hybrids. Some researchers have used the nature of naturally occurring Restriction Enzyme (RE) cleavage sites in IFN coding sequences to ligate homologous coding fragments (57, 58). A review of the generation of many hybrid IFNs has been made by Horisberger and DiMarco (11); this article provides an overview of the process of constructing such molecules. Specific examples of methods for constructing hybrid interferons are described. Some researchers have used PCR amplification to construct mutant IFN- α, creating nucleotide fragments with specific needs, and then have the possibility to link new fragments of different IFNs (59). U.S. Pat. No. 6,685,933(60) also describes PCR amplification techniques for the preparation of human IFN hybrids. The interferon hybrids may be generated within one interferon subtype, as described for example in U.S. Pat. No. 5,137,720(61) and U.S. Pat. No. 6,685,933(60), or between at least 2 different interferon classes, as described for example in U.S. Pat. No. 6174996(62) and U.S. Pat. No. 6,685,933 (60). Furthermore, the parental genes of the hybrid may be from one species (primarily from humans), such as the hybrid between HuIFN- α and HuIFN- ω, or from more than one animal species, such as the hybrid between human and murine interferon- α (63).

A second strategy for constructing interferon mutants is to use site-directed point mutagenesis by introducing one or more nucleotide changes into the IFN DNA molecule (64). More recently, systematic mutagenesis and computational methods have been used as a guide for protein mutagenesis (65).

A third strategy for constructing type I huifns is to shuffle IFN gene fragments obtained by RE digestion, PCR amplification, chemical synthesis, or dnase digestion, followed by PCR to randomly ligate the fragments, followed by amplification. The resulting PCR product is in fact a pool of rearranged interferon alpha gene fragments that can be used to construct a DNA library from which DNA clones with the desired phenotype can be isolated (66). For example, Chang et al have described a method of constructing and screening HuIFN shuffling libraries to identify HuIFN derivatives with enhanced antiviral and antiproliferative activity in mouse cells (67).

Human interferon alfacon-1 (consensus interferon) is a recombinant non-naturally occurring HuIFN-alpha, 166 amino acids. It is obtained by cloning according to known methodsTo evaluate the most highly conserved amino acids in the corresponding region of each fragment. At the amino acid level, it has 89% sequence homology with HuIFN-alpha 2b, and its specific antiviral activity is about 10⁹IU/mg. Human interferon alfacon-1 has been approved for the treatment of chronic HCV infection in patients with compensated liver disease at or above 18 years of age (68).

Although some recombinant interferon proteins are known in the art, there is still a need for novel interferon-like proteins and protein compositions with enhanced biological activity.

Summary of The Invention

In accordance with the present invention, isolated polynucleotides are disclosed which encode proteins having human interferon-like biological activity. In one embodiment, the polynucleotide comprises a nucleotide sequence identical to SEQ ID No: 1 nucleotide sequence which is at least 93% identical. In other embodiments, the nucleotide sequence is identical to SEQ ID No: 1 is at least 95% identical or at least 98% identical.

In one embodiment, the proteins encompassed by the present invention are selected from the group consisting of proteins each having a sequence identical to SEQ ID No: 2a group of proteins having an amino acid sequence which is at least 85% identical. Preferably, the protein is not naturally occurring and has enhanced antiviral and antiproliferative activity compared to human interferon alpha 2b (HuIFN-alpha 2 b). For example, the protein has at least 2-fold greater antiviral activity than HuIFN- α 2b and at least 10-fold greater antiproliferative activity than HuIFN- α 2 b. In a particular embodiment, the protein amino acid sequence is identical to SEQ ID No: 2 are at least 90% identical or at least 95% identical.

The invention includes recombinant vectors comprising the polynucleotide sequences as well as host cells comprising the vectors. The invention also includes polypeptide fragments that exhibit human interferon-like biological activity. The invention further includes protein constructs and other compositions that exhibit interferon-like biological activity, such as conjugates comprising the protein and another moiety, such as an inorganic polymer. The invention further includes methods and uses of the proteins and the compositions for therapeutic purposes, for example as antiviral or anticancer agents. The invention may also be used to treat other conditions responsive to interferon therapy.

Brief Description of Drawings

FIG. 1 depicts the complete DNA sequence (SEQ ID No: 1) encoding the novel protein of the present invention, herein designated Novaferon^TM(A) In that respect FIG. 1 also shows the expected amino acid sequence of Novaferon (SEQ ID No: 2) (B), and the alignment of the Novaferon amino acid sequence and Novaferon DNA sequence (C). The first amino acid cysteine of the mature Novaferon protein is designated as residue 1.

FIG. 2 shows a nucleotide sequence alignment (A) of the Novaferon gene and the HuIFN-. alpha.14 gene (Genbank accession No.: NM-002172), and an amino acid sequence alignment (B) of the Novaferon protein and the HuIFN-. alpha.14 protein (translated from Genbank accession No.: NM-002172). The first amino acid cysteine of the mature Novaferon protein is designated as residue 1. Novaferon shares about 93% sequence identity at the nucleotide level with HuIFN- α 14 (462/498) and about 87% sequence identity at the amino acid level (144/166). The different nucleotides are marked with a gap in the middle line.

FIG. 3 shows a nucleotide sequence alignment (A) of the Novaferon gene and the HuIFN-. alpha.2b gene (Genbank accession No.: NM-000605), and an amino acid sequence alignment (B) of the Novaferon protein and the HuIFN-. alpha.2b protein (translated from the HuIFN-. alpha.2b gene from Genbank accession No. NM-000605). The first amino acid cysteine of the mature Novaferon protein is designated as residue 1. Novaferon shares about 89% sequence identity at the nucleotide level with HuIFN- α 2b (445/498), and about 81% sequence identity at the amino acid level (135/166). The different nucleotides are marked with a gap in the middle line.

FIG. 4 is a graph showing the in vitro anti-proliferative inhibition of Daudi cells by Novaferon compared to HuIFN-. alpha.2b.

FIG. 5 is a graph showing the in vivo anti-tumor effect of Novaferon and HuIFN-. alpha.2b in nude mice bearing human prostate cancer PC-3 xenografts.

FIG. 6 is a graph showing the in vivo anti-tumor effect of Novaferon and HuIFN-. alpha.2b in nude mice bearing human liver cancer Hep G2 xenografts.

FIG. 7 is a graph showing the in vivo anti-tumor effect of Novaferon and HuIFN-. alpha.2b in nude mice bearing human melanoma A-375 xenografts.

FIG. 8 is a graph showing the in vivo anti-tumor effect of Novaferon and HuIFN-. alpha.2b in nude mice bearing LS180 xenografts of colon cancer cells.

FIG. 9 is a graph showing the in vivo anti-tumor effect of Novaferon and HuIFN-. alpha.2b in nude mice bearing human leukemia HL60(S) xenografts.

Detailed Description

Throughout the following description, specific details are provided to provide a more thorough understanding of the invention. However, the practice of the invention is not limited to these specific details. In other instances, well known elements have not been shown or described in detail to avoid unnecessarily obscuring the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense.

Definition of terms

Before the present invention is described in detail, it is to be understood that this invention is not limited to the particular protein molecules, methods, protocols, cell lines, vectors, and reagents described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

For a more complete understanding of the invention described herein, the following terms are used, and their definitions are set forth below. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the cell lines, vectors and methodologies that are reported in the publications, which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

The term "interferon" refers to a family of secreted proteins produced by a variety of eukaryotic cells following exposure to various environmental stimuli, including viral infection or exposure to mitogens. In addition to having antiviral properties, interferons have also been shown to affect a variety of cellular functions. All interferon units expressed herein are referenced to WHO International Standard, 94/786 (rHuIFN-. alpha. consensus)) and 95/650 (rHuIFN-. alpha.2a).

The term "interferon-like" refers to a functional and/or structural feature exhibited by or similar to a known interferon or interferon analog. For example, "interferon-like biological activity" includes antiviral and antiproliferative activity. Other examples of interferon-like biological activities are described herein and will be understood by those of skill in the art. The term "active" in its plural encompasses the singular; that is, the invention includes recombinant proteins or other protein constructs or compositions that exhibit at least one interferon-like activity.

The term "consensus interferon" refers to a type of synthetic interferon, the amino acid sequence of which is a roughly average sequence of all known human interferon-alpha subtypes. It has been reported that consensus interferon has better (about 5-fold) antiviral, antiproliferative and NK cell activating activities than any of the natural human IFN-alpha subtypes.

The term "isolated" as used herein refers to a molecule, such as DNA or RNA, that has left its natural environment. For example, a recombinant DNA molecule contained in a vector is considered to be isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or (partially or substantially) purified DNA molecules in solution. "isolated" DNA also includes DNA molecules recovered from libraries containing natural or artificial DNA fragments of interest, as well as chemically synthesized nucleic acids. Thus, an isolated nucleic acid can be produced recombinantly.

The term "nucleotide sequence" refers to a series of nucleotides comprising an oligonucleotide, polynucleotide or nucleotide molecule, and fragments or portions thereof. In the case of a DNA molecule, the sequence may comprise a series of deoxyribonucleotides, and in the case of an RNA molecule, the sequence may comprise a corresponding series of ribonucleotides. The oligonucleotide, polynucleotide or nucleic acid molecule may be single-stranded or double-stranded, while the nucleotide sequence may represent a sense or antisense strand.

The terms "oligonucleotide fragment" or "polynucleotide fragment", "portion", or "segment" or "probe" or "primer" are used interchangeably and refer to a stretch of nucleotide residues that are at least about 5 nucleotides in length. Preferably, the fragments are useful for hybridizing to a target nucleotide sequence. The primer serves as a starting point for nucleotide polymerization catalyzed by DNA polymerase, RNA polymerase or reverse transcriptase. The fragments or segments can uniquely identify each polynucleotide sequence of the invention. Preferably, the fragment comprises a sequence identical to SEQ ID NO: 1 substantially similar sequence.

The term "protein" or "peptide" or "oligopeptide" or "polypeptide" refers to a naturally occurring or synthetic molecule comprising a chain of amino acids.

The term "open reading frame" or ORF refers to a series of nucleotide triplets encoding amino acids without any stop codon and generally refers to sequences that can be translated into protein.

The term "mature protein coding sequence" refers to a sequence that encodes a protein or peptide without a signal sequence or leader sequence. The protein may be produced by processing within the cell with any leader/signal sequences removed. Proteins may be produced synthetically or using polynucleotides that encode only the mature protein coding sequence.

The term "purified" or "substantially purified" as used herein means that the indicated protein is present in the substantial absence of other biological macromolecules such as other proteins, polypeptides, and the like. Purifying the protein so that it constitutes at least 95% by weight of the indicated biomacromolecule present (although water, buffers and other small molecules, especially molecules having a molecular weight of less than 1000 daltons, may be present).

The term "recombinant expression vehicle or vector" refers to a plasmid or phage or virus or vector for expressing a protein from a dna (rna) sequence. The expression vehicle may comprise a transcriptional unit that includes (1) one or more genetic elements, such as promoters or enhancers, that have a regulatory role in gene expression, (2) structural or coding sequences that are transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences. The building blocks intended for use in yeast or eukaryotic expression systems preferably comprise a leader sequence which enables the host cell to secrete the translated protein extracellularly. Alternatively, when the recombinant protein is expressed without a leader or transporter sequence, it may contain a methionine residue at the amino terminus. This residue may or may not subsequently be cleaved from the expressed recombinant protein to obtain the final product.

The term "substantial similarity" relates to nucleic acids or fragments thereof that have a high degree of sequence identity to another nucleic acid when optimally aligned with other nucleotides or their complementary strands. Sequence identity or homology can be determined using sequence analysis software such as BLASTN. A first nucleic acid is considered substantially similar to a second nucleic acid if the two show at least about 85-95% or greater sequence identity when optimally aligned. For example, to determine sequence identity or homology between two different nucleic acids, the BLASTN program "BLAST 2 sequences" is used. The program is available to the public from the National Center for Biotechnology Information (NCBI) via the Internet (http:// www.ncbi.nlm.nih.gov/blast/b12 seq/wblatt 2.cgi) (69). As a non-limiting example, this comparison can be made using default software settings (expected 10, filter default, open gap 5, extended gap2 penalty, gap x decrease 50). Similarly, a first protein or polypeptide is considered substantially similar to a second protein or polypeptide if the two show at least about 85-95% or greater sequence identity when optimally aligned and compared using BLAST software (blastp) using default settings.

By way of further illustration, a polynucleotide having a nucleotide sequence that is at least, e.g., 95% "identical" to a reference nucleotide sequence encoding a protein means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence encoding the protein. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or up to 5% of the number of nucleotides of the total nucleotides of the reference sequence may be inserted into the reference sequence.

The term "complementary" or "complementarity" as used herein refers to the natural association of polynucleotides by base pairing under permissive salt and temperature conditions. For example, the sequence "A-G-T" binds to the complementary sequence "T-C-A". Complementarity between two single-stranded molecules may be "partial", i.e., where only some of the nucleic acids bind, or may be complete when there is full complementarity between the single-stranded molecules. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands. This is particularly important for amplification reactions that rely on binding between nucleic acid strands.

The term "transformation" means the introduction of DNA into an organism such that the DNA is capable of replication either as an extrachromosomal element or by chromosomal integration. The term "transfection" refers to the uptake of an expression vector by a suitable host cell, regardless of whether the coding sequence is actually expressed.

The term "treating" and grammatical equivalents thereof are used in the broadest sense and include the therapeutic treatment, prevention, prophylaxis and amelioration of certain undesirable symptoms or conditions.

The term "biological activity" as used herein refers to structural, regulatory, biochemical or other biological functions in a living system, e.g., similar or identical to naturally or non-naturally occurring molecules.

The terms "anti-proliferative" and "anti-proliferative" as used herein refer to slowing and/or preventing the growth and division of cells, resulting in a reduction in the total number of cells and/or a reduction in the percentage of target cells in any or all cell cycle phases. When the cell is resting in a specific cell cycle phase, the following can be further divided: g1 phase (Gap1), S phase (DNA synthesis), G2 phase (Gap2) or M phase (mitosis). The term "anti-proliferative activity" as used herein refers to the activity of a protein, protein construct or composition to inhibit the proliferation of a cell, particularly a neoplastic cell such as a cancer cell, in vitro or in vivo.

The term "anti-tumor" or "anti-cancer" as used herein refers to hindering or preventing the formation of malignant tumors. "anti-tumor activity" or "anti-cancer activity" as used herein refers to the activity of a protein, protein construct or composition to inhibit the proliferation of cells, particularly neoplastic cells such as cancer cells, in vitro or in vivo.

The term "IC₅₀"or" half maximal inhibitory concentration "means that the inhibitor, e.g., protein, required to inhibit 50% of cell growth in vitroAnd (4) concentration.

The terms "antiviral" and "antiviral" as used herein refer to a slowing and/or preventing viral infection of a cell or interfering with viral replication within a cell, resulting in slowing or stopping viral propagation, or a reduction in the total number of viral particles, in vitro and/or in vivo. "antiviral activity" as used herein means the activity of a protein, protein construct or composition to inhibit viral infection or interfere with viral replication in vitro or in vivo.

Novaferon protein

The present invention relates to the preparation and identification of novel human interferon-like proteins, referred to herein as "Novaferon"^TM. As described in detail below, the Novaferon protein exhibits enhanced antiviral and antiproliferative biological activity as compared to the naturally occurring HuIFN- α 2b, as measured in a standard in vitro assay. In particular, in the same test system, the Novaferon protein showed a 12.5-fold increase in antiviral activity when tested in the Wish-VSV system, and an increase in antiproliferative inhibition of Daudi cell growth to approximately 400-fold, compared to HuIFN- α 2 b.

In one embodiment, the Novaferon protein is encoded by a polynucleotide consisting of 498 nucleotides as set forth in SEQ ID No: 1 and FIG. 1 (A). The mature Novaferon protein consists of 166 amino acids, as shown in SEQ ID No: 2 and FIG. 1 (B). The polynucleotide and amino acid sequences encompassed by the present invention and variants thereof are described in further detail below.

For comparison, the inventors studied the homology of Novaferon and naturally occurring huifns. BLAST searches showed that Novaferon has the highest homology to HuIFN- α 14 at both the nucleotide and amino acid levels. As shown in FIG. 2, the Novaferon-encoding polynucleotide sequence (SEQ ID No: 1) has about 93% (462/498) homology to HuIFN-. alpha.14, while the amino acid sequence has about 87% (144-166) homology to HuIFN-. alpha.14. Homology at the nucleotide level is approximately 89% (445/498) and at the amino acid level is approximately 81% (135/166) compared to the most widely used human interferon product HuIFN- α 2b, as shown in figure 3.

For the synthetic IFN alfacon-1 (consensus interferon), Novaferon has about 91% sequence identity at the nucleotide level (453/498) and about 84% sequence identity at the amino acid level (140/166).

As described in detail in the experimental section below, the polynucleotide sequence (SEQ ID No: 1) was selected from a DNA shuffling library of type I human interferons. Briefly, the Novaferon protein is produced by contacting a protein with a protein comprising the entire SEQ ID No: 1 by transfecting a host cell with the recombinant vector. The Novaferon protein contained in the supernatant of the host cell line was purified and shown to exhibit human interferon-like biological activity, such as antiviral and antiproliferative functions.

Polynucleotides and variants

The novel polynucleotide sequence/nucleic acid molecule of the invention consists of 498 nucleotides, as shown in FIG. 1(SEQ ID No: 1). Using the information provided herein, e.g., nucleotide sequence, the nucleic acid molecules of the invention encoding the Novaferon protein (SEQ ID No: 2) can be obtained by recombinant expression, chemical synthesis, or by using other standard molecular biology procedures, such as those used for DNA mutagenesis.

In addition to the isolated nucleic acid molecule (SEQ ID No: 1), the present invention also includes nucleic acid molecules having a sequence other than SEQ ID No: 1, but still encodes an amino acid sequence which is identical or substantially identical to the Novaferon protein (SEQ ID No: 2) due to the degeneracy of the genetic code. The genetic code and species-specific codon preferences are known in the art. Thus, it is routine for one skilled in the art to generate sequences other than SEQ id no: 1, for example, to optimize codon expression for a particular host (e.g., changing codons of a human mRNA to codons preferred by a bacterial host such as e.

The invention further provides an isolated nucleotide molecule having the nucleotide sequence shown in FIG. 1(SEQ ID No: 1), or a nucleotide sequence having a nucleotide sequence identical to the nucleotide sequence shown in SEQ ID No: 1, or a sequence complementary to the nucleic acid sequence of 1. The invention also provides relevant information and relates to recombinant vectors comprising the isolated nucleic acid molecules of the invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and forming host cells that express the Novaferon protein and using the host cells for the production of Novaferon by recombinant techniques.

The invention includes nucleic acid molecules substantially similar thereto, e.g., to the nucleic acid sequences of SEQ ID Nos: 1, or a nucleic acid having at least about 85-95% or more sequence identity. For example, in one aspect, the peptide has a sequence identical to SEQ ID No: 1, whether or not it encodes a protein or polypeptide having a biological activity similar to Novaferon (such activities include, but are not limited to, enhanced antiviral, antiproliferative, and antitumor functions as compared to HuIFN) is within the scope of the invention. Such nucleic acid molecules can be used, for example, as probes for detecting mRNA in cells that have been transfected with a vector comprising the nucleotide sequence of the present invention to produce Novaferon. In other words, these are similar to SEQ ID No: 1 can be used as a marker for determining expression of a heterologous gene in a host cell.

Furthermore, the invention includes a polynucleotide comprising the sequence of SEQ ID No: 1, preferably at least about 50 contiguous nucleotides.

More generally, the invention includes and encompasses fragments of any and all isolated nucleic acid molecules that are identical to a partial sequence of the nucleotide sequence depicted in FIG. 1(SEQ ID No: 1). In one embodiment, these fragments are at least about 15 nucleotides in length and are useful as diagnostic probes and primers as discussed herein. In addition, the invention includes and encompasses larger fragments that are about 50 nucleotides in length or longer.

In addition to SEQ ID No: 1, the present invention also includes, but is not limited to, nucleic acid sequences encoding the amino acid sequence of the entire Novaferon protein as well as additional amino acids/peptides/polypeptides such as an added secretion leader sequence.

The invention also includes nucleic acid sequences having the sequence of SEQ ID Nos: 1, as well as additional non-coding sequences, including, for example, but not limited to, introns and 5 'and 3' non-coding sequences such as transcribed but not translated sequences that play a role in transcription, mRNA processing (i.e., splicing and polyadenylation signals, ribosome binding and stabilizing mRNA), and additional coding sequences that encode additional amino acids with or without function.

The invention further relates to variants of the nucleotide molecules of the invention (SEQ ID No: 1) which encode parts, analogues or derivatives of the Novaferon protein. Variants can be obtained by screening interferon shuffling libraries or by using mutagenesis techniques or/and other known techniques described in the art.

As noted above, such variants may include those produced by nucleotide insertions, deletions, or substitutions. Insertions, deletions or substitutions may involve one or more nucleotides. These mutations can occur at the 5 'or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed individually among the nucleotides of the reference sequence, or interspersed in one or more contiguous groups within the reference sequence. The changes may result in conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and/or deletions which do not alter the properties and activity of the Novaferon protein or parts thereof. Also particularly preferred in this respect are conservative substitutions.

The invention also provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 93% identical, more preferably at least about 95%, 96%, 97%, 98% or 99% identical to a polynucleotide selected from the group consisting of: (a) encodes a polypeptide having the sequence of SEQ ID No: 2 (i.e. positions 1-166 of SEQ ID No: 2) of the complete amino acid sequence of Novaferon protein; and (b) a nucleotide sequence encoding a biologically active fragment of the protein of (a); and (c) a nucleotide sequence complementary to any of the nucleotide sequences of (a) or (b) above.

Due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of nucleic acid molecules having a sequence that is at least about 93%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence shown in FIG. 1(SEQ ID NO: 1) will encode proteins having similar or identical activity to the Novaferon protein. In fact, since degenerate variants all encode the same protein, it will be clear to the skilled person, even if no comparative assay is performed. It will be further recognized in the art that for such nucleic acid molecules that are not degenerate variants, there will also be a significant number of proteins that will encode interferon-like biological activity. This is because those skilled in the art are fully aware of amino acid substitutions that are less likely or less likely to significantly affect protein function (e.g., substitution of one aliphatic amino acid with another aliphatic amino acid), as described further below. For example, Bowie et al (70) provide guidance on how to perform phenotypically silent amino acid substitutions, where the authors indicate that many proteins are tolerant of amino acid substitutions.

Protein and polypeptide variants and constructs

The invention includes SEQ ID No: 2 and substantially similar proteins or polypeptides thereto, such as proteins or polypeptides substantially similar to SEQ ID nos: 2a non-naturally occurring protein having at least about 85-95% or more amino acid sequence identity. For example, a peptide similar to SEQ ID No: 2, a non-naturally occurring protein having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity is within the scope of the invention. Furthermore, the Novaferon protein of the invention may be structurally modified by fusing it to other proteins or protein fragments or other molecules in order to enhance its function and properties. Examples include, but are not limited to, fusing them to other proteins/protein fragments to increase their expression or to further stabilize the Novaferon protein.

In one embodiment, the Novaferon-encoding nucleic acid sequences and/or Novaferon proteins of the invention may be labeled with a label other than a scaffold. Here, "labeled" means that a compound of the nucleic acid sequence (SEQ ID No: 1) or Novaferon protein (SEQ ID No: 2) has attached at least one element, isotope, or other chemical (label) to enable detection of the compound. In general, markers fall into three categories: a) an isotopic label, which can be a radioisotope or a heavy isotope; b) an immunological marker, which may be an antibody or an antigen; and c) a coloured or fluorescent dye. The label may be incorporated at any position of the compound.

Once prepared, the Novaferon protein may also be covalently modified. One type of covalent modification involves treating the Novaferon protein with an organic derivatizing agent (derivitizing agent) capable of reacting with selected side chains or N-or C-terminal residues of the Novaferon protein. Derivatization with bifunctional reagents may be used, for example, to crosslink the Novaferon protein to a water-insoluble support matrix or surface for purification of anti-Novaferon antibodies or screening assays. Commonly used crosslinking agents include 1, 1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters (e.g., esters formed with 4-azidosalicylic acid), homobifunctional imidoesters, including disuccinimidyl esters such as 3, 3' -dithiobis (succinimidyl propionate), bifunctional maleimides such as bis-N-maleimido-1, 8-octane, and reagents such as methyl-3- [ (p-azidophenyl) dithio ] propioimidate.

Other modifications of the Novaferon protein include: deacylation of glutaminyl and asparaginyl residues to glutamyl and aspartyl residues, respectively; hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of seryl and threonyl residues; methylation of the amino groups of lysine, arginine, and histidine side chains (71); acetylation of the N-terminal amine; and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the Novaferon proteins of the invention involves altering the natural glycosylation pattern of the protein. This can be achieved, for example, by: (1) deletion and/or addition of one or more sugar moieties found in the native sequence of the Novaferon protein, or (2) addition and/or deletion of one or more glycosylation sites not present in the native sequence of the Novaferon protein.

The addition of glycosylation sites to the Novaferon protein can be accomplished by altering the amino acid sequence of the Novaferon protein. Such changes (for O-linked glycosylation sites) are achieved, for example, by the addition or substitution of one or more serine or threonine residues in the native sequence of the Novaferon protein. Alteration of the amino acid sequence of the Novaferon protein can be achieved by changes at the DNA level, particularly by mutating the DNA sequence encoding the Novaferon protein at preselected nucleotide bases so that the altered codons can be translated into the desired amino acids.

Another way to increase the number of sugar moieties in the Novaferon protein is to couple glycosides to the protein either chemically or enzymatically. Such methods have been described in the prior art, for example, as early as 1981, Aplin JD and Wriston JC Jr have described the preparation, properties and use of glycoconjugates of proteins and lipids (72).

Removal of the sugar moiety present in the Novaferon protein can be accomplished chemically or enzymatically, or by mutational substitution of codons encoding amino acid residues used as glycosylation targets. Chemical deglycosylation techniques are known in the art and are described, for example, by Edge AS et al (73). Enzymatic cleavage of the sugar moiety on the polypeptide can be achieved by using a variety of endo-and exoglycosidases, as described by Thotakura et al (74).

Such derivatized constructs may include moieties that improve solubility, absorption, permeability across the blood-brain barrier, biological half-life, and the like. Such portions or modifications of the Novaferon protein may alternatively clear or attenuate any possible unwanted side effects of the protein, and the like. Moieties capable of mediating such effects have been disclosed, for example in Remington: the science and Practice of Pharmacy (75).

Another type of covalent modification of Novaferon includes, for example, modification in the order of u.s.pat.nos.: 4,640,835 (76); 4,496,689 (77); 4,791,192(78) or 4,179,337(79) to attach the Novaferon protein to one of a variety of non-protein polymers, such as polyethylene glycol, polypropylene glycol or polyalkylene oxide.

In addition, the Novaferon proteins of the invention can be modified to form chimeric molecules comprising the Novaferon protein fused to another heterologous polypeptide or amino acid sequence. In one embodiment, such a chimeric molecule comprises a fusion compound of the Novaferon protein with a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is typically located at the amino or carboxy terminus of the Novaferon protein. The presence of such epitope tagged forms of the Novaferon protein can be detected with antibodies against the tagged polypeptide. Furthermore, the provision of an epitope tag enables the Novaferon protein to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. In an alternative embodiment, the chimeric molecule may comprise a fusion compound of the Novaferon protein with an immunoglobulin or a specific region/fragment of an immunoglobulin. For example, to form a bivalent form of the chimeric molecule, the Novaferon protein may be fused to the Fc region of an IgG molecule.

A variety of tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; flu HA-tag polypeptide and its antibody 12CA5 (80); the C-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies directed thereto (81); and herpes simplex virus glycoprotein d (gd) tags and antibodies thereto (82). Other tag polypeptides include Flag-peptide (83); tubulin epitope peptide (84); and T7 gene 10 protein peptide tag (85).

Furthermore, the Novaferon protein of the invention can be produced by chemical synthesis procedures known to those of ordinary skill in the art. For example, polypeptides up to about 80-90 amino acid residues in length can be produced on a commercially available 433A model peptide synthesizer (Applied Biosystems, Inc., Foster City, Calif.). In addition, longer chemically synthesized peptides of up to 120 residues are also commercially available, for example, from Bio-synthesis, inc. Thus, it is readily understood that the full-length mature Novaferon protein can be produced synthetically (e.g., by first synthesizing fragments and then ligating the fragments together).

Thus, the Novaferon protein of the invention (SEQ ID No: 2) includes all proteins having a sequence identical to that of SEQ ID No: 2, whether or not these Novaferon proteins and protein derivatives are produced by chemical synthetic procedures, and/or by recombinant techniques from prokaryotic or eukaryotic host cells or other cells and hosts, including but not limited to bacterial, yeast, plant, insect and mammalian cells. Depending on the host employed in the recombinant production method, the protein of the invention may be glycosylated or non-glycosylated, pegylated or non-pegylated. In addition, the proteins of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processing. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon is typically removed efficiently from any protein post-translationally in all eukaryotic cells. In most prokaryotes, the N-terminal methionine of most proteins is also effectively removed, and for some proteins, this prokaryotic removal process is ineffective, depending on the nature of the amino acid to which the N-terminal methionine is covalently attached.

Generating

The invention also relates to recombinant vectors comprising the isolated DNA molecules of the invention, host cells genetically engineered/transfected with said recombinant vectors, and the production of Novaferon protein or fragments thereof by recombinant techniques. The vector may be, for example, a plasmid, phage, viral or retroviral vector. Retroviral vectors may be replication-competent or replication-defective. In the latter case, viral propagation usually occurs only in the complementing host cells. Detailed description examples of Novaferon generation are as follows.

Preferred vectors for expressing the Novaferon proteins of the invention include, but are not limited to, vectors comprising cis-acting regulatory regions effective for expression in a host operably linked to the polynucleotide to be expressed. Suitable trans-acting factors are provided by the host, by a complementing vector, or by the vector itself after introduction into the host.

The nucleic acid sequence disclosed in the present invention (SEQ ID No: 1) can be operably linked to a suitable promoter. By "promoter" herein is meant any nucleic acid sequence capable of binding RNA polymerase and initiating transcription of an exon (typically downstream (3')) of the coding sequence of the Novaferon protein into mRNA. Bacterial promoters have a transcriptional initiation region that is generally near the 5' end of the coding sequence. The transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. Sequences encoding metabolic pathway enzymes may provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar-metabolizing enzymes such as galactose, lactose and maltose-metabolizing enzymes, and sequences derived from biosynthetic enzymes such as tryptophan synthase. Promoters from bacteriophages, which are known in the art, may also be utilized. In addition, synthetic promoters and hybrid promoters are also useful; for example, the tac promoter is a hybrid of trp and lac promoter sequences. In addition, bacterial promoters may include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. Preferred bacterial promoters include, but are not limited to, the E.coli laci, trp, phoA and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, PL promoter, and the trp promoter.

Eukaryotic promoters have a transcriptional initiation region, usually near the 5' end of the coding sequence, and a TATA box, usually 25-30 base pairs (bp) upstream of the transcriptional initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. Mammalian promoters also contain upstream promoter elements (enhancer elements), which are typically located within 100 to 200 base pairs upstream of the TATA box. Upstream promoter elements determine the rate of transcription initiation and can function in either direction. Particularly useful as mammalian promoters are promoters from mammalian viral genes, since viral genes are generally highly expressed and have a broad host range. Examples include the SV40 early promoter, the mouse mammary tumor virus LTR promoter. Preferred animal cell promoters include, but are not limited to, adenovirus major late promoter, herpes simplex virus promoter, and CMV promoter. Among the known eukaryotic promoters suitable in this regard are the CMV immediate early promoter, the elongation factor 1 α (EF1A) promoter, the HSV thymidine kinase promoter, the SV40 early and late promoters, and promoters of retroviral LTRs such as those of the rous sarcoma virus ("RSV"). Preferred promoter sequences for expression in yeast include the inducible GAL1/10 promoter, promoters from the genes for alcohol dehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and acid phosphatase.

Vectors for propagation and expression also typically include one or more selectable markers. Such labels are suitable for amplification, or the vector contains further labels for this purpose. In this regard, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transfected host cells, although one of skill in the art will recognize that certain systemic selectable markers may be provided by separate vectors. For culturing in E.coli and other bacteria, preferred markers include, for example, ampicillin (Amp), tetracycline (Tet), or Hygromycin (HYG) resistance genes. Yeast selectable markers include ADE2, HIS4, LEU2, TRP1, and ALG7, which confer resistance to tunicamycin; neomycin phosphotransferase gene, which confers resistance to G418; and the CUP1 gene, which enables yeast to grow in the presence of copper ions. Animal cell selectable markers include the dihydrofolate reductase (DHFR) gene, neomycin (neo) or Hygromycin (HYG) resistance gene.

In addition, vectors for propagation and expression typically contain one or more sites for transcription initiation, termination, and a ribosome binding site for translation in the transcribed region. The coding part of the transcript expressed by the construct preferably contains a translation initiation codon at the beginning and a stop codon (UAA, UGA or UAG) suitably at the end of the DNA sequence to be translated. The choice of promoter, terminator, selectable marker, vector and other elements is a matter of routine design within the ability of one skilled in the art. Many such elements are described in the literature and are available through commercial suppliers.

The following vectors are commercially available and are preferably used in bacteria: pBV220(86) from shanghai sangon and derivatives thereof; pQE series from Qiagen; pET vector from Qiagen; pBS vector, Phagescript vector, Bluescript vector, pNH8A, pNH16a, pNH18A, pNH46A from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 from Pharmacia. Among the preferred eukaryotic vectors are the pCI vectors from Promega; pcDNA vector from Invitrogen; pSV2CAT, pOG44, pXT1 and pSG from Stratagene; and pSVK3, pBPV, pMSG, and pSVL from Pharmacia. These vectors are listed individually as examples to demonstrate that many commercially available well known vectors are available to those skilled in the art for use in the production of the Novaferon proteins disclosed herein by genetic/recombinant means.

In certain preferred embodiments in this regard, the vector provides a means for specific expression. Such specific expression may be inducible expression or expression in only certain cell types, or may be inducible and cell-specific. Particularly preferred inducible vectors are those which are capable of being induced to express by readily manipulated environmental factors such as temperature and nutrient supplements. A variety of vectors suitable for this application are well known and are commonly employed by those skilled in the art, including constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts.

For example, a polypeptide comprising SEQ ID No: 1, as well as suitable promoters and other suitable regulatory sequences, into a suitable host cell suitable for expression of the desired protein. Representative of such suitable hosts include bacterial cells, such as E.coli, Bacillus subtilis, Streptomyces (Streptomyces) cells; yeast cells, such as Pichia pastoris (Pichia pastoris) cells; insect cells, such as Drosophila (Drosophila) S2 and Trichoplusia ni (Spodoptera) Sf9 cells; mammalian cells, such as CHO and COS; and plant cells. Hosts for a variety of expression constructs are well known and, using the information disclosed herein, one of skill in the art can readily select a host for expressing the amino acid sequence of SEQ ID No: 2, or a Novaferon protein as shown in figure 2.

Host cells may be genetically engineered to integrate Novaferon-encoding polynucleotides and express the Novaferon proteins of the invention. For example, Novaferon-encoding polynucleotides can be introduced into host cells using transfection techniques known in the art. Such methods are described in many standard laboratory manuals, such as those discussed by Kingston (87). Novaferon-encoding polynucleotides may be introduced/transfected alone or with other polynucleotides. Such other polynucleotides may be introduced independently, or in combination with SEQ ID nos: 1 or a combination thereof.

For example, for co-transfection and selection of a marker in mammalian cells, the Novaferon-encoding polynucleotides of the invention may be transfected into host cells along with a separate polynucleotide encoding a selectable marker. Alternatively, for inducing proliferation in a host cell, the Novaferon-encoding polynucleotide may be incorporated into a vector containing a DNA sequence encoding a selectable marker.

The engineered host cells transfected with a vector comprising a Novaferon-encoding polynucleotide may be cultured in conventional nutrient media specially modified for promoter activation, transformant selection, or target gene amplification. Culture conditions such as temperature, pH, etc., are adjusted to suit the host cell selected for expression of the Novaferon protein of the invention.

To facilitate secretion of the translated protein polypeptide into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be combined and co-expressed with the Novaferon protein.

Purification of

The selection of an appropriate host cell type for expressing a recombinant target protein is generally based on the nature of the target protein and considering other conditions such as production cost, ease of scale-up, scale-up of industrial production, and the like. Then, transfected cell clones expressing the target protein at the highest yield were selected, and the final clone with the best expression was named the cell line expressing the target protein and used for production of the target protein. Cell lines expressing the target protein are grown in media containing various nutrients. To obtain optimal growth of the cell and/or optimal expression of the target protein, various agents or conditions are used to induce a selective promoter, which is integrated with the cDNA sequence of the target protein in the transfection vector. If the host cell type/expression system is bacterial, the cultured cells are typically harvested from the culture medium by centrifugation. The harvested cell bodies are disrupted by physical or chemical means, and the harvested crude extract containing the synthesized target protein is retained for further purification of the protein. Methods applied to disrupt microbial cells include, but are not limited to, freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysing agents. Such methods are well known to those skilled in the art.

The present inventors utilized such bacteria of escherichia coli as host cells for expression of recombinant Novaferon protein. Coli was transfected with a vector containing a Novaferon-encoding polynucleotide sequence and one of the strains of e.coli with optimal expression of the Novaferon protein was selected for production of the Novaferon protein as described below. Once synthesized, the protein may be retained in the cytoplasm in the form of insoluble particles, or may be secreted into the cytoplasm in soluble form. In the former case, the particles are recovered after lysis of the cell bodies and denatured, for example with guanidinium isothiocyanate or urea. Then, refolding of the denatured polypeptide/Novaferon protein was obtained by: the denaturant is diluted with an excess of a diluting solution, or dialysis is performed against a solution of urea in combination with reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter case, the protein can be recovered in soluble and functional form directly from the periplasmic space without denaturation after disruption of the harvested cells. By avoiding denaturation and refolding processes, the soluble Novaferon protein is not damaged and does not contain distorted or misfolded protein molecules.

The present inventors have found that a significant portion of the synthetic Novaferon protein produced in an e.coli cell line is secreted into the cytoplasm. This fraction was then purified as described below.

Activity assays and medical uses

As shown above, the Novaferon protein shows sequence homology to many members of the interferon family, particularly to interferon proteins translated from the mRNA of HuIFN- α 14 (fig. 2). HuIFN- α has shown a wide range of biological activities, including antiviral, antiproliferative and immunomodulatory activity (10).

Because of the homology to HuIFN- α, Novaferon is expected to exhibit similar biological functions as HuIFN- α, including, but not limited to, inhibition of tumor proliferation, antiviral activity, activation of NK cells, and modulation of the immune system. Of particular importance is not only the retention of HuIFN-alpha like functional properties, but also the enhanced potency of these biological functions of the Novaferon protein compared to HuIFN-alpha. To verify and determine the efficacy of its functional properties, the biological activity of the Novaferon protein was thus determined using standard conventional in vitro assays that were designed to test for antiviral and antiproliferative properties. As described in the experimental section below, in some experiments, the in vivo efficacy of the antiproliferative properties of Novaferon protein was further observed in animal models of various human cancer types and compared to HuIFN- α and chemo-anticancer agents.

Many suitable assays for determining HuIFN activity are well known in the art. The present inventors used a cell-based in vitro assay system to determine antiviral and antiproliferative activity. The same in vitro assay is used for all procedures and experiments relevant to the present invention, including but not limited to screening of human type I interferon gene shuffling libraries, selection of Novaferon from expressed proteins of human type I interferon gene shuffling libraries, and determination of the biological activity of pure recombinant Novaferon proteins.

There are many assays for measuring the antiviral activity of test samples/agents by observing the degree of resistance of cells to viruses (88). There are 3 major bioassays for measuring the antiviral activity of huifns and their hybrids. Viruses are classified according to the method used to determine various aspects of their behavior on cultured cells.

An assay for determining inhibition of virus-induced cytopathic effect measures the extent of reduction of virus-induced lytic cytopathic effect in cultured cells pretreated with IFN. This assay can be performed in 96-well plates (89) and has been widely used for recombinant HuIFN- α because it provides a simple method for screening large numbers of samples.

Inhibition of viral plaque formation is another method to quantify HuIFN antiviral activity in tissue culture. The results of the plaque reduction assay were independent of multiplicity of infection. Furthermore, a 50% reduction in plaque formation was measurable with high accuracy. For example, the use of ubiquitous Vesicular Stomatitis Virus (VSV) to induce plaque formation, a specific recombinant IFN can be tested for cross-species activity by screening a large number of cell lines from different animal species (90).

The third assay is based on determining the reduction in virus production. Viral production is typically measured by the number of viruses released during a single cell growth cycle. The assay is particularly useful for testing the antiviral activity of IFN against viruses that do not cause cytopathic effects or viruses that do not form plaques in target cell cultures. However, in this test, multiplicity of infection affects the apparent degree of protection induced by a fixed concentration of IFN (91).

The antiviral activity of Novaferon was measured by a standard cytopathic effect-inhibition assay using WISH cells and Vesicular Stomatitis Virus (VSV). Standard reference samples using WHO international standard: 95/650 (rhhuifn- α 2a) and 94/786 (rhhuifn- α complex), to determine and calibrate antiviral activity. 1 unit of antiviral activity is defined as the amount of protein required to achieve 50% inhibition of the cytopathic effect of VSV on cultured cells. The activity of the Novaferon protein is 2.5X 10, as described further below⁹IU/mg, which is about 12.5 times the activity of HuIFN-. alpha.2b. These tests show that the antiviral properties of Novaferon are greatly enhanced compared to HuIFN- α 2 b. The Novaferon protein exhibits this enhanced antiviral efficacy, which provides the basis for predicting enhanced antiviral effects in humans. Based on the properties of huifns, it is reasonable to expect Novaferon to have a very broad antiviral profile. In other words, Novaferon should be more effective than the native HuIFN for a wide range of viruses. The enhanced antiviral efficacy of Novaferon may translate into better antiviral or better therapeutic effects in clinical settings for patients with multiple viral diseases.

As explained above, IFNs also inhibit cell proliferation and exhibit potential anti-tumor effects through a variety of mechanisms. Cultivation using cellsTrophic systems, several in vitro antiproliferative tests have been established, which are well described in the prior art. In these assays, cell proliferation can be measured by: counting the cells; crystal violet bioassay (92, 93); chemical sensitivity to neutral red dye (94-96); incorporation of a radiolabeled nucleotide (97); incorporating 5-bromo-2' -deoxyuridine into the DNA of proliferating cells (98); using tetrazolesSalt (99, 100).

The human lymphoblast Daudi cell line is very sensitive to the antiproliferative effects of HuIFN- α, and its growth in suspension culture helps to quantify its cell number (101). This cell line has been used for many years to measure the antiproliferative activity of HuIFN-alpha and hybrids (102). Other cell lines may also be used to test the antiproliferative activity of the test agent.

The antiproliferative activity of Novaferon protein was observed in vivo by observing the inhibition of tumor mass growth due to the administration of Novaferon to animal models with various human tumor xenografts. The in vivo anti-tumor effects of Novaferon were compared to HuIFN- α 2b and also to chemical anti-tumor agents in some xenograft models.

As described in detail below, the present inventors have discovered that Novaferon's in vitro anti-proliferative activity, as measured by standard Daudi cell methods, is 400 times more potent than native HuIFN- α 2b, which, of all native huifns, is likely to exhibit the most potent anti-proliferative activity. The enhanced anti-proliferative efficacy of Novaferon is widespread and widespread, as it exhibits more effective or enhanced inhibition than native HuIFN- α 2b for all human cancer cell lines tested in vitro by the present inventors. This indicates that Novaferon is not selective for effective inhibition of human cancer. Although Novaferon differs in the degree of enhanced antiproliferative activity against all tested types of human cancer cell lines, it has the potential to be a broad spectrum anticancer agent in clinical settings. This is a significant advantage over chemo-anti-cancer agents, monoclonal antibodies and other target-specific anti-cancer agents.

The xenograft animal model described below has further established that:

(1) the in vivo anti-proliferative effects of Novaferon were greatly enhanced or more effective compared to native HuIFN- α 2 b.

(2) The in vivo antiproliferative effect of Novaferon at much lower doses was better than the test chemical 5-fluorouracil (5-FU) in the same xenograft model.

(3) Novaferon was able to achieve over 90% inhibition of cancer growth in the xenograft model, but did not cause weight loss, altered vigor, and other negative side effects in the test animals, in sharp contrast to the significant weight loss and decreased vigor in the 5-FU treated animals.

These results indicate that the in vitro and in vivo anti-proliferative properties of Novaferon are greatly enhanced compared to native HuIFN- α 2 b. The enhanced antiproliferative efficacy of Novaferon translates into an effective inhibition (> 90%) of human tumor growth in mouse animal models, and this inhibition appears to act very broadly on all human cancer types tested and is better than the traditional anticancer agent 5 FU. These results also indicate that the effective inhibition of growth of cancer cells by Novaferon is very specific for the cancer cells and not for normal cells, which supports the observation that normal diet and action behavior is observed and body weight is not reduced in Novaferon treated animals. Thus, Novaferon has the potential to play a role in all or most human cancers.

In a preferred embodiment, the whole or part of a Novaferon protein (SEQ ID No: 2) molecule (prepared by recombinant techniques or chemically synthesized using the polynucleotide sequence of SEQ ID No: 1) can be used in human and/or non-human species for the treatment and/or prevention of any and/or all human or non-human derived tumors and cancers. Such tumors include, for example, but are not limited to, osteogenic sarcomas; multiple myeloma; hodgkin's disease; nodular poorly differentiated lymphomas; acute lymphocytic leukemia; acute myeloid leukemia; breast cancer; melanoma; papilloma; and nasopharyngeal carcinoma, colon cancer, liver cancer and melanoma.

In another embodiment, a whole or partial Novaferon protein (SEQ ID NO: 2) molecule (prepared by recombinant techniques or chemically synthesized using the polynucleotide sequence of SEQ ID NO: 1) can be used in the treatment and/or prevention of any and/or all viral diseases in human and/or non-human species. Examples of susceptible viral infections include, but are not limited to, viral encephalomyocarditis, influenza and other respiratory viral infections, rabies and other viral animal infections, and arboviral infections, as well as herpes simplex keratitis, acute hemorrhagic conjunctivitis, varicella zoster, and hepatitis b and c, SARS and avian influenza, human immunodeficiency syndrome (AIDS, HIV).

In another embodiment, a whole or partial Novaferon protein (SEQ ID NO: 2) molecule (prepared by recombinant techniques or chemically synthesized using the polynucleotide sequence of SEQ ID NO: 1) can be used in humans for the treatment and/or prevention of any and/or all immune system related disorders. Examples of immune disorders include, but are not limited to, rheumatoid arthritis, multiple sclerosis, and Sjogren's syndrome diabetes. The Novaferon protein may also be used to prevent graft versus host rejection.

In another embodiment, the whole or part of the Novaferon protein (SEQ ID NO: 2) molecule (prepared by recombinant techniques or chemically synthesized using the polynucleotide sequence of SEQ ID NO: 1) can be used as an immunoadjuvant for the treatment and/or prevention of any and/or all angiogenic diseases. Examples of angiogenic diseases include, but are not limited to, hemangiomas, tumor-induced neovasculature, age-related macular degeneration, and diabetic retinopathy.

The Novaferon protein may be administered to human and/or non-human species, alone or with any other protein/carrier material or other construct, in any pharmaceutically acceptable formulation/formulation, by any route/method of administration/delivery including, but not limited to, oral ingestion, inhalation, intranasal spray, intraperitoneal, intravenous, intramuscular, intralesional or subcutaneous injection.

Pharmaceutical formulations/formulations comprising Novaferon protein as active ingredient may be prepared by incorporating suitable solid or liquid carriers in the form of liquids, solids, semisolids and/or any other clinically acceptable form such as tablets, pills, powders, liquid solutions or suspensions, liposomes, suppositories, injectable solutions and infusible solutions. Novaferon-containing formulations/formulations can be prepared using conventional carriers, materials, methods described in the art or generally accepted by practice in the pharmaceutical industry. Novaferon-containing formulations/formulations can also be prepared using unconventional methods, materials, not described in the art or used by the pharmaceutical industry.

For example, parenteral formulations are generally injection solutions composed of pharmaceutically and physiologically acceptable materials, such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol, and the like. Furthermore, the injection solution may contain, in addition to the Novaferon protein, other proteins, such as carriers, for example human serum albumin or plasma preparations. The pharmaceutical preparations/formulations may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives and pH buffering agents (e.g. sodium acetate or sorbitan monolaurate). Formulation methods are well known in the art and are described, for example, in Remington: the Science and Practice of pharmacy, pharmaceutical Sciences (75).

The particular formulation/formulation of Novaferon protein may be determined by the desired clinical application and/or method of use, and may be prepared by any one of skill in the art using known techniques. For example, in addition to injection solutions, topical and oral formulations may be employed. Topical formulations may include, but are not limited to, eye drops, ointments, and sprays. Oral formulations include, but are not limited to, liquid forms (e.g., syrups, solutions or suspensions), or solid forms (e.g., powders, pills, tablets or capsules). For solid formulations/formulations, conventional non-toxic solid carriers include, but are not limited to, pharmaceutical grades of mannitol, lactose, starch or magnesium stearate. The actual processes and/or methods for preparing these formulations/formulations are known or will be apparent to those skilled in the art (75).

The formulation/formulation of pharmaceutically acceptable Novaferon protein can be administered to human and/or non-human species by a variety of routes including, but not limited to, oral, subcutaneous, intravenous, intranasal, transdermal, intraperitoneal, intramuscular, intrapulmonary, vaginal, rectal or intraocular delivery, and direct topical application in the treatment of wounds.

According to clinical practice, the concentration/amount of Novaferon protein in the formulation/formulation may be from > 0 to 1.0M and/or from > 0 to 100% (weight/weight). The exact dosage, interval of administration, and duration of treatment of each and/or all Novaferon formulations/formulations are determined by clinical trials, disease conditions, patient status, and health care providers. In a preferred embodiment, due to protein degradation, differences in systemic versus local delivery, rates of synthesizing novel proteases, as well as age, body weight, general health, sex, diet, time of administration, drug interactions and the severity of the condition, among others, it may be desirable to adjust the administration of Novaferon, including but not limited to single and/or total doses, interval of administration, duration of treatment, and necessary course of treatment, and can be determined by one skilled in the art through routine experimentation.

In a preferred embodiment, the circulating half-life of the Novaferon protein may be altered following administration to a human and/or non-human species in vivo. These changes include, but are not limited to, an extension or shortening of the in vivo half-life of Novaferon. The in vivo half-life extension of the Novaferon protein can be achieved by a variety of means, including but not limited to:

(1) complexes between the Novaferon molecule and the monoclonal antibody are formed. Such antibodies are preferably linked to the Novaferon protein at a site that does not materially impair its therapeutic function (103).

(2) Fusion complexes of Novaferon with other proteins/polypeptides. The Novaferon molecule can be recombinantly fused to other proteins/polypeptides, such as the constant region fragment (Fc) of an immunoglobulin (104).

(3) Conjugation of Novaferon protein. For example, the Novaferon protein may be conjugated with a non-antigenic polymer, such as polyethylene glycol or related polyalkylene glycol moieties (105-108).

In another preferred embodiment, the therapeutic compound may be conjugated to an antibody, preferably an anti-Novaferon protein antibody. The therapeutic compound may be a cytotoxic agent. In this method, the cytotoxic agent may be targeted to the tumor tissue or cells by binding of the conjugated antibody to the Novaferon molecule, thereby destroying the diseased cells and reducing the number of diseased cells to achieve a reduction in the symptoms of cancer. Cytotoxic agents include, but are not limited to, cytotoxic drugs, toxins or active fragments of such toxins, and radioactive chemical agents. Suitable toxins and their corresponding fragments include diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, jatrophin, crotin, phenomycin, enomycin, and the like.

In a preferred embodiment, the full length sequence, partial sequence and/or regulatory sequences of the polynucleotide sequence encoding the Novaferon protein (SEQ ID No: 1) may be used by any one of skill in the art for gene therapy related administration. Antisense applications based on the polynucleotide sequence encoding the Novaferon protein (SEQ ID No: 1) can also be used as gene therapy (i.e., integrated into the genome) or as antisense compositions, as will be appreciated by those skilled in the art.

In gene therapy applications, genes are introduced into cells to obtain in vivo synthesis of target proteins encoded by these genes. Conventional gene therapy achieves sustained therapeutic efficacy through single or repeated administration of therapeutically effective DNA or mRNA. On the other hand, antisense RNA and DNA can also be used as therapeutic agents to block the expression of certain genes in vivo. It has been shown that short antisense oligonucleotides can be delivered to cells where they act as inhibitors (109).

In a preferred embodiment, full or partial length polynucleotide sequences encoding the Novaferon protein (SEQ ID No: 1) are useful as DNA vaccines. Naked DNA vaccines are generally known in the art (110). Methods for applying full or partial length Novaferon-encoding genes (SEQ ID No: 1) as DNA vaccines are well known to those of ordinary skill in the art and include, but are not limited to, placing the Novaferon gene or partial Novaferon gene under the control of a promoter for expression of full or partial length Novaferon protein in human and/or non-human species.

Examples

The following examples are presented to more fully describe the manner in which the above-described invention may be practiced, and to set forth the best mode contemplated for carrying out various aspects of the invention. It should be understood that these examples in no way limit the true scope of the invention, but are given for illustrative purposes only. All references cited herein are incorporated by reference in their entirety.

Example 1 PCR amplification of human IFN-. alpha.Gene from human leukocyte cDNA

Total mRNA was extracted from human peripheral blood leukocytes. Using Advantage^TMRT-for-PCR kit (Clontech, Mountain View, CA, US) and the manufacturer's recommended cDNA synthesis primer (oligo dT 18).

Human IFN-alpha cDNA was amplified by PCR on an MG PTC thermal cycler using the following conditions: 2.5. mu.l of 10 XPfx amplification buffer (Invitrogen, Carlsbad, CA, US), 0.75. mu.l of 10mM dNTP, 0.5. mu.l of 25mM MgSO₄Mu.l platinumfx DNA polymerase (2.5U/. mu.l; Invitrogen,carlsbad, CA, US), 0.75 μ l cDNA, 0.75 μ l 5' primer (10 μ M; IFNaO 5: 5 '-TGGTGCTCAGCT (A/G) CAAGTC-3') (SEQ ID No: 3) 0.75 μ l 3' primer mix (1.7 μ M each; IFNaO 3-1: 5 '-AATCATTTCCATGTTG (A/G) ACCAG-3' (SEQ ID No: 4); IFNaO 3-2: 5'-AATCATTTCCCGGTTGTACCAG-3' (SEQ ID No: 5); IFNaO 3-3: 5'-AATCATTTCCATGTTGAAACAG-3' (SEQ ID No: 6); IFNaO 3-4: 5'-AATCATTTCAAGATGAGCCCAG-3' (SEQ ID No: 7); IFNaO 3-5: 5'-AATGATTTTCATGTTGAACCAG-3' (SEQ ID No: 8); IFNaO 3-6: 5 '-AATCATTT (C/G) (C/G) ATGTTGAACCAG-3' (SEQ ID No: 9); IFNaO 3-7: 5'-GATCATTTCCATGTTGAATGAG-3' (SEQ ID No: 10); IFNaO 3-8: 5'-GAGTCGTTTCTGTGTTGGATCAG-3' (SEQ ID No: 11)).

The amplified PCR products were electrophoresed on a 1.0% agarose gel, excised, gel purified, and cloned into pCRII-TOPO or pCR-4-TOPO vectors (Invitrogen, Carlsbad, Calif., US) according to the manufacturer's recommendations. Automated sequencing was performed in a Prism Ready Reaction Dye Termination mixture on an ABI automated sequencer (PE applied biosystems, CA, US).

Since the desired inserts for the IFNa6, IFNa7 and IFNa16 coding sequences were not found in the above clones, PCR was performed again under the above conditions, except for the type-specific primers. For specific amplification of IFNa6, the 5 'and 3' primers were IFNaO 5: 5 '-TGGTGCTCAGCT (A/G) CAAGTC-3' (SEQ ID NO: 3), and IFNaO 3-8: 5'-GAGTCGTTTCTGTGTTGGATCAG-3' (SEQ ID No: 11). For specific amplification of IFNa7, the 5 'and 3' primers were IFNaO7 UO: 5'-ATGCCCCTGTCCTTTTCTTTAC-3' (SEQ ID No: 12), and an equimolar mixture of IFNaO3-5 and IFNaO 3-6. For specific amplification of IFNa16, the 5 'and 3' primers used were IFNa7UO and IFNaO 3-7: 5'-GATCATTTCCATGTTGAATGAG-3' (SEQ ID No: 10). The amplified fragment was cloned into pCRII-TOPO or pCR-4-TOPO vector and sequenced as above.

All cloned human type I IFN-alpha genes were individually aligned to those DNA sequences in Genebank. The GeneBank nucleotide accession numbers for these genes referred to herein are: NM-024013 (IFN-. alpha.1), NM-000605 (IFN-. alpha.2), NM-010504 (IFN-. alpha.4), NM-010505 (IFN-. alpha.5), NM-008335 (IFN-. alpha.6), NM-008334 (IFN-. alpha.7), NM-008336 (IFN-. alpha.8), NM-002171 (IFN-. alpha.10), NM-002172 (IFN-. alpha.14), NM-002173 (IFN-. alpha.16), NM-021268 (IFN-. alpha.17), NM-002175 (IFN-. alpha.21).

Example 2 construction of a shuffling library of plasmids carrying type I HuIFN

To construct a plasmid carrying the coding sequence for one of the human type I IFN- α, a 15 base pair oligonucleotide with a BamHI nuclear EcoRI restriction site was synthesized based on the individual cDNA coding region of the mature human type I IFN protein (Genentech, South SanFrancisco, Calif., US). The GeneBank nucleotide accession numbers for these proteins referred to herein are: NM-024013 (IFN-. alpha.1), NM-000605 (IFN-. alpha.2), NM-010504 (IFN-. alpha.4), NM-010505 (IFN-. alpha.5), NM-008335 (IFN-. alpha.6), NM-008334 (IFN-. alpha.7), NM-008336 (IFN-. alpha.8), NM-002171 (IFN-. alpha.10), NM-002172 (IFN-. alpha.14), NM-002173 (IFN-. alpha.16), NM-021268 (IFN-. alpha.17), NM-002175 (IFN-. alpha.21). The plasmid constructed in example 1 was used as a template, together with primers, for standard PCR (111). The resulting product was digested with the restriction enzymes BamHI and EcoRI and cloned into the E.coli expression vector pBVB, a pBV 220-derived expression plasmid containing BamHI sites and EcoRI sites in its polyclonal region (86). These final constructs were verified by DNA sequence analysis (PE applied biosystems, US).

Using a pair of oligonucleotides BVF 4: 5'-AGGGCAGCATTCAAAGCAG-3' (SEQ ID NO: 13) and BVR 3: 5'-TCAGACCGCTTCTGCGTTCTG-3' (SEQ ID No: 14), and a DNA fragment containing the human IFN ORF was amplified by PCR using the previously constructed plasmid carrying type I HuIFN. The resulting products were mixed in equal amounts and digested with DNase I and assembled by PCR according to the procedure described by Stemmer (112).

The assembled PCR product was further amplified by a pair of inner primers: BVF: 5'-GAAGGCTTTGGGGTGTGTG-3' (SEQ ID No: 15) and BVR: 5'-AATCTTCTCTCATCCGC-3' (SEQ ID No: 16), followed by digestion with BamHI and EcoRI and recloning into the E.coli expression vector pBVB digested with RE BamHI and EcoRI. These final constructs were verified by DNA sequence analysis. The plasmid carrying the shuffled HuIFN-alpha gene was transformed into e.coli DH5 alpha competent cells.

In all of the above PCR procedures, conventional DNA polymerase (New England Biolab, MA, US) was used, not high fidelity DNA polymerase, whether PCR amplification or PCR assembly.

Example 3 screening of shuffled libraries

Freshly transfected E.coli DH5 alpha cells were cultured overnight at 37 ℃ on LB plates. Individual colonies were picked separately and inoculated into 100. mu.l of LB medium containing 50. mu.g/ml ampicillin in a 96-well plate. The colonies were shaken at 30 ℃ at 250 rpm. After overnight incubation, 10. mu.l of the bacterial culture was inoculated in duplicate into 100. mu.l of LB medium containing 50. mu.g/ml ampicillin in 96-well plates. The initial plate (the so-called stock plate) is temporarily stored at 4 ℃. Cells in duplicate plates were cultured at 30 ℃ until the OD600 became 0.4, and then induced with 42 ℃. After 4 hours of heat induction, the bacterial cultures were transferred directly to a-80 ℃ freezer to begin the freeze-thaw cycle. After 2 cycles of freeze-thaw, the bacterial suspension/lysate was diluted to the desired concentration and exposed to Daudi cell culture for antiproliferative testing (101) or to Wish cell culture for antiviral testing (113).

In each round of screening, 20,000 colonies were subjected to a primary screening and approximately 100 colonies with the highest antiproliferative or antiviral activity were selected for further validation testing. Selected bacterial cultures on the stock plates were streaked on LB plates containing 50. mu.g/ml ampicillin. Individual colonies were cultured overnight at 37 deg.C, picked and inoculated in 1ml LB medium containing 50. mu.g/ml ampicillin in a test tube. The bacteria in the test tube were incubated overnight at 30 ℃ with shaking at 250 rpm. Then, 40. mu.l of the cultured bacteria were inoculated into one of another set of tubes containing 1ml of LB with ampicillin (50. mu.g/ml). The samples were then subjected to the following steps as described above in relation to the primary screening step: induction of expression, collection of cell cultures, freeze-thaw cycling, and antiproliferative or antiviral testing.

In each round of screening, approximately 20 colonies with the highest antiproliferative or antiviral activity were picked after the validation test to perform automated sequencing of plasmids and their inserts. The insert with the unique DNA sequence was further amplified with a pair of PCR primers, i.e. BVBF: 5'-ACCATGAAGGTGACGCTC-3' (SEQ ID No: 17); and BVR: 5'-AATCTTCTCTCATCCGC-3' (SEQ ID No: 16), which are flanking sequences upstream and downstream, respectively, of the multiple cloning site of the pBVB vector. The amplified PCR product was used for the next round of shuffling library construction.

Based on the antiproliferative or antiviral activity increase, 5 cycles of screening steps were performed.

Example 4 expression and purification of recombinant Novaferon protein in E.coli

The Novaferon protein (SEQ ID No: 2) was expressed in E.coli. The SEQ ID No: 1 are cloned into a temperature-inducible pBVB vector and placed under the control of the PRPL promoter (114). The Novaferon expression plasmid pBVBNF was transformed into DH5 α cells. Individual colonies were picked, inoculated into 2ml of LB medium containing 50. mu.g/ml ampicillin, and cultured at 30 ℃ for 8 hours. Then, 2ml of the cultured bacteria were taken and further cultured overnight at 30 ℃ with 50ml of a medium containing 50. mu.g/ml ampicillin under agitation. The next morning, inoculating the bacteria cultured overnight at a ratio of 1: 10-1: 20 to the bacteria containing 50 μ g/mlAmpicillin in a large volume of LB medium and cultured with agitation at 30 ℃. When the culture reached mid-log phase of growth (a550 ═ 0.5-0.6), the culture temperature was rapidly raised to 42 ℃ and maintained for 4 hours, in order to induce expression of Novaferon. After 4 hours of heat induction, the bacteria were centrifuged and treated with PBS (137mM NaCl, 2.7mM KCl, 10mM Na)₂HPO₄，2mM KH₂PO₄) Washed 3 times and then stored at-80 ℃ until purification.

Most of the Novaferon protein molecules are soluble in the e.coli production system described herein, although they are overexpressed in the cytoplasm. Thus, the cells were disrupted by lysozyme digestion in cell lysis buffer I (50mM Tris-Cl (pH8.0), 1mM EDTA (pH8.0), 100mM NaCl). The lysate is further sonicated to disrupt the remaining intact cells and splice the DNA molecules. The lysate is then centrifuged.

The soluble Novaferon protein molecules in the supernatant are purified by hydrophobic, ion exchange chromatography and gel filtration in sequence. First, the supernatant was loaded onto and passed through a Phenyl Sepharose6 fast flow column (GE Healthcare, US). Next, the Novaferon protein containing fraction was Applied to a POROS 50D column (Applied biosystems, US). Third, the Novaferon molecule containing fractions were purified using POROS 50 HS columns (applied biosystems, US). Finally, the collected Novaferon molecules were further purified with HiLoad26/100 Superdex 75pg (Amersham, US).

The purity of the pure Novaferon protein was verified by 15% SDS-PAGE analysis. The pure recombinant Novaferon protein showed a single band with a Molecular Weight (MW) of 19-20 kDa. Mass spectrometry analysis showed that the purified Novaferon molecule was > 98% pure and had a molecular weight of 19313 daltons, consistent with a molecular weight of 19315 daltons, which is presumed from its amino acid sequence.

Example 5 expression and purification of recombinant HuIFN-. alpha.2b in E.coli

Expression plasmid pBV2b for HuIFN- α 2b contains the cDNA coding region of the mature HuIFN- α 2b protein (GeneBank nucleotide accession No.: NM-000605), which is heat inducible

Under the control of the PRPL promoter. Expression of HuIFN- α 2b was performed according to the protocol described by Joseph S and DavidWR (116).

Expression plasmid pBV2bF was transformed into DH5 α cells. Individual colonies were picked, inoculated into 2ml of LB medium containing 50. mu.g/ml ampicillin, and cultured at 30 ℃ for 8 hours. Then, 2ml of the cultured bacteria were taken out and further cultured overnight at 30 ℃ with 50ml of LB medium containing 50. mu.g/ml ampicillin under agitation. The next morning, the bacterial culture was inoculated in a large volume of LB medium containing 50. mu.g/ml ampicillin at a ratio of 1: 10 to 1: 20 and cultured with stirring at 30 ℃. When the culture reached mid-log growth (a550 ═ 0.5-0.6), the culture temperature was rapidly raised to 42 ℃ and maintained for 4 hours, in order to induce HuIFN- α 2b expression. After 4 hours of heat induction, cells were centrifuged and washed with PBS (137mM NaCl, 2.7mM KCl, 10mM Na)₂HPO₄，2mM KH₂PO₄) Washed 3 times and then stored at-80 ℃ until purification.

HuIFN-. alpha.2b is expressed in an insoluble form in the E.coli expression system described herein, and recovery and washing procedures for inclusion bodies are therefore performed according to the protocol described by Molecular Cloning (115). Briefly, the harvested bacterial cells were resuspended in cell lysis buffer I (50mM Tris-Cl (pH8.0), 1mM EDTA (pH8.0), 100mM NaCl) and lysed by lysozyme and sonication. The contents were washed 3 times with ice-cold cell lysis buffer II (cell lysis buffer I supplemented with 0.5% (v/v) Triton X-100).

The recovered inclusion bodies were disrupted by suspending in 7N guanidine for 4 hours at room temperature with agitation. After centrifugation at 4 ℃ for 15 minutes, the denatured protein was refolded in 0.15M Borex buffer pH9.5 at 4 ℃ for 48 hours. In the final refolding step, the pH was adjusted to 7.4 with HCl.

The solution containing the folded HuIFN- α 2b was then purified by hydrophobic, ion exchange chromatography and gel filtration. First, the solution was loaded onto and passed through a Phenyl Sepharose6 fast flow column (GE Healthcare, US). Next, the HuIFN-. alpha.2b containing fraction was Applied to a POROS 50D column (Applied Biosystem, US). Third, the HuIFN- α 2b containing fraction was purified using POROS 50 HS column (Applied Biosystems, US). Finally, the collected HuIFN- α 2b molecules were further purified with HiLoad26/100 Superdex 75pg (Amersham, US). In 15% SDS-PAGE analysis, the pure HuIFN-. alpha.2b protein appears as a single band and is > 98% pure as confirmed by mass spectrometry.

Example 6 measurement of antiviral Activity of Novaferon

Antiviral activity was determined using the WISH-VSV system in the conventional protocol described in Armstrong JA (113). The first day, WISH cells (ATCC, catalog number CCL25) were seeded at a density of 14,000 cells/well in 96-well plates and incubated at 37 ℃. After 24 hours, 2-fold serial dilutions of Novaferon, HuIFN- α 2b, WHO IFN international standard or blank medium were added to each well and incubated for an additional 24 hours at 37 ℃. On the third day, the medium was removed and replaced with a medium containing 1,000PFU of vesicular stomatitis virus (VSV, ATCC, Cat. No. VR-1421). The cells were again incubated at 37 ℃ for 24 hours, followed by washing with 0.85% NaCl to remove dead cells. Then, the culture plate was soaked in dye-fixing solution (dye-fixer solution) (0.5% crystal violet, 5% formalin (V/V), 50% ethanol (V/V), and 0.85% NaCl) for 1 hour. Next, the dye fixing solution was decanted and the microplate was rinsed thoroughly with tap water and allowed to dry. The stained cells were lysed with 0.2ml 2-methoxyethanol. For the crystal violet bioassay, plates were read at 550nm in a ModelOpsys MR (Thermo Labsystems, US).

All experiments were repeated 3 times and Novaferon and HuIFN- α 2b samples were tested in the same plate. For a parallel analysis of the antiviral activity of Novaferon and HuIFN- α 2b prepared herein, the antiviral units (international units or IU) were determined with reference to WHO international Standards 94/786 (rhufn- α complex) and 95/650 (rhufn- α 2a), which are commercially available from National Institute for Biological Standards and control (NIBSC, USA).

The antiviral activity of the purified Novaferon protein against VSV in WISH was measured to be 2.5X 10⁹IU/mg, and the antiviral activity of HuIFN-. alpha.2b is 2.0X 10⁸IU/mg. This data indicates that the antiviral activity of the Novaferon protein is about 12.5 times that of HuIFN-. alpha.2b.

Example 7 antiproliferative Activity of Novaferon

The antiproliferative activity assay was performed essentially as described by Esinger and Pestka (101).

A. Cell culture of human tumor cell lines

Human tumor Cell lines were purchased from different institutions (Table 1 below), namely ATCC (American type Culture Collection, P.O.Box 1549, Manassas, VA 20108, USA), DSMZ (German National Resource Centre for biologica Material, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig, Germany), JCRB (Japanese Collection of research Bioresources-Cell Bank, National Institute of biomedical Innovation, 7-6-8 Saito-Asagi, Ibaraki-shi, Osa 7-567-0085, Japan).

TABLE 1 human tumor cell lines

DSMZ: deutsche Sammlung von Mikroorganismen Zellkulturen, Germany

ATCC: american Type Culture Collection, USA

JCRB：Japanese Collection of Research Bioresources-Cell Bank，Japan

At 37 ℃ in a medium containing 5% CO₂All cells tested for antiproliferative activity were cultured in a humid atmosphere. Cells were grown in basal growth medium according to the growth manual for each cell, e.g., DMEM, MEM, F12K and 1640 or 1640 plus F12 (all from Gibco BRL, US) supplemented with 5-20% heat-inactivated fetal bovine serum FBS from Gibco BRL, US. The basal growth medium for each cell line is listed in table 2 below. All cell lines were examined daily in an inverted microscope in culture plates. At its logarithmic growth phase (viability over 90% as determined by trypan blue dye exclusion), cells were harvested and used for experiments. Cell number and viability were checked in a standard hemocytometer.

TABLE 2 culture and measurement of human tumor cell lines

B. Procedure for antiproliferative assays

Cell lines in logarithmic growth phase were gently suspended in warm (36 ℃) medium at a density of 2X 10³-6×10⁴Individual cells/ml (varied from cell line to cell line, see table 2). In 96-well platesInto each well 100. mu.l of the cell suspension was inoculated, followed by incubation at 37 ℃ for 6-8 hours. Then, the same volume (100 μ l) of Novaferon or HuIFN- α 2b diluted in culture medium was added to the wells in triplicate. The plates were gently shaken for 4-5 seconds to mix the contents and incubated at 37 ℃ for 6 days. Novaferon and HuIFN- α 2b samples were tested in the same plate to ensure comparability.

Two methods were used to determine the number of cells in the cell well and the antiproliferative activity of Novaferon and HuIFN- α 2b was calculated from the number of cells.

Direct cell counting was used to determine the cell number of the suspended cells. After 6 days of culture, the suspension cell culture was diluted with trypan blue (final concentration: 0.02%) and the number of cells was directly counted with a hemocytometer.

Crystal violet bioassay was used to determine the cell number of adherent cells (93). After 6 days of culture, dead cells were removed by pipetting PBS in culture wells. Next, the wells were filled with a dye fixing solution to stain the living cells for 1 hour. The dye fixing solution contained 0.5% crystal violet, 5% formalin (V/V), 50% ethanol (V/V), and 0.85% NaCl in distilled water. The microplate was then rinsed thoroughly with tap water and allowed to dry. The stained cells were lysed with 0.2ml 2-methoxyethanol. The optical density at 550nm (OD550) was measured (ModelOpsys plate reader, Thermo Labsystems, US) and used as a relative indicator of the number of cells.

The growth inhibition rate was calculated by the following formula: percent inhibition ═ 1- (E-B)/(C-B)) × 100, where E is the number of cells or OD in Novaferon or HuIFN- α 2B treated wells on day 6₅₅₀A value; b is the cell number or OD of the cultured cells in the cell culture on day 0₅₅₀A value; c is the cell number or OD in untreated wells at day 6₅₅₀The value is obtained.

The inhibition rate is expressed together with the concentration of the compound. Estimation of IC of Novaferon or HuIFN-. alpha.2b by Using a series of sample concentrations₅₀. Fitting the data into a sigmoidal curve (117) havingThere is the Hill slope equation: y ═ Min + (Max-Min)/(1+10^ (IC)₅₀-X)), wherein X is the logarithm of the drug concentration; y is the inhibition rate; min or Max is the minimum or maximum inhibition rate platform. The IC of various compounds can be compared for a particular target₅₀Wherein IC₅₀Lower indicates more effective compounds.

The concentrations of Novaferon and HuIFN-. alpha.2b and the corresponding cell growth inhibition rates for the Daudi cell lines are given in FIG. 4. According to the data, the IC of the Novaferon and HuIFN-alpha 2b for inhibiting the growth of Daudi cells is calculated₅₀0.0174pmol and 6.9550 pmol. Thus, Novaferon's IC₅₀Is about 1/400 for HuIFN- α 2b, which indicates that Novaferon has about 400 times greater anti-proliferative efficacy than HuIFN- α 2 b.

Novaferon was evaluated for antiproliferative activity and compared with the antiproliferative activity of HuIFN-. alpha.2b in 23 tumor cell lines, including 4 melanoma-derived cell lines (A-375, IGR-1, IGR-37, IPC-298), 5 colorectal adenocarcinoma cell lines (HCT-8, SW1116, LS180, DLD-1, LS174T), 4 liver cancer cell lines (Hep G2, Hep 3B, HuH-7, PLC/PRF/5), 3 lymphoma cell lines (HL-60(S), Daudi, L-428), 2 prostate cancer cell lines (DU 145, PC-3), 2 cervical cancer cell lines (HeLa, C-33A), 1 gastric adenocarcinoma cell line (MKN1), 1 lung cancer cell line (A) and KY1 esophageal cancer cell line (SE 30). Novaferon showed much stronger antiproliferative activity against all cancer cell lines tested than HuIFN-. alpha.2b. The degree of increase in potency varied from 16 to 1134-fold in different cancer cell lines (table 3 below).

TABLE 3 IC of Novaferon and HuIFN-. alpha.2b₅₀Value of

And fold increase in tumor cell inhibition by Novaferon relative to HuIFN-. alpha.2b

Example 8 in vivo tumor model experiments

A. Cell culture and in vivo human tumor xenograft models

The colon cancer cell line (LS180), melanoma cell line (A-375), and liver cancer cell line (Hep G2) were obtained from the American type culture Collection (ATCC, Rockville, Md.). The prostate cancer cell line (PC-3) was obtained from Deutsche Sammlung von Mikroorganismen and Zellkulturen, Germany. The lymphocyte Cell line (HL 60(S)) was purchased from Japanese Collection of research Bioresources Cell Bank (JCRB, Japan). All cells were cultured according to their protocol (see table 1). Briefly, LS180 and Hep G2 were cultured in MEM medium. A-375 was cultured in DMEM. Both media were supplemented with 10% Fetal Bovine Serum (FBS), 2mM glutamine, 100U/ml penicillin, 100mg/ml streptomycin, 0.1mM non-essential amino acids, and 1.0mM sodium pyruvate. PL-3 and HL60(S) cells were cultured in RPMI1640 supplemented with 10% FBS, 100U/ml penicillin and 100mg/ml streptomycin. All cells were maintained at 37 ℃ at 5% CO₂In an atmosphere.

A model of human cancer transplantation was established using the method described by Beverly et al (118). Logarithmically growing cancer cells were harvested from tissue culture plates, washed and resuspended in phosphate buffered saline (PBS, pH 7.5, 20 mM). In the two ribs of a 6-week-old athymic Balb/c nude mouse, 6X 10 mice are injected subcutaneously⁶Cells/0.3 ml (PC-3, HepG2), 4X 10⁶Cells/0.3 ml (LS180), 2X 10⁷Cells/0.3 ml (HL 60(s)) or 8X 10⁶Individual cells/0.3 ml (a-375), a subcutaneous tumor graft was generated. For each in vivo tumor model, tumor-bearing mice (tumor volume of about 100 mm) were treated on day 6 after tumor cell inoculation³) Randomized into 7 or 8 groups of the same number of animals per group and treatment was initiated.

Novaferon and HuIFN-. alpha.2b were formulated in PBS solution. PBS alone, various doses of Novaferon, or HuIFN-. alpha.2b were injected subcutaneously daily for 30 days (PC-3, HepG2, A-375), 28 days (LS180), or 21 days (HL 60(s)) starting on the day of mice grouping. For the treatment of 5-FU, 30mg/kg of 5-FU was administered intravenously every two days for a total of 5 times. Groups and therapeutic doses are summarized below:

group 1 (control): PBS was used daily.

Group 2 (low dose Novaferon): 1.25. mu.g/kg per day.

Group 3 (medium dose Novaferon): 12.5. mu.g/kg per day.

Group 4 (high dose Novaferon): 125. mu.g/kg per day.

Group 5 (low dose HuIFN- α 2 b): 1.25. mu.g/kg per day.

Group 6 (moderate dose HuIFN- α 2 b): 12.5. mu.g/kg per day.

Group 7 (high dose HuIFN- α 2 b): 125. mu.g/kg per day.

Group 8 (5-FU): 30mg/kg, once every 2 days, 5 times.

Once treatment was initiated, tumors were measured weekly using calipers. Tumor volume was calculated using the following formula: volume is 0.5 × width²Length x. On the day of treatment cessation (day 30 after treatment initiation), mice were sacrificed. Solid tumors were isolated, photographed, and measured.

The growth inhibition rate was calculated using the following formula: inhibition ═ 1-T/C × 100%, where T is the mean weight of tumors in the Novaferon, HuIFN- α 2b or 5-FU treated groups; c is the average weight of the tumor in the control group after treatment.

B. Human prostate cancer xenograft model

Prostate cancer PC-3 xenografts were treated by subcutaneous injection of 1.25, 12.5, or 125 μ g/kg novaferon for 30 days. Novaferon showed a strong, dose-dependent inhibition of PC-3 tumor growth (P < 0.05). As shown in figure 5 and table 4 below, PC-3 tumor growth was greatly inhibited in the Novaferon treated group compared to the PBS treated control group. For example, the mean weight of the PC-3 xenograft tumor mass in the Novaferon treated group (125. mu.g/kg) was 0.091. + -. 0.081g, which is a very significant reduction (P < 0.001) compared to 1.948. + -. 0.567g in the control group of animals (Table 4). In other words, 95.3% inhibition of PC-3 tumor growth was obtained with treatment at 125. mu.g/kg for 30 days (Table 4).

TABLE 4 human prostate cancer PC-3 treated with Novaferon and HuIFN-. alpha.2b

Tumor weight and growth inhibition rate of xenografts (n ═ 10)

Note: p < 0.05, p < 0.001: compared to the control group; p < 0.001: compared with the high-dose HuIFN-alpha 2b group.

At 6X 10⁶Balb/c nude mice were treated for 30 days by daily subcutaneous injection of Novaferon (1.25. mu.g/kg, 12.5. mu.g/kg or 125. mu.g/kg) after subcutaneous introduction of live PC-3 cells into the mice. Results are expressed as mean tumor volume (mm)³). FIG. 5 shows that all 3 doses of Novaferon exhibited dose-dependent inhibition of PC-3 tumor growth (P < 0.05) compared to the PBS control group. Novaferon at 125. mu.g/kg induced much stronger or almost complete inhibition of PC-3 tumor growth (95.3% vs 75.6%, P < 0.01) than HuIFN-. alpha.2b at the same dose (Table 4).

It is interesting to note that longer treatment with Novaferon or HuIFN- α 2b resulted in greater differences in tumor growth inhibition in the high dose (125 μ g/kg) treatment group. The mean volume of the PC-3 tumor mass in the Novaferon-treated group was 107.9. + -. 68.7mm on day 28³And 620.7 + -296.6 mm in the HuIFN-alpha 2b treatment group³(P is less than 0.001) and the diameters of the particles are respectively 122.1 +/-100.7 mm on day 30³691.9 + -428.3 mm³(P < 0.001). This also occurred after the end of the observation taking into account the mean tumor weight (0.091. + -. 0.081g in the high dose Novaferon group, while HuIFN-0.476. + -. 0.271g in the group. alpha.2b, P < 0.001). This suggests that longer treatment of the dose of Novaferon may exhibit better or complete inhibition of PC-3 tumor growth in the xenograft model.

C. Human liver cancer xenograft model

The in vivo anti-tumor activity of Novaferon was also evaluated in a liver cancer Hep G2 xenograft model. Novaferon showed effective, dose-dependent inhibition of Hep G2 tumor growth (P < 0.001) compared to the control group. Mean tumor volumes in the Novaferon-treated groups (1.25, 12.5 or 125. mu.g/kg subcutaneously per day for 30 days) were 783.2 + -270.0, 459.3 + -414.3 and 104.6 + -56.5 mm, respectively³And 2125.8 + -743.1 mm in PBS control group³. The highest inhibition of Hep G2 (96.6%) was achieved with Novaferon treatment at 125. mu.g/kg for 30 days, which was significantly better than the inhibition of HuIFN-. alpha.2b at 125. mu.g/kg (89.2%, P < 0.01). Longer treatment with Novaferon at this dose showed a trend of better or complete inhibition. At the end of the observation period, the mean tumor weight at 125 μ g/kg for the Novaferon treated group was 0.074 ± 0.083g, which is significantly lower than the mean tumor weight for the 125 μ g/kg HuIFN- α 2b treated group (0.235 ± 0.199g, P < 0.001) (table 5 below).

At 6X 10⁶Balb/c nude mice were treated for 30 days by daily subcutaneous injection of Novaferon (1.25. mu.g/kg, 12.5. mu.g/kg and 125. mu.g/kg) after subcutaneous introduction of live Hep G2 cells into the mice. Results are expressed as mean tumor volume (mm)³). Figure 6 shows that all 3 doses of Novaferon showed dose-dependent inhibition of Hep G2 tumor growth (P < 0.001) compared to the PBS control group. Novaferon at 125. mu.g/kg induced much stronger or almost complete tumor growth inhibition by Hep G2 (96.6% vs. 89.2%, P < 0.05) than HuIFN-. alpha.2b at the same dose (Table 5).

TABLE 5 human hepatoma cells Hep G2 treated with Novaferon and HuIFN-. alpha.2b

Tumor weight and growth inhibition rate of xenografts (n ═ 10)

Note: p < 0.01, p < 0.001: compared to the control group; p < 0.01: compared with the high-dose HuIFN-alpha 2b group.

D. Human melanoma xenograft model

Novaferon was further evaluated for in vivo anti-tumor activity in a malignant melanoma A-375 xenograft model. The A-375 cell line (ATCC accession number: CRL-1619) was derived from a human malignant solid tumor. Novaferon showed effective, dose-dependent inhibition of A-375 tumor growth (P < 0.001) compared to the control group. The inhibition in the Novaferon treated group(s) (subcutaneous injections of 1.25, 12.5 or 125 μ g/kg per day for 28 days) was 40.1%, 75.0% and 82.8%, respectively, as compared to the PBS control group (P < 0.001) (table 6 below). The highest inhibition of A-375 (82.8%) was achieved with Novaferon treatment at 125. mu.g/kg for 30 days, which was significantly better than the inhibition of HuIFN-. alpha.2b at 125. mu.g/kg (69.9%, P < 0.001).

Significantly, Novaferon showed more effective growth inhibition of melanoma cells a-375 than the chemotherapeutic agent 5-FU (table 6). For example, on day 30, the mean tumor weights were 0.763. + -. 0.187g (P < 0.01) and 0.527. + -. 0.149g (P < 0.001) in the groups treated with 12.5. mu.g/kg or 125. mu.g/kg Novaferon, whereas the mean tumor weight was 1.004. + -. 0.105g in the group treated with 30mg/kg5-FU (Table 6). This suggests that Novaferon may be more effective than 5-FU for treating human melanoma A-375.

At 8X 10⁶Balb/c nude mice were treated for 28 days by daily subcutaneous injection of Novaferon (1.25. mu.g/kg, 12.5. mu.g/kg and 125. mu.g/kg) after a single A-375 cell was subcutaneously introduced into the mice. Results are expressed as mean tumor volume (mm)³). FIG. 7 shows that all 3 doses of Novaferon exhibited dose-dependent inhibition of A-375 tumor growth (P < 0.001) compared to the PBS control group. Novaferon at 125. mu.g/kg induced a stronger tumor growth inhibition of A-375 (82) than HuIFN-. alpha.2b at the same dose.8% vs 69.9%, P < 0.001) (FIG. 7). Both 12.5. mu.g/kg and 125. mu.g/kg Novaferon showed better tumor growth inhibition (75.0% and 82.8%, respectively) than 5-FU (67.2%, P < 0.01 and P < 0.001) (FIG. 7).

TABLE 6 human melanoma cells treated with Novaferon and HuIFN-. alpha.2b A-375

Tumor weight and growth inhibition rate of xenografts (n ═ 10)

Note: p < 0.001, compared to control; $ p < 0.001: compared to a medium dose (12.5) of HuIFN- α 2 b; p < 0.001: compared to the high dose (125) HuIFN-. alpha.2b group; and &: p < 0.01, & & & &: p < 0.001, compared to the 5-FU group.

E. Human colon cancer xenograft model

Novaferon was evaluated for in vivo anti-tumor activity in a colon cancer LS180 xenograft model. The LS180 cell line (ATCC accession number: CL-187) was derived from human colon adenocarcinoma. Novaferon showed effective, dose-dependent inhibition of colon cancer LS180 tumor growth (P < 0.001) compared to the control group. The inhibition rates in the Novaferon treated groups (subcutaneous injections of 1.25, 12.5 or 125 μ g/kg per day for 28 days) were 75.0%, 80.5% and 92.5%, respectively, in comparison to the PBS control group (P < 0.001, table 7 below). The highest inhibition of LS180 tumor growth (92.5%) was achieved with 125. mu.g/kg of Novaferon for 28 days, which was significantly better than the inhibition of 125. mu.g/kg of HuIFN-. alpha.2b (82.3%, P < 0.001).

After 28 days of treatment, 12.5 μ g/kg Novaferon inhibited LS180 cancer xenograft growth similar to 5-FU (30mg/kg) (0.815. + -. 0.221g vs. 0.758. + -. 0.227g) in mean tumor weight. 125 μ g/kg Novaferon significantly inhibited LS180 tumor growth better than 30mg/kg5-FU (92.5% vs. 81.8%, P < 0.001) (Table 7 and FIG. 8). These observations are very significant given the routine clinical use of 5-FU in standard chemotherapy in colon cancer patients. The better inhibition of LS180 tumor growth by Novaferon in animal models suggests that Novaferon has the potential to be a very effective anti-colon cancer agent in clinical settings.

At the place of 4X 10⁶Balb/c nude mice were treated for 28 days by daily injection of Novaferon (1.25. mu.g/kg, 12.5. mu.g/kg and 125. mu.g/kg) after subcutaneous introduction of individual viable LS180 cells into the mice. Results are expressed as mean tumor volume (mm)³). Figure 8 shows that all 3 doses of Novaferon showed dose-dependent LS180 tumor growth inhibition (P < 0.001) compared to the PBS control group. Novaferon at 125. mu.g/kg induced a greater inhibition of LS180 tumor growth (92.5% vs. 82.3%, P < 0.001) than HuIFN-. alpha.2b at the same dose (Table 7). Both 1.25. mu.g/kg and 12.5. mu.g/kg Novaferon obtained similar tumor growth inhibition (75.0% and 80.5%, respectively) as 5-FU (81.8%) (Table 7, FIG. 8). However, 125. mu.g/kg Novaferon showed much better LS180 tumor growth inhibition than 5-FU (92.5% vs. 81.8%, P < 0.001).

TABLE 7 human colon carcinoma cells LS180 treated with Novaferon and HuIFN-. alpha.2b

Tumor weight and growth inhibition rate of xenografts (n ═ 10)

Note: p < 0.001, compared to control; p < 0.001: compared with high-dose HuIFN-alpha 2 b; and &, p < 0.001, compared with the 5-FU group.

F. Human leukemia xenograft model

Novaferon was also evaluated for its in vivo anti-tumor activity in an HL60(s) lymphocytic leukemia xenograft model. Novaferon showed effective, dose-dependent inhibition of HL60(s) tumor growth (P < 0.001) compared to the control group. The inhibition rates in the Novaferon treated groups (subcutaneous injections of 1.25, 12.5 or 125 μ g/kg per day for 28 days) were 43.8%, 55.2% and 80.4%, respectively, compared to the PBS control group (P < 0.001, table 8 below). The highest inhibition of HL60(s) tumor growth (80.4%) was achieved with 125. mu.g/kg of Novaferon for 21 days, which was significantly better than the 125. mu.g/kg HuIFN-. alpha.2b inhibition (69.8%, P < 0.05).

At 2X 10⁷After subcutaneous introduction of live HL60(s) cells into mice, Balb/c mice were treated for 21 days by daily subcutaneous injections of Novaferon (1.25. mu.g/kg, 12.5. mu.g/kg and 125. mu.g/kg). Results are expressed as mean tumor volume (mm)³). Figure 9 shows that all 3 doses of Novaferon showed dose-dependent HL60(s) tumor growth inhibition (P < 0.001) compared to the PBS control group. Novaferon at 125. mu.g/kg induced a stronger suppression of HL60(s) tumor growth (80.4% vs. 69.8%, P < 0.05) than HuIFN-. alpha.2b at the same dose, and a similar suppression as 5-FU (FIG. 9, Table 8).

TABLE 8 human leukemia cells HL60(S) treated with Novaferon and HuIFN-. alpha.2b

Tumor weight and growth inhibition rate of xenografts (n ═ 10)

Note: p < 0.001, compared to control; p < 0.05: compared to the high dose (125) HuIFN-. alpha.2b group.

G. General status of mice during Novaferon treatment

Mice bearing various human cancer xenografts were closely observed during treatment with Novaferon, HuIFN- α 2b or 5-FU. Unlike in the 5-FU treated group, mice in all Novaferon or HuIFN- α 2b treated groups generally diet and behave normally and without weight loss. Mice treated with 5-FU showed typical dietary and behavioral changes, and weight loss. These observations suggest that while exhibiting similar or better anti-cancer efficacy than 5-FU in xenograft animal models, Novaferon may be more specific with respect to inhibition of tumor cells and have much less impact on normal cellular and/or physiological function. These translate into better tolerability and superior therapeutic efficacy in human applications.

As will be apparent to those skilled in the art in the light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without departing from the scope or spirit thereof. The scope of the invention should, therefore, be determined with reference to the spirit of the invention as defined by the appended claims.

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Claims (21)

1. An isolated polynucleotide encoding a protein having human interferon-like biological activity, wherein said polynucleotide consists of the nucleotide sequence of seq id no:

(a) the coded amino acid sequence is SEQ ID No: 2, the nucleotide sequence of the Novaferon protein of 2; or

(b) A nucleotide sequence which is fully complementary to the nucleotide sequence in (a) above.

2. The polynucleotide of claim 1, wherein the sequence of the polynucleotide is SEQ id no: 1.

3. the polynucleotide of any one of claims 1-2, wherein the protein is not naturally occurring.

4. A recombinant vector comprising the polynucleotide of claim 1.

5. A host cell comprising the recombinant vector of claim 4.

6. A non-naturally occurring protein exhibiting human interferon-like biological activity, wherein the amino acid sequence of said protein is SEQ ID No: 2.

7. the protein of claim 6, wherein the protein is recombinant.

8. A protein construct comprising the protein of claim 6 coupled to another moiety, wherein said construct exhibits said human interferon-like biological activity.

9. The protein construct of claim 8, wherein the protein is glycosylated.

10. The protein construct of claim 8, wherein the moiety is a polypeptide.

11. The protein construct of claim 8, wherein the moiety is a non-polypeptide.

12. The protein construct of claim 11, wherein the moiety is a polymer molecule.

13. A protein construct according to claim 12, wherein the polymer molecule is linear or branched polyethylene glycol.

14. The protein construct of claim 8, wherein the moiety is a labeling molecule.

15. A composition comprising the protein of claim 6 and a pharmaceutically acceptable carrier, diluent or excipient.

16. Use of a protein of claim 6 in the manufacture of a medicament for treating cancer in a subject, wherein the cancer is selected from melanoma, colorectal adenocarcinoma, liver cancer, lymphoma, prostate cancer, gastric adenocarcinoma, esophageal cancer, lung cancer, and cervical cancer.

17. Use of the protein of claim 6 in the preparation of a medicament for treating hepatocellular carcinoma in a subject.

18. Use of a protein of claim 6 in the manufacture of a medicament for treating cervical adenocarcinoma in a subject.

19. Use of a protein according to claim 6 in the manufacture of a medicament for treating a viral disease caused by vesicular stomatitis virus in a subject.

20. The use of any one of claims 16-19, wherein the subject is a human.

21. The use of any one of claims 16-19, wherein the medicament comprises a pharmaceutically acceptable carrier, diluent or excipient.