RU2788736C2 - Interferon prodrug for cancer treatment - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: group of inventions relates to the field of pharmaceuticals and medicine. 1 object is an interferon prodrug for the treatment of cancer containing: (a) interferon alpha and beta receptor 2 domain (IFNAR2), which retains IFN-binding activity; (b) interferon type 1 domain (IFN), which retains the activity of interferon type 1 when it is not bound by the specified IFNAR2 domain; (c) the Fc domain of immunoglobulin (Ig), (d) the first linker fused at one end with the N-end of the specified IFN and fused at the other end with the specified IFNAR2, and the specified first linker is G4S-PVGLIG-G4S; and (e) a second linker fused at one end with the C-end of the specified IFN and fused at the other end with the N-end of the specified Fc-domain Ig. 2-4 objects are a nucleic acid construct encoding an interferon prodrug, a recombinant cell containing a nucleic acid construct, and a method for expressing an interferon prodrug. 5-7 objects suggest the use of the interferon prodrug for the treatment of cancer and in the preparation of a drug, a method for the treatment of cancer, including the administration of the interferon prodrug to the patient.

EFFECT: effectiveness and reduced side effects of an IFN prodrug based on IFNAR2.

26 cl, 7 dwg, 1 tbl, 1 ex

Description

PRIORITY CLAIM

This application claims priority to U.S. Provisional Application Serial No. 62/522564, filed June 20, 2017, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Technical field

The present invention generally relates to the fields of medicine, oncology and immunotherapy. More specifically, it relates to the development and use of immunoreagents for the treatment of cancer.

2. State of the art

It is believed that type I interferons (IFN) directly suppress the proliferation of tumor cells. They have been successfully used in the treatment of several types of cancer, including hematologic tumors (chronic myeloid leukemia, hairy cell leukemia, multiple myeloma, and non-Hodgkin's lymphomas) as well as solid tumors (melanoma, kidney cancer, and Kaposi's sarcoma) (Ferrantini et al., 2007, Moschos & Kirkwood, 2007, Zitvogel et al., 2015, and Antonelli et al., 2015). Indeed, according to accumulating data, endogenous type I IFN plays a key role in "teaching" dendritic cells (DCs) to increase the efficiency of cross-priming against tumor antigens to enhance antitumor activity in chemotherapy, radiotherapy, targeted therapy and immunotherapy (Schiavoni et al., 2011, Burnette et al., 2011; Stagg et al., 2011 and Woo et al., 2014).

A particular benefit of type I IFN treatment is due to its ability to be activated at many points in the generation of antitumor immune responses, including stimulation of populations of innate and adaptive cytotoxic lymphocytes, downregulation of suppressor-type cells, effects on tumor cells by inhibiting proliferation and modulating apoptosis, differentiation, migration, and antigen expression on the cell surface (Parker et al., 2016). Importantly, some of these actions may represent a potential strategy for using type I IFN to overcome cancer resistance to immunotherapy. One of the mechanisms of recurrence in cancer patients is the inability of T cells to recognize due to suppression of the expression of class I MHC molecules and peptide transporter genes for tumor antigen presentation (Sharma et al., 2017). Type I IFN can be used to induce class I MHC expression, such as LMP2/7 and TAPA-1/2 expression in tumor cells, which together is an ideal combination therapy strategy to combat immunotherapy resistance (Khanna, 1998). In addition, type I IFN can be used to suppress Tregs and MDSCs by downregulating Tregs proliferation (Pace et al., 2010 and Srivastava et al., 2014) and reducing MDSC accumulation and downregulation (Zoglmeier et al., 2011) , both of which directly suppress the activity of cytotoxic T-lymphocytes (Schmidt et al., 2012 and Gabrilovich et al., 2009). These multiple antitumor effects have generated interest in type I IFNs as anticancer drugs, either alone or in combination with other therapies.

However, one of the biggest obstacles to the use of type I IFN in the clinic is the serious side effects associated with such treatment. The most commonly reported side effects are flu-like symptoms, haematological toxicity, elevated transaminase levels, nausea, fatigue, and psychiatric effects. These side effects prevent achievement and maintenance of doses required to obtain maximum therapeutic effect, since the occurrence of these side effects can completely invalidate the clinical benefit of type I IFN treatment (Kreutzer et al., 2004, Sleijfer et al., 2005 and Lotrich, 2009). Therefore, the ability to provide specific delivery of type I IFN to the tumor microenvironment is a necessary condition for long-term clinical use of type I IFN. Strategies for modifying type I IFN are needed to provide new forms of the drug that are only active within the tumor and to prevent serious side effects outside the tumor.

SUMMARY OF THE INVENTION

Thus, the present invention provides an interferon prodrug comprising (a) an interferon alpha and beta receptor (IFNAR) domain that retains IFN-binding activity; (b) a type 1 interferon (IFN) domain that retains type 1 interferon activity when not associated with said IFNAR domain; (c) an immunoglobulin (Ig) Fc domain, (d) a first linker fused at one end to the N-terminus of said IFN and fused at the other end to said IFNAR, said first linker being cleavable by a protease; and (e) a second linker fused at one end to the C-terminus of said IFN and fused at the other end to the N-terminus of said Ig Fc domain. The Ig may be an IgG such as IgG1 or IgG2. An interferon prodrug may contain two copies of said type 1 IFN domain. An interferon prodrug may contain more than two copies of the specified type 1 IFN domain. The first linker may be cleavable by one or more matrix metalloproteinases such as MMP1, MMP3, MMP9, MMP10, MMP11, MMP12, MMP13 or MMP14. The first linker may be cleavable by UPA, FAPa and/or cathepsin B. The linker may be G4S-SUB1-G4S-SUB2-G4S-SUB3-G4S, where SUB1-3 are different enzyme cleavage sites. IFNAR may be IFNAR1 or IFNAR2. The IFN may be IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω or IFN-ζ.

In another embodiment, the invention relates to a nucleic acid construct encoding an interferon prodrug comprising (a) an interferon alpha and beta receptor (IFNAR) domain that retains IFN-binding activity; (b) a type 1 interferon (IFN) domain that retains type 1 interferon activity when not associated with said IFNAR domain; (c) an immunoglobulin (Ig) Fc domain, (d) a first linker fused at one end to the N-terminus of said IFN and fused at the other end to said IFNAR, said first linker being cleavable by a protease; (e) a second linker fused at one end to the C-terminus of said IFN and fused at the other end to the N-terminus of said Ig Fc domain; and (f) a promoter located 5' to the 5' end of said IFNα domain. The Ig may be an IgG such as IgG1 or IgG2. The interferon prodrug contains two copies of the specified type 1 IFN domain. An interferon prodrug may contain more than two copies of the specified type 1 IFN domain. The first linker may be cleavable with a matrix metalloproteinase such as MMP1, MMP3, MMP9, MMP10, MMP11, MMP12, MMP13 and/or MMP14. The first linker may be cleavable by UPA, FAPa and/or cathepsin B. The linker may be G4S-SUB1-G4S-SUB2-G4S-SUB3-G4S, where SUB1-3 are different enzyme cleavage sites. IFNAR may be IFNAR1 or IFNAR2. The IFN may be IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω or IFN-ζ.

The invention also relates to a recombinant cell expressing an interferon prodrug as defined above; a recombinant cell containing the nucleic acid construct as defined above; a method for expressing an interferon prodrug, comprising culturing a cell as defined above; a method for expressing an interferon prodrug, comprising culturing a cell as defined above; and the use of the above-defined interferon prodrug (a) in the preparation of a medicament for the treatment of cancer; or (b) for the treatment of cancer.

In another embodiment, the invention relates to a method of treating cancer comprising administering to a patient an interferon prodrug as defined above. The method may further include the step of assessing protease expression in a cancer cell obtained from said patient. The cancer cell may be obtained from a biopsy or may be a circulating tumor cell. The cancer may be lung cancer, breast cancer, brain cancer, oral cavity cancer, esophageal cancer, head and neck cancer, skin cancer, gastric cancer, liver cancer, pancreatic cancer, kidney cancer, ovarian cancer, prostate cancer, bladder cancer, colon cancer, testicular cancer, uterine cancer, cervical cancer, lymphoma, or leukemia. Cancer can be primary, recurrent, metastatic or multidrug resistant. The patient may have previously undergone surgery, chemotherapy, radiation therapy, hormonal therapy, or immunotherapy.

The method may further include receiving said patient a second cancer therapy, such as surgery, chemotherapy, radiation therapy, hormonal therapy, or immunotherapy. The patient may be a human or a non-human mammal. The method may further include administering said interferon prodrug more than once, for example daily, every other day, weekly, every other week, or monthly. The prodrug can be administered systemically or intratumorally, locally to the tumor, or regionally to the tumor. Treatment may include one or more of: slowing tumor growth, arresting tumor growth, reducing tumor size or volume, increasing survival compared to an untreated patient, inducing cancer remission, inducing tumor cell apoptosis, or inducing tumor necrosis.

It is assumed that any method or composition described in this application can be implemented in relation to any other method or composition described in this application.

The use of the singular in conjunction with the term "comprising" in the claims and/or description may mean "one", but also includes "one or more", "at least one", and "one or more than one". The word "about" means plus or minus 5% of the specified number.

Other objects of the invention, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while pointing to specific embodiments of the invention, are provided by way of illustration only, since various changes and modifications will be apparent from the detailed description to those skilled in the art and are within the spirit and scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are part of the present description and are included to further demonstrate certain aspects of the present disclosure. The present invention may be better understood with reference to one or more of these drawings in conjunction with a detailed description of the specific embodiments provided herein.

Fig. 1. Schematic structure of an IFN prodrug. IFNAR-ECD (blue), substrate linker (red), IFN (yellow), tumor specific enzyme (green), IgG Fc (grey). The left N-terminal arm is the intact form of the IFN prodrug fused to IFNAR-ECD and coupled to IFN, while the right N-terminal arm is the activated form of the IFN prodrug from which IFNAR-ECD has been cleaved.

Fig. 2A-C. The activated form of the IFN prodrug exhibits higher IFN activity than the reduced form. (FIG. 2A) A 5-fold serial dilution of 20 nM hIg, IFNa4-Fc, IFNAR1 based IFN prodrug and IFNAR2 based IFN prodrug was performed. (FIG. 2B-C) IFN prodrug was diluted to 1 μM in assay buffer (50 mM Tris, 10 mM CaCl ₂ , 150 mM NaCl, 0.05% Brij-35 (w/v), pH 7, 5 (TCNB)). Added rmMMP-9 to a final concentration of 1 ng/μl and incubated at 37°C for 6 hours. (FIG. 2B) A 5-fold serial dilution of 20 nM IFNa4-Fc, R1-NSUB with or without rmMMP-9 treatment and R1-SUB (MMP-2/9 substrate) with or without rmMMP-9 treatment was performed. (FIG. 2C) A 5-fold serial dilution of 20 nM IFNa4-Fc, R2-NSUB (MMP-2/9 substrate) was performed with or without rmMMP-9 treatment and R2-SUB with or without rmMMP-9 treatment. Diluted fusion protein solutions were added to RAW-Lucia-ISG reporter cells to stimulate luciferase secretion. Conditioned supernatants were collected 24 h after stimulation for analysis of luciferase activity. Error bars represent means ± standard error of the mean (SEM) for three repetitions.

Fig. 3. The IFNAR2-based IFN prodrug has a stronger antitumor effect compared to the IFNAR1-based IFN prodrug . C57BL/6 mice (n=5 per group) were injected subcutaneously with 5×10 ⁵ B16 cells and injected ip with 1 nmol hIg, IFNa4-Fc, R1-SUB (MMP2/9 substrate) or R2-SUB (MMP2/9 substrate) per days 11, 15 and 21. Tumor growth was monitored twice a week and reported as mean tumor size ± standard error of the mean versus time.

Fig. 4A-D. Expression of enzymes in mouse normal and tumor tissues. C57BL/6 mice (n=4) were injected subcutaneously with 1×10 ⁶ MC38 cells or 5×10 ⁵ B16 cells. These normal tissues and tumors were harvested on day 11. Intracellular RNA was extracted for analysis by RT-qPCR (RT-qPCR) to determine the number of mRNA copies (Fig. 4A) uPA, (Fig. 4B) MMP-2, (Fig. 4C) MMP-9 and (Fig. 4D) MMP-14. The results are presented as a percentage relative to 18sr. Error bars represent means ± standard error of the mean for triplicates.

Fig. 5A-E. The IFN prodrug improves safety without sacrificing efficacy. C57BL/6 mice (n=3 per group) were injected subcutaneously with 5×10 ⁵ B16 cells and injected intraperitoneally with 1 mM hIg, IFNa4-Fc or R2-SUB (MMP-14 substrate) on days 10, 13 and 16. Mice were taken blood and serum was collected on day 17 (FIG. 5A). Serum concentrations of the inflammatory cytokines IL-6, TNF, MCP-1, and IFN-g in inflammatory mice were measured using a bead cytometric array (CBA). (FIG. 5B) Serum ALT activity was measured using the Reflotron Plus® system. (FIG. 5C) Tumor growth and (FIG. 5D) body weight were recorded twice a week. (FIG. 5E) Built a survival curve due to toxicity. Mice with more than 30% body weight loss were considered dead. Error bars represent means ± standard error of the mean.

Fig. 6A-H. The level of expression of human proteases between the tumor and adjacent normal tissues. DiffExp TIMER module (Tumor IMmune Esribution Resource Resource) website for online analysis comparing gene expression levels for all samples from TCGA (Cancer Genome Atlas). (Fig. 6A) MMP-1, (Fig. 6B) MMP-3, (Fig. 6C) MMP-9, (Fig. 6D) MMP-10, (Fig. 6E) MMP-11, (Fig. 6F) MMP-12, (Fig. 6G) MMP-13 and (Fig. 6H) MMP-14.

Fig. 7A-C. Activation of human ProIFN in vitro . (Fig.7A) Performed a 10-fold serial dilution of 50 μm hIFNa2-Fc, ProIFN-based hIFNAR1 and ProIFN-based hIFNAR2. IFN activity was measured using reporter cells 293T-Dual ^TM hSTING-R232. (FIG. 7B) hIFNAR2-based human ProIFN with or without substrate (hProIFN-Sub) was diluted to 1 μM in assay buffer (50 mM Tris, 10 mM CaCl ₂ , 150 mM NaCl, 0.05 % Brij-35 (w/v), pH 7.5 (TCNB)). Added rmMMP-9 to a final concentration of 1 ng/μl and incubated at 37°C for 6 hours. IFN activity was measured using reporter cells 293T-Dual ^TM hSTING-R232. (FIG. 7C) Human hProIFN-Sub based on hIFNAR2 or hProIFN-Nsub was diluted to 1 μM in assay buffer (50 mM Tris, 10 mM CaCl ₂ , 150 mM NaCl, 0.05% Brij-35 (w/v .), pH 7.5 (TCNB)). Added rmMMP-9 to a final concentration of 1 ng/μl and incubated at 37°C for 0, 0.5, 2 or 6 hours. Cleaved samples were separated on a Stain ^FreeTM gel. The gel was visualized with a dye-free imaging device. Error bars represent means ± standard error of the mean for triplicates.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Prodrugs are pharmacologically inactive chemical derivatives of drugs that must undergo transformation in the body to become active. They are intended to overcome pharmaceutical and/or pharmacokinetic problems associated with the parent drug that would otherwise limit the clinical utility of that drug (Stella et al., 1985). A protease-activated antibody (pro-antibody) targeting the vascular endothelial adhesion molecule type 1 (VCAM-1), a marker of atherosclerotic plaques, was recently constructed by attaching a binding site masking peptide to the antibody via a matrix metalloprotease (MMP) receptive linker. . The activity of such disease-associated proteases can be used to site-specifically target antibody activity in vivo (Erster et al., 2012). In one example of cancer therapy, a screening-identified peptide that can block anti-EGFR binding to an EGFR target is coupled to an anti-EGFR antibody. The resulting pro-antibody directed to the epidermal growth factor receptor (EGFR) had a markedly improved safety and extended half-life in non-human primates, allowing safe administration of much higher doses of this substance compared to cetuximab (Desnoyers et al., 2013). However, such peptides often have low affinity, resulting in incomplete blockade of the drug protein, as well as high immunogenicity, which precludes longer-term treatment. Therefore, the inventors have developed a novel strategy based on a naturally occurring receptor with the correct affinity, without host immunogenicity, which allows suppression of drug binding to its receptor in non-tumor tissues. In particular, emphasis has been placed on type I interferons due to their important role in the antitumor immune response to many types of cancer ⁸ .

We have constructed an IFN prodrug using immunoglobulin constant regions (IgG) and IFNAR extracellular domains (IFNAR1 or IFNAR2) as an IFN activity blocker fused at the N-terminus to IFN. IFNAR is associated with a specific linker that is selectively cleaved by a protease overexpressed in the tumor microenvironment. This allows the toxic activity of the IFN domain to be masked until the prodrug reaches the tumor, while the Fc fragment fused to the C-terminal end of IFN improves its in vivo half-life. Such an IFN prodrug has a greatly reduced IFN activity until the linker is cleaved, with said activity being restored after enzymatic cleavage. The inventors have also demonstrated the efficacy and improved safety of the IFN prodrug in vivo in a mouse model of B16-OVA melanoma.

Thus, the advantage of using this IFN prodrug as an anti-cancer therapeutic agent is: 1) low toxicity; 2) unexpectedly higher activity of IFN in activated form compared to the original IFN-Fc; 3) easy to obtain and clean with high yield; 4) specific targeting of tumor tissues; 5) use of a non-immunogenic blocking reagent; and 6) personalized design of the IFN prodrug depending on the expression level of the tumor enzyme, which may vary. Thus, such an IFN prodrug may improve the safety profile of interferons without compromising their efficacy, and thus may enable wider use of modern interferon therapy formats such as bispecific antibody-cytokine fusion proteins. These and other aspects of the invention are described in more detail below.

I. Type 1 interferons

A. Types of interferon

Human type I interferons (IFNs) are a large subgroup of proteins, the interferons, that help regulate the activity of the immune system. Interferons bind to interferon receptors. All type I IFNs bind to a specific cell surface receptor complex known as the IFN-α receptor (IFNAR), which consists of the IFNAR1 and IFNAR2 chains. Type I IFNs are found in all mammals, and homologous (similar) molecules are found in bird, reptile, amphibian and fish species. Types in mammals are designated as: IFN-α (alpha), IFN-β (beta), IFN-κ (kappa), IFN-δ (delta), IFN-ε (epsilon), IFN-τ (tau), IFN- ω (omega) and IFN-ζ (zeta, also known as limitin).

IFN-α proteins are produced by leukocytes. They are mainly involved in the innate immune response against viral infection. The genes responsible for their synthesis are included in 13 subtypes, which are known as IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21. These genes are clustered together on chromosome 9.

IFN-α is also produced synthetically as a drug for the treatment of hairy cell leukemia. The international non-proprietary name (INN, INN) of the product is interferon alfa. Recombinant type - interferon alfacon-1. The pegylated types are pegylated interferon alfa-2a and pegylated interferon alfa-2b.

IFN-β proteins are produced in large quantities by fibroblasts. They have antiviral activity, which is mainly involved in the innate immune response. Two types of IFN-β have been described: IFN-β (IFNB1) and IFN-β3 (IFNB3) (the gene designated IFN-β2 is actually IL-6). IFN-β1 is used in the treatment of multiple sclerosis because it reduces the recurrence rate. IFN-β1 is not suitable for the treatment of patients with progressive non-relapsing forms of multiple sclerosis.

IFN-ε, -κ, -τ and -ζ in humans appear to be one isoform, IFNK. Only in ruminants, IFN-τ, a variant of IFN-ω, is encoded. To date, IFN-ζ has only been found in mice, while the structural homologue of IFN-δ has been found in a wide range of non-primate and non-rodent placental mammals. Most, but not all, placental mammals code for functional IFN-ε and IFN-κ genes.

IFN-ω, although it has only one functional form described to date (IFNW1), there are several pseudogenes in humans: IFNWP2, IFNWP4, IFNWP5, IFNWP9, IFNWP15, IFNWP18, and IFNWP19. Many non-primate placental mammals express multiple subtypes of IFN-ω.

IFN-ν has recently been described as a pseudogene in humans, but is potentially functional in the domestic cat genome. In all other non-feline placental mammal genomes, IFN-ν is a pseudogene; in some species this pseudogene is well preserved, while in others it has undergone strong transformation or is undetectable. Moreover, the IFN-ν promoter carries deleterious mutations in the cat genome. It is likely that the IFN-ν gene family became useless before the advent of diverse mammalian species. Its presence at the edge of the mammalian type I IFN locus may have protected it from destruction, leading to its discovery.

IFN-α and IFN-β are secreted by many cell types, including lymphocytes (NK cells, B cells, and T cells), macrophages, fibroblasts, endothelial cells, osteoblasts, and others. They provide stimulation of both macrophages and NK cells, which cause an antiviral response, and also exhibit antitumor activity. Plasmacytoid dendritic cells have been identified as the most potent type I IFN producers in response to antigen action and thus have been termed natural IFN producing cells. According to the results of current research, it is assumed that by increasing the expression of IFN-α in tumor-infiltrating macrophages, more efficient activation of dendritic cells and cytotoxicity of immune effector cells can be induced.

IFN-ω is released by leukocytes at the site of viral infection or tumors. IFN-α acts as a pyrogenic factor, altering the activity of thermosensitive neurons in the hypothalamus, thereby causing fever. This occurs through binding to opioid receptors, causing the release of prostaglandin-E2 (PGE2). A similar mechanism is used by IFN-α to reduce pain; IFN-α interacts with the μ-opioid receptor, acting as an analgesic.

In mice, IFN-β inhibits the production of growth factors in immune cells, thereby slowing down tumor growth, and prevents other cells from producing growth factors that activate vessel formation, thereby blocking tumor angiogenesis and preventing the connection of the tumor with the blood vessel system.

B. Interferon receptors

The interferon-α/β receptor (IFNAR) is a receptor that binds type I interferons, including interferon-α and -β. It is a heteromeric cell surface receptor consisting of a single chain with two subunits called IFNAR1 and IFNAR2. After IFN type I binding, IFNAR activates the JAK-STAT signaling pathway. Stimulation of interferons traditionally leads to stimulation of the antiviral immune response.

Its structure was obtained using NMR. Initially, 35 conformers were calculated, which were reduced to 22 according to the low energy criterion. This was the first helical structure of a cytokine receptor determined in solution. The molecule has one polymer. The structure reveals the nature of the binding. The IFNAR2 model revealed a predominantly hydrophobic site on the receptor that interacts with the corresponding hydrophobic surface on IFN-alpha. An adjacent motif of charged side chains directs proteins to form a strong complex. The binding surface helps explain the cross-reactivity and specificity of the receptor ligand. The source for the experiments was human, but the expression was carried out in Escherichia coli .

IFNAR is classified as a beta protein based on its secondary structure. The folds of the IFNAR chain show a clear evolutionary relationship to the immunoglobulin beta sandwich. Given the structural and functional similarities that suggest a likely common evolution, it is included in the superfamily and the type III fibronectin family. The domain, or evolutionary unit, seen in nature is the beta chain of the interferon-alpha/beta receptor. The species was a person.

II. Interferon prodrugs

A. General information

Prodrugs are generally defined as molecules that are capable of being therapeutic agents but are in a form that requires some modification to be truly therapeutically useful. Such reagents are particularly useful when the delivery of the active dosage form has some of its inherent limitations, such as toxicity or lack of stability, by designing a prodrug form that avoids these disadvantages and provides effective in vivo activation at the appropriate time and/or site.

The prodrugs of the present invention have five different components. The first component is interferon. The second component is the masking domain, which blocks the ability of interferon to exercise its normal activity while in the bound state. The third component is a stabilizing agent, such as the constant domain of an antibody. These three components are connected by linkers, and the linker located between the interferon and the masking domain is selectively cleaved, for example, by a protease that is expressed by the cancer cell or in the tumor environment.

As discussed above, type 1 interferons are IFN-α (alpha), IFN-β (beta), IFN-κ (kappa), IFN-δ (delta), IFN-ε (epsilon), IFN-τ (tau) , IFN-ω (omega) and IFN-ζ (zeta). Any of these molecules can be included in the designs described in this application. It is also possible to obtain heterodimeric constructs with two or even four different type 1 interferons. The interferons used in this application to obtain the data disclosed in the present description are full-length proteins, but do not contain signal peptides.

An important part of the design, of course, is the masking area. For this purpose, the inventors used natural receptors for type 1 interferons, and not non-natural sequences. The advantage of using a masking domain based on the structure of natural receptors includes both (a) a high affinity for type 1 interferon and (b) a lower likelihood of an immune response against the sequences. Sequences:

Mouse IFNAR1-ECD:

1 mlavvgaaal vlvagapwvl psaaggenlk ppenidvyii ddnytlkwss hgesmgsvtf

61 saeyrtkdea kwlkvpecqh ttttkcefsl ldtnvyiktq frvraeegns tsswnevdpf

121 ipfytahmsp pevrleaedk ailvhisppg qdgnmwalek psfsytiriw qksssdkkti

181 nstyyvekip ellpettycl evkaihpslk khsnystvqc isttvankmp vpgnlqvdaq

241 gksyvlkwdy iasadvlfra qwlpgyskss sgsrsdkwkp iptcanvqtt hcvfsqdtvy

301 tgtfflhvqa segnhtsfws eekfidsqkh ilppppvitv tamsdtllvy vncqdstcdg

361 lnyeiifwen tsntkismek dgpeftlknl qpltvycvqa rvlfrallnk tsnfseklce

421 ktrpgsfst (SEQ ID NO: 1)

Murine IFNAR2-ECD :

1 mrsrctvsav gllslclvvs asletitpsa fdgypdepct initirnsrl ilswelenks

61 gppanytlwy tvmskdenlt kvkncsdttk sscdvtdkwl egmesyvvai vivhrgdltv

121 crcsdyivpa napleppefe ivgftdhinv tmefppvtsk iiqekmkttp fvikeqigds

181 vrkkhepkvn nvtgnftfvl rdllpktnyc vslyfdddpa iksplkcivl qpgqesglse

241sa (SEQ ID NO: 2)

Human IFNAR1-ECD :

1 mmvvllgatt lvlvapwv lsaaaggknl kspqkvevdi iddnfilrwn rsdesvgnvt

61 fsfdyqktgm dnwiklsgcq nitstkcnfs slklnvyeei klriraeken tsswyevdsf

121 tpfrkaqigp pevhleaedk aivihispgt kdsvmwaldg lsftyslviw knssgveeri

181 eniysrhkiy klspettycl kvkaalltsw kigvyspvhc ikttvenelp ppenievsvq

241 nqnyvlkwdy tyanmtfqvq wlhaflkrnp gnhlykwkqi pdcenvkttq cvfpqnvfqk

301 giyllrvqas dgnntsfwse eikfdteiqa fllppvfnir slsdsfhiyi gapkqsgntp

361 viqdypliye iifwentsna erkiiekktd vtvpnlkplt vycvkaraht mdeklnkssv

421 fsdavcektk pgntsk (SEQ ID NO: 3)

Human IFNAR2-ECD :

1 mllsqnafif rslnlvlmvy islvfgisyd spdytdesct fkislrnfrs ilswelknhs

61 ivpthytlly timskpedlk vvkncanttr sfcdltdewr stheayvtvl egfsgnttlf

121 scshnfwlai dmsfeppefe ivgftnhinv mvkfpsivee elqfdlslvi eeqsegivkk

181 hkpeikgnms gnftyiidkl ipntnycvsv ylehsdeqav iksplkctll ppgqesesae

241 sak (SEQ ID NO: 4)

Further, the constructs include stabilization domains which, for example, will increase the in vivo half-life. The inventors decided to use the Ig constant domain. Although the Fc domain of IgG1 has been used, other Fc domains can also be used. A particular advantage of using IgA Fc domains is their ability to dimerize, which means that up to four different interferons can be included in such a construct. One example of multi-interferon constructs is the use of an FcA-FcB IgG1 heterodimer with one blocking receptor and one interferon fused to an FcA or FcB using separate cleavable substrates specific for different enzymes embedded in FcA and FcB linkers, respectively. Other stabilizing proteins may also be used, including human serum albumin and transferrin.

Finally, the above domains are connected to each other by short peptide regions or "linkers". One of these linkers, located between the interferon molecule and the masking domain, undergoes cleavage when the prodrug reaches or approaches its target, the tumor or cancer cell. In a particular embodiment, the linker contains a protease target site in which, when the prodrug is exposed to the environment containing the protease, the linker is cleaved, releasing the masking agent from the prodrug, thereby activating the interferon molecule. Ideally, the linker can be selected for proteases overexpressed in cancer cells or at the site of tumor formation, and can even be adapted to the profile of the protease expressed in cancer cells/tumor in a particular individual through preliminary testing. The following are a few examples of enzymes that are selectively expressed or overexpressed in cancer or tumor environments.

TABLE 1 - PROTEASE SUBSTRATES

Enzyme Substrate amino acid sequence MMP2, MMP9 PVGLIG (SEQ ID NO: 5) MMP11 GGYAELRMGG (SEQ ID NO: 6) MMP13 GPRPFNYL (SEQ ID NO: 7) MMP13 GGALGLSL (SEQ ID NO: 8) MMP13 GPMSYNAL (SEQ ID NO: 9) MMP14 SGRSENIRTA (SEQ ID NO: 10) FAPa TSGPNQEQK (SEQ ID NO: 11) Cathepsin B GFLG (SEQ ID NO: 12)

An example of a linker form is GGGGS-substrate-GGGGS (GGGGS=SEQ ID NO: 13). Other two examples are (GGGGS) _n -substrate-(GGGGS) _n and G _n -substrate-G _n (n can be any number) (GGGGS=SEQ ID NO: 13).

B. Development and expression of nucleic acid constructs.

Various genetic constructs are available that contain the interferon prodrug components described above and that can be introduced into expression vectors. Nucleic acids of the present invention that encode prodrug molecules may optionally be linked to other protein sequences. In the context of the present application, the term "expression construct" is intended to refer to any type of genetic construct containing a nucleic acid encoding a gene product in which part or all of the sequence encoding the nucleic acid is capable of being transcribed. The transcript may be translated into a protein, but this is not required. In some embodiments, expression includes both transcription of the gene and translation of the mRNA into the gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding the gene of interest.

The term "vector" is used to refer to a carrier nucleic acid molecule intended to be introduced into a cell, where it can be replicated, into which the nucleic acid sequence can be inserted. The nucleic acid sequence may be "exogenous", which means that the sequence is foreign to the cell into which the vector is introduced, or that the sequence is homologous to the sequence found in the cell, but is located within the nucleic acid of the host cell at a position in which usually this sequence is missing. Vectors include plasmids, cosmids, viruses (bacteriophage, animal and plant viruses) and artificial chromosomes (eg YAC). A person skilled in the art has the necessary skills to construct a vector using standard recombinant methods, which are described in the works of Sambrook et al. (1989) and Ausubel et al. (1994), both of which are incorporated herein by reference.

The term "expression vector" refers to a vector containing a nucleic acid sequence encoding at least a portion of a gene product capable of being transcribed. In some cases, the RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, such as in the production of antisense molecules or ribozymes. Expression vectors may contain a plurality of "control sequences", which refer to nucleic acid sequences necessary for transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that direct transcription and translation, vectors and expression vectors may contain nucleic acid sequences that also perform other functions and are described below.

1. Regulatory elements

A "promoter" is a control sequence, which is a region of a nucleic acid sequence that controls the initiation and rate of transcription. It may contain genetic elements to which regulatory proteins and molecules such as RNA polymerase and other transcription factors can bind. The phrases "operably located", "operably linked", "under control" and "under transcriptional control" mean that the promoter is in the correct functional position and/or orientation relative to the nucleic acid sequence to control the initiation of transcription and/or expression of this sequence. A promoter may or may not be used in conjunction with an "enhancer", which refers to a cis-acting regulatory sequence involved in activating the transcription of a nucleic acid sequence.

The promoter may be a promoter that is naturally associated with a gene or sequence that can be obtained by isolating 5' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter may be referred to as "endogenous". Similarly, an enhancer can be an enhancer that is naturally associated with a nucleic acid sequence located upstream or downstream of that sequence. Alternatively, certain advantages may be obtained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter related to a promoter that is not naturally associated with the nucleic acid sequence in the natural environment.

Recombinant or heterologous enhancer also refers to an enhancer that is not naturally associated with a nucleic acid sequence in the natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other prokaryotic, viral or eukaryotic cell, and promoters or enhancers that are not "naturally occurring", i. containing other elements of other transcriptional regulatory regions and/or mutations that alter expression. In addition to synthetically derived promoter and enhancer nucleic acid sequences, sequences can be obtained by recombinant nucleic acid cloning and/or amplification techniques, including ^TMα PCR, in relation to the compositions disclosed herein (see US Patent 4,683,202, US Patent 5,928,906, each of which is incorporated herein by reference). Furthermore, it is contemplated that control sequences can also be used for direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like.

Naturally, it is important to note the use of a promoter and/or enhancer that effectively directs the expression of a DNA segment in the particular cell type, organelle, and organism that is chosen for expression. Those skilled in the art of molecular biology are generally aware of the use of combinations of promoters, enhancers, and cell types for protein expression, for example, see Sambrook et al. (1989), included in the present description by reference. The promoters used can be constitutive, tissue-specific, inducible and/or useful under appropriate conditions to obtain a high level of expression of the introduced DNA segment, which is an advantage in large scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.

The identity of tissue-specific promoters or elements, as well as assays to characterize their activity, are well known to those skilled in the art. Examples of such regions include the human LIMK2 gene (Nomoto et al. 1999), the somatostatin 2 receptor gene (Kraus et al., 1998), the mouse epididymal retinoic acid-binding gene (Lareyre et al., 1999), human CD4 (Zhao-Emonet et al. al., 1998), mouse alpha2 (XI) collagen (Tsumaki, et al., 1998), dopamine D1A receptor gene (Lee, et al., 1997), insulin-like growth factor II (Wu et al., 1997), molecule human platelet-1 endothelial cell adhesion (Almendro et al., 1996). Tumor-specific promoters can also be used in the present invention.

Efficient translation of coding sequences may also require a specific initiation signal. These signals include the initiation codon ATG or adjacent sequences. It may be necessary to provide exogenous signals for the regulation of translation, including the initiation codon ATG. A person skilled in the art will easily be able to determine this and provide the necessary signals. It is well known that in order to ensure translation of the entire insert, the initiation codon must be "in-frame" with the reading frame of the desired coding sequence. Exogenous translational control signals and initiation codons may be natural or synthetic. Expression efficiency can be increased by including appropriate transcriptional enhancer elements.

2. IRES

In some embodiments of the present invention, elements such as internal ribosome entry sites (IRES) are used to create multigene or polycistronic messages. IRES elements are able to bypass the 5'-methylated Cap ribosome scanning pattern and initiate translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements derived from two members of the picornavirus family (poliomyelitis and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well as IRES from mammalian mRNA (Macejak and Sarnow, 1991). IRES elements may be associated with heterologous open reading frames. Several open reading frames can be co-transcribed, with each reading frame separated by an IRES, thus generating polycistronic mRNAs. Thanks to the IRES element, each open reading frame is accessible to ribosomes, which ensures efficient translation. With a single promoter/enhancer, multiple genes can be efficiently expressed to ensure transcription of a single mRNA (see US Pat. Nos. 5,925,565 and 5,935,819, incorporated herein by reference).

3. Multipurpose cloning sites

Vectors may include a multiple cloning site (MCS), which is a region of nucleic acid containing multiple restriction enzyme sites, any of which can be used in conjunction with a standard recombinant method to cleave the vector. See Carbonelli et al., 1999, Levenson et al., 1998 and Cocea, 1997, incorporated herein by reference. "Restriction enzyme cleavage" refers to the catalytic cleavage of a nucleic acid molecule by an enzyme that functions only at specific locations in the nucleic acid molecule. Many of these restriction enzymes are commercially available. The use of such enzymes is well known to those skilled in the art. Often, the vector is linearized or fragmented using a restriction enzyme that cuts the MCS internally, allowing exogenous sequences to be ligated to the vector. "Ligation" refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be adjacent to each other. Techniques involving restriction enzymes and ligation reactions are well known to those skilled in the art of recombinant technology.

4. Site splicing

Most transcribed eukaryotic RNA molecules undergo RNA splicing to remove introns from primary transcripts. Vectors containing eukaryotic genomic sequences may require donor and/or acceptor splicing sites to ensure proper transcript processing for protein expression (see Chandler et al., 1997, incorporated herein by reference).

5. Termination signals

The vectors or constructs of the present invention typically contain at least one termination signal. A "termination signal" or "terminator" consists of DNA sequences involved in the specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments, a termination signal is considered that terminates the production of an RNA transcript. An in vivo terminator may be needed to obtain the desired level of mRNA.

In eukaryotic systems, the terminator region may also contain specific DNA sequences that provide site-specific cleavage of the new transcript, exposing the polyadenylation site. This is the signal for a specialized endogenous polymerase to add a region of approximately 200 A residues (polyA) to the 3' end of the transcript. RNA molecules modified with such a polyA tail are more stable and more efficiently translated. Thus, in other embodiments that use eukaryotes, it is preferred that the terminator contains a signal for RNA cleavage, and more preferably, the terminator signal stimulates mRNA polyadenylation. The terminator and/or polyadenylation site elements may serve to increase mRNA levels and/or to minimize reading of the cassette into other sequences.

Terminators contemplated for use in the present invention include any known transcription terminator described in this application or known to a person skilled in the art, including, without limitation, for example, gene termination sequences such as, for example, the bovine growth hormone terminator, or viral sequences terminations, such as, for example, the SV40 terminator. In some embodiments, the termination signal may be the absence of a transcribed or translated sequence, for example due to sequence truncation.

6. Polyadenylation signals

When expressed, especially when expressed in a eukaryotic cell, a polyadenylation signal is usually included to ensure correct polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be critical to the success of the invention, and/or any such sequence may be used. Preferred embodiments include an SV40 polyadenylation signal and/or a bovine growth hormone polyadenylation signal convenient and/or known to function well in various target cells. Polyadenylation may increase transcript stability or may promote cytoplasmic transport.

7. Start point of replication

To propagate a vector in a host cell, it may contain one or more origins of replication (often referred to as "ori"), which are the specific nucleic acid sequence at which replication is initiated. Alternatively, an autonomously replicable sequence (ARS) can be used if the host cell is yeast.

8. Selectable and screenable markers

In some embodiments, cells containing the nucleic acid construct of the present invention can be identified in vitro or in vivo by incorporating a marker into an expression vector. Such markers provide an identifiable change in the cell, allowing the cells containing the expression vector to be easily identified. Typically, a selectable marker is a marker that imparts a property that allows selection to be made. In the case of a positive selectable marker, its presence is selected, while in the case of a negative selectable marker, its presence excludes the selection. An example of a positive selectable marker is a drug resistance marker.

Typically, the inclusion of a drug susceptibility selection marker aids in the cloning and identification of transformants; for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selectable markers. In addition to markers conferring a phenotype that distinguishes transformants based on the realization of conditions, other types of markers are also contemplated, including screenable markers such as GFP, which are based on colorimetric analysis. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) can be used. One of skill in the art is also aware of how to use immunological markers, possibly in combination with FACS analysis. The marker used is considered to be of no importance, provided that it is capable of being expressed simultaneously with the nucleic acid encoding the gene product. Additional examples of selectable and screenable markers are well known to those skilled in the art.

9. Viral vectors

The ability of some viral vectors to efficiently infect or enter cells, integrate into the host cell genome, and stably express viral genes has led to the development and use of a number of different viral vector systems (Robbins et al., 1998). Virus systems are currently being developed for use as ex vivo and in vivo gene transfer vectors. For example, the use of vectors based on adenovirus, herpes simplex virus, retrovirus and adeno-associated viruses for the treatment of diseases such as cancer, cystic fibrosis, Gaucher disease, renal failure and arthritis is currently being evaluated (Robbins and Ghivizzani, 1998; Imai et al., 1998 ; US Pat. No. 5,670,488). The various viral vectors described below have certain advantages and disadvantages depending on the particular application of gene therapy.

10. Non-viral transformation

It is believed that suitable nucleic acid delivery methods for transforming an organelle, cell, tissue, or organism suitable for use in the present invention include virtually any method by which a nucleic acid (e.g., DNA) can be introduced into an organelle, cell, tissue, or organism described in this application or known to a person skilled in the art. Such methods include, without limitation, direct DNA delivery such as injection (US Pat. Nos. 5,994,624; 5,981,274; 5,945,100; 5,780,448; Harland and Weintraub, 1985; US Pat. No. 5,789,215 incorporated herein by reference); by electroporation (US patent 5384253, included in the present description by reference); calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); with DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonication (Fechheimer et al., 1987); liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); microparticle bombardment (PCT Application Nos. WO 94/09699 and 95/06128; US Pat. Nos. 5,610,042; 5,322,783; 5,563,055; 5,550,318; by mixing with silicon carbide fibers (Kaeppler et al., 1990; US Pat. Nos. 5,302,523 and 5,464,765, each of which is incorporated herein by reference); or by PEG-mediated protoplast transformation (Omirulleh et al., 1993; US Pat. Nos. 4,684,611 and 4,952,500, each of which is incorporated herein by reference); by desiccation/inhibition mediated DNA uptake (Potrykus et al., 1985). Through the use of such technologies, a stable or temporary transformation of the organelle(s), cell(s), tissue(s) or organism(s) is provided.

Injection . In some embodiments, the nucleic acid can be delivered to an organelle, cell, tissue, or organism via one or more injections (i.e., needle injections), such as, for example, either subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal injections. Methods for administering vaccines are well known to those skilled in the art (eg, injection of a saline composition). Additional embodiments of the present invention include the introduction of a nucleic acid by direct microinjection. Direct microinjection has been used to introduce nucleic acid constructs into Xenopus oocytes (Harland and Weintraub, 1985).

Electroporation . In some embodiments, implementation of the present invention, the nucleic acid is introduced into the organelle, cell, tissue or organism by electroporation. Electroporation involves exposing cell suspension and DNA to a high-voltage electrical discharge. Some embodiments of this method use certain cell wall degrading enzymes, such as pectin degrading enzymes, making recipient target cells more susceptible to transformation by electroporation compared to untreated cells (U.S. Patent 5,384,253, incorporated herein by reference) . Alternatively, recipient cells can be made more susceptible to transformation by mechanical injury.

Transfection of eukaryotic cells by electroporation is quite successful. Mouse pre-B lymphocytes have been transfected with human immunoglobulin genes (Potter et al., 1984) and rat hepatocytes have been transfected in this manner with the chloramphenicol acetyltransferase gene (Tur Kaspa et al., 1986).

To effect transformation in cells, such as, for example, plant cells, either loose tissues such as suspension cell culture or embryogenic callus can be used by electroporation, or alternatively immature embryos or other organized tissues can be directly transformed. In this method, it is possible to partially destroy the cell walls of selected cells by exposing them to pectin-degrading enzymes (pectoliases) or to inflict controlled mechanical damage. Examples of some species that have been transformed by electroporation of intact cells include corn (U.S. Patent 5,384,253; Rhodes et al., 1995; D'Halluin et al., 1992), wheat (Zhou et al., 1993), tomato (Hou and Lin, 1996), soy (Christou et al., 1987) and tobacco (Lee et al., 1989).

Protoplasts can also be used to transform plant cells by electroporation (Bates, 1994; Lazzeri, 1995). For example, the production of transgenic soybean plants by electroporation of protoplasts derived from cotyledons is described by Dhir and Widholm in International Patent Application No. WO 92/17598, incorporated herein by reference. Other examples of species for which protoplast transformation has been described include barley (Lazerri, 1995), sorghum (Battraw et al., 1991), maize (Bhattacharjee et al., 1997), wheat (He et al., 1994), and tomato ( Tsukada, 1989).

calcium phosphate . In other embodiments of the present invention, the nucleic acid is introduced into cells using calcium phosphate precipitation. Human KB cells were transfected with adenovirus 5 DNA (Graham and Van Der Eb, 1973) using this method. Mouse L (A9) cells, mouse C127 cells, CHO, CV 1, BHK, NIH3T3, and HeLa cells were transfected in the same manner with the neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes were transfected with various marker genes ( Rippe et al., 1990).

DEAE dextran . In another embodiment, the nucleic acid is delivered to the cell using DEAE dextran followed by polyethylene glycol. Thus, reporter plasmids were introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985).

Ultrasonication . Additional embodiments of the present invention include the introduction of a nucleic acid by direct sonication. LTK fibroblasts were transfected with the thymidine kinase gene using sonication (Fechheimer et al., 1987).

Liposomal mediated transfection . In another embodiment of the invention, the nucleic acid can be captured by a lipid complex, such as, for example, a liposome. Liposomes are vesicular structures characterized by a bilayer phospholipid membrane and an internal aqueous environment. Multilamellar liposomes have several lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in excess aqueous solution. Before the formation of closed structures, self-organization of lipid components and the capture of water and solute components between lipid bilayers occur (Ghosh and Bachhawat, 1991). Also considered is a nucleic acid that forms a complex with lipofectamine (Gibco BRL) or superfect (Qiagen).

Liposome-mediated delivery of nucleic acids and expression of foreign DNA in vitro has been very successful (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987). The possibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryos, HeLa cells, and hepatoma cells has also been demonstrated (Wong et al., 1980).

In some embodiments, implementation of the present invention, the liposome can form a complex with hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and facilitate the entry of liposome-encapsulated DNA into the cell (Kaneda et al., 1989). In other embodiments, the implementation of the liposome can be complexed or used together with nuclear non-histone chromosomal proteins (HMG 1) (Kato et al., 1991). In other embodiments, the liposome may be complexed with or used in conjunction with HVJ and HMG 1. In other embodiments, the delivery vehicle may comprise a ligand and a liposome.

Receptor-mediated transfection . The nucleic acid can also be delivered to the target cell using carriers for receptor-mediated delivery. Their advantage lies in the selective uptake of macromolecules through receptor-mediated endocytosis that occurs in the target cell. In view of the specific distribution of different receptors depending on the cell type, this delivery method adds another degree of specificity to the present invention.

Some receptor-mediated gene-targeting carriers contain a cell receptor-specific ligand and a nucleic acid binding agent. Others contain a cell receptor-specific ligand to which the nucleic acid to be delivered is operatively attached. Several ligands have been used for receptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al., 1990; Perales et al., 1994; Myers, EPO 0273085), demonstrating the operational suitability of this method. Specific delivery in the context of another mammalian cell type has been described (Wu and Wu, 1993; incorporated herein by reference). In some aspects of the present invention, a ligand is selected that corresponds to a receptor specifically expressed in a population of target cells.

In other embodiments, a carrier component for delivering a nucleic acid, a carrier targeting a specific cellular nucleic acid of a cell, may contain a specific binding ligand in combination with a liposome. The nucleic acid(s) to be delivered is contained in a liposome and the specific binding ligand is operably incorporated into the liposomal membrane. Thus, the liposome will specifically bind to the receptor(s) of the target cell and deliver the contents to the cell. Such systems have been shown to function by systems in which, for example, in the case of receptor-mediated nucleic acid delivery to cells that exhibit EGF receptor activation, epidermal growth factor (EGF) is used.

In other embodiments, the carrier component for delivering the nucleic acid of the targeted delivery carrier may be the liposome itself, which preferably will contain one or more lipids or glycoproteins that direct binding to specific cells. For example, lactosylceramide, a galactose-terminal asialganglioside, was introduced into liposomes and an increase in uptake of the insulin gene by hepatocytes was observed (Nicolau et al., 1987). It is contemplated that the tissue-specific transform constructs of the present invention can be specifically delivered to a target cell in a similar manner.

11. Expression systems

Numerous expression systems exist that include at least some or all of the compositions disclosed above. In the present invention, prokaryotic and/or eukaryotic systems can be used to obtain nucleic acid sequences or their related polypeptides, proteins and peptides. Many such systems are widely used and commercially available.

An insect cell/baculovirus system can provide a high level of protein expression in a heterologous ^nucleic acid segment as described in US Pat. Invitrogen® and ^BacPackTM baculovirus expression system from Clontech®.

Other exemplary expression systems include the Stratagene® Complete ControlTM ^mammalian inducible expression system, which includes a synthetic ecdysone-inducible receptor or pET expression system expressed in E. coli . Another example of an inducible expression system is available from Invitrogen®, which carries the T- ^RexTM (Tetracycline Regulated Expression) System, which is a mammalian inducible expression system that uses the full length CMV promoter. Invitrogen® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for highly efficient production of recombinant proteins in the methylotrophic yeast Pichia methanolica . The pGEM-T Easy vectors, pCon Vectors ^TM , Lonza pConIgG1 or pConK2 plasmid vectors, and 293 Freestyle cells or Lonza CHO cells can also be used to express the disclosed prodrug constructs.

Primary cultures of mammalian cells can be obtained in various ways. In order for the cells to be viable in vitro and in contact with the expression construct, it is necessary to ensure that the cells are maintained under conditions of the correct ratio of oxygen and carbon dioxide and nutrients, and at the same time protect them from microbial contamination. Cell culture methods are well documented.

One embodiment of the foregoing involves using gene transfer to immortalize cells to produce proteins. The gene of interest for the protein can be transferred by the method described above into appropriate host cells, followed by culturing the cells under appropriate conditions. Thus, the gene of virtually any polypeptide can be used. The creation of recombinant expression vectors and the elements included therein has been discussed above. Alternatively, the protein produced may be an endogenous protein normally synthesized by the cell in question.

Examples of useful mammalian host cell lines are Vero and HeLa cells and Chinese Hamster Ovary, W138, BHK, COS-7, 293, HepG2, NIH3T3, RIN and MDCK cell lines. In addition, a host cell strain can be selected that modulates the expression of the introduced sequences or modifies and processes the gene product in the desired manner. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important for protein function. Various host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins. Appropriate cell lines or host systems can be selected to ensure correct modification and processing of the expressed foreign protein.

A number of selection systems can be used, including, without limitation, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase genes in tk , hgprt , or aprt cells, respectively. In addition, metabolite resistance can be used as a basis for selecting a dhfr that provides resistance to; gpt providing resistance to mycophenolic acid; neo providing resistance to the aminoglycoside G418; and hygro providing resistance to hygromycin.

E. Cleaning

In some embodiments, the implementation of the interferon prodrugs of the present invention can be purified. In the context of the present invention, the term "purified" means a composition, isolated from other components, in which the protein is purified to any extent compared to its state when obtained naturally. Therefore, a purified protein also refers to a protein that is free from the environment in which it may occur naturally. When the term "substantially purified" is used, this designation refers to a composition in which a protein or peptide forms the main component of the composition, for example, proteins in the composition are about 50%, about 60%, about 70%, about 80%, about 90% , about 95% or more.

Protein purification methods are well known to those skilled in the art. These methods include, at one level, coarse fractionation of the cell medium to obtain polypeptide and non-polypeptide fractions. After separating the polypeptide from other proteins, the polypeptide of interest can be further purified by chromatography and electrophoresis to achieve partial or complete purification (or purification to a homogeneous state). Analytical methods particularly suitable for obtaining a pure peptide include ion exchange chromatography, size exclusion chromatography; electrophoresis in polyacrylamide gel; isoelectric focusing. Other protein purification methods include precipitation with ammonium sulfate, PEG, antibodies, and the like. or thermal denaturation followed by centrifugation; gel filtration, reverse phase, hydroxyapatite and affinity chromatography; and a combination of these and other methods.

Purification of an interferon prodrug of the present invention may require expression of the polypeptide in a prokaryotic or eukaryotic expression system and extraction of the protein under denaturing conditions. The polypeptide can be purified from other cellular components using an affinity column that binds the labeled portion of the polypeptide. It is well known in the art that reversing the order of the various purification steps, or skipping some steps, will also provide a suitable method for obtaining a substantially purified protein or peptide. Typically, antibodies are fractionated with agents (ie, protein A) that bind to the Fc portion of the antibody. In cases where the interferon prodrug contains such a domain, this approach can be used.

Those skilled in the art are aware of various methods for quantifying the degree of purification of a protein or peptide used in the context of the present invention. These include, for example, determining the specific activity of the active fraction, or estimating the amount of polypeptides in the fraction using an analysis based on SDS/PAGE methods. Another method for assessing the purity of a fraction is to calculate the specific activity of the fraction, compare it with the specific activity of the original extract, and thus calculate the degree of purity. The actual units used to represent the amount of activity will, of course, depend on the particular assay method chosen for subsequent purification and whether the expressed protein or peptide exhibits detectable activity.

It is known that polypeptide migration can vary, sometimes significantly, under different SDS/PAGE conditions (Capaldi et al., 1977). Therefore, it should be understood that under different electrophoresis conditions, the apparent molecular weight of the purified or partially purified expression products may vary.

III. Pharmaceutical formulations and cancer treatment

A. Cancers

Cancer results from the growth of a clonal population of cells from a tissue. The development of cancer, called carcinogenesis, can be modeled and characterized in several ways. The link between the development of cancer and inflammation has long been established. The inflammatory response is involved in host defense against microbial infection and also promotes tissue repair and regeneration. There is ample evidence that points to a link between inflammation and cancer risk, ie. chronic inflammation can lead to dysplasia.

Cancer cells to which the methods of the present invention may be applied generally include any cell that selectively expresses proteases, and in particular that overexpresses such proteases over normal cells. A suitable cancer cell may be breast cancer, lung cancer, colon cancer, pancreatic cancer, kidney cancer, stomach cancer, liver cancer, bone cancer, hematological cancer (eg leukemia or lymphoma), nervous tissue cancer, melanoma, ovarian cancer. , testicular cancer, prostate cancer, cervical cancer, vaginal cancer, or bladder cancer. In addition, the methods of the invention can be applied to a wide range of species, such as humans, non-human primates (e.g. monkeys, baboons or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats and mice. Cancer may also be recurrent, metastatic and/or multidrug resistant, and the methods of the present invention may be particularly applicable to such cancers to enable resection, prolong or reinduce remission, suppress angiogenesis, prevent or limit metastasis, and/ or for the treatment of multi-drug resistant cancer. At the cellular level, this can lead to the destruction of cancer cells, inhibition of cancer cell growth, or reversal or reduction of the malignant phenotype of tumor cells.

B. Composition and introduction

The present invention relates to pharmaceutical compositions containing interferon prodrugs. In a particular embodiment, the term "pharmaceutically acceptable" means approved by a federal or state government regulatory agency or listed in the USP or other generally accepted pharmacopeia for use in animals, and more specifically, in humans. The term "carrier" refers to a diluent, excipient, or carrier vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers may be sterile liquids such as water and oils, including petroleum, animal, vegetable or synthetic oils such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, saline, dextrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerin, propylene glycol, water , ethanol, etc.

The compositions may be prepared in neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions, such as those derived from hydrochloric, phosphoric, acetic, oxalic and tartaric acids, etc., as well as salts formed with cations, such as those derived from sodium, potassium hydroxides. , ammonium, calcium, ferric iron, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The antibodies of the present invention may include classical pharmaceutical preparations. The introduction of these compositions of the present invention can be carried out by any conventional route, provided that this route provides access to the target tissue. Such a route includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous. Such compositions are typically administered as the pharmaceutically acceptable compositions described above. Of particular interest is administration directly into the tumor, perfusion of the tumor, or administration locally or regionally relative to the tumor, for example, into the local or regional vasculature or lymphatic system, or into the bed of the resected tumor.

The active compounds can also be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

C. Combination therapy

In the context of the present invention, it is also contemplated that the interferon prodrugs described herein can be used similarly in combination with immuno-, chemo- or radiotherapy intervention or other treatments. It may also be effective, in particular, to combine an interferon prodrug with other therapies that target different aspects of cancer cell function.

In order to kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis, or alternatively achieve reversal or reduction of the malignant phenotype of tumor cells using the methods and compositions of the present invention, it is generally necessary to contact the target cell with the interferon prodrugs of the present invention and with at least one other agent. These compositions may be provided in a combined amount effective to kill or inhibit cell proliferation. This process may include bringing the cells into contact with the interferon prodrugs of the present invention and other agent(s) or factor(s) at the same time. This can be achieved by contacting the cell with one composition or formulation that includes both agents, or by contacting the cell simultaneously with two different compositions or formulations, one composition comprising the interferon prodrugs of the present invention and the other comprising the other agent. .

Alternatively, therapy with an interferon prodrug may precede or follow treatment with another agent at intervals ranging from minutes to weeks. In embodiments in which the other agent and the interferon prodrugs are administered separately to the cell, it is generally provided that the deliveries are not separated by a significant amount of time, allowing the combined effect of both agents on the cell. In such instances, contacting the cell with both modalities is expected to occur with an interval between administrations of between about 12-24 and more preferably within about 6-12 hours, most preferably with a delay time of only about 12 hours. In some situations, a significant extension of the treatment period may be required, however, in this case, the interval between the respective injections ranges from several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6 , 7 or 8).

More than one administration of interferon prodrugs or other agent may also be required. Various combinations may be used where the interferon prodrug of the present invention is "A" and the other therapy is "B", as shown below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B

A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A

A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B

Other combinations are also provided. Again, it should be noted that in order to kill cells, both agents are delivered to the cell in a combined amount effective to kill the cell.

Agents or factors suitable for the treatment of cancer include any chemical compound or treatment that causes DNA damage when introduced into a cell. Such agents and factors include DNA damaging radiation and waves such as irradiation, microwaves, electron radiation, and the like. You can use a variety of chemical compounds, also called "chemotherapeutic" or "genotoxic agents". This can be achieved by irradiating the localized area with the tumor; alternatively, the tumor cells can be brought into contact with the agent by administering to the patient a therapeutically effective amount of the pharmaceutical composition.

Various classes of chemotherapeutic agents are provided for use in the present invention. For example, selective estrogen receptor antagonists ("SERMs") such as tamoxifen, 4-hydroxy tamoxifen (afimoxfen), falsodex, raloxifene, bazedoxifene, clomiphene, femarel, lasofoxifene, ormeloxifene, and toremifene.

Chemotherapeutic agents contemplated for use include, for example, camptothecin, actinomycin D, mitomycin C. The invention also includes the use of a combination of one or more DNA damaging agents, either radioactive or actual compounds, such as the use of x-rays with cisplatin or the use of cisplatin with etoposide. The agent may be formulated and used as a combination therapeutic composition.

Heat shock protein 90 is a regulatory protein found in many eukaryotic cells. HSP90 inhibitors have been shown to be useful in the treatment of cancer. Such inhibitors include geldanamycin, 17-(allylamino)-17-demethoxygeldanamycin, PU-H71 and rifabutin.

Also contemplated are agents that directly cross-link DNA or form adducts. Agents such as cisplatin and other DNA alkylating agents may be used. Cisplatin is widely used in the treatment of cancer, with doses effective for clinical use being 20 mg/m ² for 5 days every three weeks for all three courses. Cisplatin is not absorbed orally and therefore must be administered by intravenous, subcutaneous, intratumoral, or intraperitoneal injection.

DNA damaging agents also include compounds that interfere with DNA replication, mitosis, and chromosome segregation. Such chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin, and the like. Widely used clinically for the treatment of neoplasms, these compounds are administered intravenously as bolus doses ranging from 25-75 mg/m ² at 21-day intervals for doxorubicin, to 35-50 mg/m ² for etoposide administered intravenously or orally at a dose twice the intravenous dose. Also contemplated is the administration of microtubule inhibitors such as taxanes. These molecules are diterpenes produced by plants of the genus Taxus , and include paclitaxel and docetaxel.

Epidermal growth factor receptor inhibitors such as iressa, mTOR, the mammalian target of rapamycin, also known as the FK506-binding protein 12 complex with rapamycin-associated protein 1 (FRAP1), which is a serine/threonine protein kinase, regulate cell growth, cell proliferation , cell motility, cell survival, protein synthesis and transcription. Therefore, rapamycin and its analogues ("rapalogues") are also contemplated for use in cancer therapy in accordance with the present invention.

Another possible therapeutic agent is TNF-α (tumor necrosis factor-alpha), a cytokine involved in systemic inflammation and a member of a group of cytokines that stimulate the acute phase response. The main role of TNF is in the regulation of immune cells. TNF is also capable of inducing apoptotic cell death, inflammation, and inhibiting oncogenesis and viral replication.

Agents that interfere with the synthesis and fidelity of nucleic acid precursors and subunits also cause DNA damage. As such, a number of nucleic acid precursors have been developed. Particularly useful are agents that have been extensively tested and are readily available. As such, agents such as 5-fluorouracil (5-FU) are preferentially utilized by the tumor tissue, making this agent particularly useful for targeting tumor cells. Although 5-FU is quite toxic, it is used in a wide range of vehicles, including topical, but intravenous administration at doses of 3 to 15 mg/kg/day is commonly used.

Other commonly used factors that cause DNA damage include factors commonly referred to as γ-rays, x-rays and/or targeted delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors such as microwaves and UV radiation are also contemplated. It is most likely that all these factors provide a wide range of damage to DNA, DNA precursors, DNA replication and repair, as well as damage to the assembly and maintenance of chromosomes. The range of doses of x-rays used varies from daily doses of 50 to 200 roentgens for extended periods of time (3 to 4 weeks) to single doses of 2000 to 6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and absorption by tumor cells.

In addition, the possible use of immunotherapy, hormone therapy, toxin therapy and surgical intervention is also envisaged. In particular, targeted therapies such as Avastin, Erbitux, Gleevec, Herceptin and Rituxan can be used.

One of skill in the art may use Remington's Pharmaceutical Sciences, 15th edition, chapter 33, in particular pp. 624-652. As a rule, the dose is adjusted depending on the condition of the patient receiving treatment. The person responsible for administration is in any case able to select the appropriate dose for the individual patient. In addition, for human administration, preparations must meet the standards of sterility, pyrogenicity, general safety, and purity as defined by the FDA Department of Biological Substances Standards.

IV. Protease detection methods

In other embodiments, it may be necessary to determine the nature and amount of proteases expressed in the target cancer cell or tumor, which will allow the selection of a particular interferon prodrug that is highly likely to be activated in the patient's tumor. In general, there are three approaches to such detection: immunological detection, for example, using protease-specific antibodies, mRNA detection, and analysis of protease activity.

A. Immunodetection

Immunodetection methods for identifying and quantifying proteases may include, among others, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescence assay, bioluminescence assay, and Western blot assay. The steps of various useful immunodetection methods are described in the scientific literature, see, for example, Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al. (1993) and Nakamura et al. (1987). Western blot methods typically involve obtaining a sample and contacting the sample with a first antibody, according to the embodiments discussed herein, under conditions effective to form immunocomplexes.

Contacting a selected biological sample with an antibody under effective conditions and for a period of time sufficient to form immune complexes (primary immune complexes) usually consists of simply adding the antibody composition to the sample and incubating the mixture for a period of time sufficient for the antibodies to formed immune complexes, i.e. bound to the protease present. After this time, the sample-antibody composition, such as on a tissue section, in an ELISA plate, dot blot, or Western blot, is typically washed to remove any non-specifically bound antibody species, ensuring that only those antibodies that are specifically bound in the primary are detected. immune complexes.

Detection of immunocomplex formation is generally well known in the art and can be accomplished using numerous approaches. These methods are usually based on the detection of a label or marker, such as any of the radioactive, fluorescent, biological, and enzyme labels. Patents relating to the use of such labels include US Pat. may provide additional benefits.

The antibody used for detection can itself be associated with a detectable label, which can then be easily detected, thereby allowing the amount of primary immune complexes in the composition to be determined. Alternatively, the first antibody that becomes bound in the primary immune complexes can be detected by a second binding ligand that has a binding affinity for the antibody. In these cases, the second binding ligand may be associated with a detectable label. The second binding ligand itself is often an antibody, which can appropriately be called a "secondary" antibody. Primary immune complexes are contacted with a labeled, secondary binding ligand or antibody under effective conditions and for a period of time sufficient to form secondary immune complexes. Secondary immune complexes are then typically washed to remove any non-specifically bound labeled secondary antibodies or ligands, and then detection of the remaining label in the secondary immune complexes is performed.

Additional methods include the detection of primary immune complexes using a two-step approach. A second binding ligand, such as an antibody that has a binding affinity for the antibody, is used to form the secondary immune complexes described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to form immune complexes (tertiary immune complexes). The third ligand or antibody binds to the detectable label, allowing detection of the thus formed tertiary immune complexes. If necessary, this system can provide signal amplification.

One method of immunodetection uses two different antibodies. The first biotinylated antibody is used to detect the target antigen and then the second antibody is used to detect the biotin associated with the complexed biotin. In this method, the test sample is first incubated in a solution containing the first step antibody. If the target antigen is present, the antibody portion binds to the antigen to form a biotinylated antibody/antigen complex. The antibody/antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and/or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody/antigen complex. The amplification steps are repeated until a suitable level of amplification is reached, after which the sample is incubated in a solution containing second step anti-biotin antibody. This second step antibody is labeled with, for example, an enzyme that can be used to detect an antibody/antigen complex by histoenzymatic assay using a chromogenic substrate. With suitable amplification, a macroscopically visible conjugate can be obtained.

Another known immunodetection method takes advantage of the immuno-PCR (polymerase chain reaction) method. The PCR method is similar to the Cantor method up to incubation with biotinylated DNA, however, instead of using several cycles of incubation with streptavidin and biotinylated DNA, the DNA/biotin/streptavidin/antibody complex is washed out with a low pH or high salt buffer to release the antibody. The resulting wash solution is then used to carry out the PCR reaction with the appropriate primers with appropriate controls. At least theoretically, the powerful amplification ability and specificity of PCR allows detection of a single antigen molecule.

In one example of an ELISA, antibodies of the invention are immobilized on a selected surface that exhibits affinity for a protein, such as a well in a polystyrene microtiter plate. Then the test composition, presumably containing the protease, is added to the wells. After binding and washing to remove non-specifically bound immune complexes, bound antigen can be detected. Detection can be achieved by adding another anti-protease antibody that binds to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection can also be achieved by adding a second antibody to the protease followed by adding a third antibody that has a binding affinity for the second antibody, the third antibody being bound to a detectable label.

In another exemplary ELISA method, samples suspected of containing a protease are immobilized on the surface of a well and then contacted with an anti-protease antibody. After binding and washing to remove non-specifically bound immune complexes, detection of bound anti-protease antibodies is performed. If the parent anti-protease antibodies are bound to a detectable label, this allows direct detection of immune complexes. Again, immune complexes can be detected with a second antibody that has a binding affinity for the first antibody for the protease, the second antibody binding to the detectable label.

Regardless of the format used, ELISA methods share certain common features such as coating, incubation and binding, washing to remove non-specifically bound species, and detection of bound immune complexes. They are described below.

When coating a plate with an antigen or antibody, typically the wells of the plate are incubated with a solution of the antigen or antibody, either overnight or for a specified period of time. The wells of the plate are then washed to remove incompletely adsorbed material. Any remaining accessible well surfaces are then "coated" with a non-specific protein that is antigenically neutral to the antiserum being tested. These include bovine serum albumin (BSA), casein or powdered milk solutions. The coating makes it possible to block non-specific adsorption sites on the immobilizing surface and thus reduces the background caused by non-specific binding of antiserum to the surface.

In ELISA methods, it seems more common to use secondary or tertiary means of detection rather than a direct procedure. Thus, after protein or antibody has been bound to the well, coated with non-reactive material to reduce background, and washed to remove unbound material, the immobilizing surface is brought into contact with the biological specimen under test under conditions effective to form an immune complex (antigen/antibody). The detection of the immune complex then requires a labeled secondary binding ligand or antibody and a secondary binding ligand or antibody in combination with a labeled tertiary antibody or third binding ligand.

"Under conditions effective to form an immune complex (antigen/antibody)" means that the conditions preferably include dilution of antigens and/or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS)/ twin. These added agents also contribute to the reduction of non-specific background.

"Suitable" conditions also mean that the incubation is carried out at a temperature or for a period of time sufficient to ensure effective binding. The incubation steps typically last from about 1-2 to 4 hours or so, at temperatures preferably from 25 to 27°C, or may last overnight at about 4°C or so.

After all incubation steps in the ELISA method, the contact surface is washed to remove uncomplexed material. A preferred washing procedure includes washing with a solution such as PBS/tween or borate buffer. After the formation of specific immune complexes between the test sample and the initially bound material and subsequent washing, the appearance of even small amounts of immune complexes can be determined.

To provide a means of detection, the second or third antibody typically contains an associated label to allow detection. Preferably, such a label is typically an enzyme that allows color development upon incubation with an appropriate chromogenic substrate. Thus, for example, it is necessary to contact or incubate the first and second immune complex with antibodies conjugated to urease, glucose oxidase, alkaline phosphatase, or hydrogen peroxidase for a period of time and under conditions that promote the development of further immune complex formation (for example, incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-tween).

After incubation with labeled antibody and after washing to remove unbound material, the label is quantified, for example by incubation with a chromogenic substrate such as urea or bromocresol purple, or 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) or H ₂ O ₂ , in the case of peroxidase, as an enzymatic label Quantification is achieved by measuring the degree of color development, for example with a spectrophotometer in the visible spectrum.

B. mRNA detection

An mRNA detection method can be used to evaluate protease activity in a cancer cell or tumor. Typically, mRNA detection is based on the hybridization of one nucleic acid (probe or primer) with another nucleic acid (target).

The use of a probe or primer 13 to 100 nucleotides in length, preferably 17 to 100 nucleotides in length, or in some aspects up to 1-2 kilobases or more in length, allows the formation of a duplex molecule that is both stable and selective. Molecules having complementary sequences in contiguous regions longer than 20 bases are generally preferred in terms of increasing the stability and/or selectivity of the resulting hybrid molecules. It is generally preferred to design hybridization nucleic acid molecules having one or more complementary sequences of 20 to 30 nucleotides, or even more where desired. Such fragments can be easily obtained, for example, by direct synthesis of the fragment by chemical means or by introducing selected sequences into recombinant vectors for production by recombinant methods.

Accordingly, nucleotide sequences can be used to selectively generate duplex molecules with complementary mRNA regions or provide primers for amplifying mRNA from samples. Depending on the intended application, different hybridization conditions can be used to achieve different degrees of probe or primer selectivity for the target sequence.

For applications requiring high selectivity, it is generally desirable to use relatively highly stringent hybridization conditions. For example, relatively low salt and/or high temperature conditions such as provided by about 0.02M to about 0.10M NaCl at temperatures from about 50°C to about 70°C. Such high stringency conditions allow for little, if any, mismatch between the probe or primers and the template or target strand, and may be particularly suitable for isolating specific genes or for detecting specific mRNA transcripts. As a rule, it should be understood that more stringent conditions can be provided by adding increasing amounts of formamide.

It is understood that in some cases lower stringency conditions are preferred. Under these conditions, hybridization can occur even if the sequences of the hybridizing strands are not exactly complementary and do not match at one or more positions. Less stringent conditions can be achieved by increasing the salt concentration and/or decreasing the temperature. For example, medium stringency conditions can be provided with a NaCl content of from about 0.1 to 0.25 M at temperatures from about 37°C to about 55°C, while low stringency conditions can be provided with a salt content of from about 0.15 M to about 0.9 M at temperatures ranging from about 20°C to about 55°C. Hybridization conditions can be easily manipulated depending on the desired results.

In other embodiments, hybridization can be achieved under conditions such as 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl ₂ , 1.0 mM dithiothreitol, at temperatures from about 20° C. to about 37 °C. Other hybridization conditions used may include about 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ at temperatures ranging from about 40°C to about 72°C.

In some embodiments, it is generally advantageous to use nucleic acids of certain sequences of the present invention in combination with suitable means, such as a label, to determine hybridization. A wide variety of suitable indicator agents are known in the art, including fluorescent, radioactive, enzymatic or other ligands such as detectable avidin/biotin. In preferred embodiments, a fluorescent label or an enzyme label, such as urease, alkaline phosphatase, or peroxidase, can be used instead of radioactive or other environmentally undesirable reagents. In the case of enzyme labels, colorimetric indicator substrates are known that are used to identify specific hybridization with complementary nucleic acid containing samples, which can be used as a means of providing visual or spectrophotometric detection.

In general, the probes or primers described herein are expected to be useful as reagents in solution hybridization, as in the case of ^TM PCR, to detect the expression of relevant genes, as well as in embodiments that use a solid phase. In embodiments that use a solid phase, the mRNA to be tested is adsorbed or otherwise attached to a selected template or surface. This fixed single-stranded nucleic acid is then subjected to hybridization with selected probes under the required conditions. The conditions chosen will depend on the particular circumstances (depending on, for example, G+C content, type of target nucleic acid, nucleic acid source, hybridization probe size, etc.). Optimizing hybridization conditions for a particular application of interest is well known to those skilled in the art. After washing the hybridized molecules to remove non-specifically bound probe molecules, hybridization is detected and/or quantified by determining the amount of bound label. Typical solid phase hybridization methods are disclosed in US Pat. The relevant portions of these and other references cited in this section of the description are incorporated herein by reference.

Target nucleic acids can be isolated from cells, tissues or other samples by standard methods (Sambrook et al., 2001). In some embodiments, analysis is performed on whole cell or tissue homogenates or body fluid samples without significant purification of the nucleic acid template. In other cases, purification and/or amplification may be required.

Amplification typically includes primer pairs designed to selectively hybridize to nucleic acids that match the target, under conditions that allow selective hybridization. Depending on the desired application, high stringency hybridization conditions can be selected that ensure hybridization only with sequences that are fully complementary to the primers. In other embodiments, hybridization may occur under less stringent conditions to allow amplification of nucleic acids containing one or more primer sequence mismatches. Following hybridization, the template-primer complex is coupled to one or more enzymes that facilitate template-dependent nucleic acid synthesis. Several cycles of amplification, also referred to as "cycles", are carried out until a sufficient amount of amplification product is obtained.

The amplification product can be detected or quantified. In some cases, detection can be performed using visual means. Alternatively, detection may include indirect identification of the product by chemiluminescence, radioactive scintigraphy of an embedded radioactive label or fluorescent label, or even by a system that uses electrical and/or thermal pulse signals (Bellus, 1994).

There are several template dependent processes for amplifying oligonucleotide sequences present in a given template template. One of the best known amplification methods is the polymerase chain reaction (called PCR ^TM ), which is detailed in US Pat. as a link.

To quantify the amplified mRNA, a reverse transcriptase PCR ^TM amplification procedure can be performed. Methods for reverse transcription of RNA into cDNA are well known (see Sambrook et al., 2001). Alternative reverse transcription methods use thermostable DNA polymerases. These methods are described in WO 90/07641. Polymerase chain reaction methods are well known in the art. Typical RT-PCR methods are described in US Pat. No. 5,882,864.

Reverse transcription (RT) of RNA into cDNA followed by quantitative PCR (RT-PCR) can be used to determine the relative concentrations of specific mRNA species isolated from a cell. By determining that the concentration of particular mRNA species varies, it is shown that the gene encoding particular mRNA species is differentially expressed. When plotting with the cycle number on the x-axis and the logarithm of the amplified target DNA concentration on the y-axis, by connecting the points plotted on the graph, a curved line of characteristic shape is obtained. Starting from the first cycle, the slope of the line is positive and constant. This is called the linear part of the curve. After the reactant becomes the limiting factor, the slope of the line begins to decrease and eventually becomes zero. At this point, the concentration of the amplified target DNA becomes asymptotic to some fixed value. This part of the curve is called the plateau.

The target DNA concentration in the linear part of the PCR amplification is directly proportional to the initial target concentration before the start of the reaction. By determining the concentration of amplified target DNA products in PCR reactions that have gone through the same number of cycles and are in linear ranges, one can determine the relative concentrations of a particular target sequence in the original DNA mixture. If the DNA mixtures are cDNAs synthesized from RNA isolated from different tissues or cells, then the relative copy numbers of the specific mRNAs from which the target sequence was derived can be determined for the respective tissues or cells. This direct relationship between the concentration of PCR products and the relative content of mRNA is true only in the linear range of the PCR reaction.

The final concentration of the target DNA in the part of the curve represented by the plateau is determined by the presence of reagents in the reaction mixture and does not depend on the initial concentration of the target DNA. Thus, the first condition that must be met before RT-PCR can determine the relative copy number of mRNA species for a collection of RNA populations is that sampling to determine the concentration of amplified PCR products must be performed when the reactions PCR are in the linear part of the corresponding curve.

The second condition of the RT-PCR experiment is to determine the relative content of specific mRNA species. Typically, the relative concentrations of amplified cDNA are normalized against some independent standard. The purpose of the RT-PCR experiment is to determine the amount of specific mRNA species relative to the average amount of all mRNA species in the sample.

Most protocols for competitive PCR use internal PCR standards that have approximately the same copy number as the targets. These strategies are effective if PCR amplification products are sampled during their linear phases. If the products are withdrawn as the reactions approach a plateau phase, then an excess of the less abundant product appears. This leads to misrepresentation of relative abundance comparisons made for many different RNA samples, for example in the case of examining RNA samples for differential expression, such as reducing differences in relative RNA abundance, which is not true. This is not a serious problem if the internal standard has a much higher copy number than the target. If the internal standard has a higher copy number than the target, direct linear comparisons can be made between RNA samples.

The RT-PCR method can be performed as relative quantitative RT-PCR with an internal standard, where the internal standard is an amplified cDNA fragment that is longer than the target cDNA fragment, and where the copy number of the mRNA encoding the internal standard is approximately 5-100 times higher than the mRNA , which encodes the target. This assay allows one to measure the relative content, rather than the absolute amount, of the respective mRNA species.

Another amplification method is the ligase chain reaction ("LCR") disclosed in European application No. 320308, fully incorporated in the present description by reference. US Pat. No. 4,883,750 describes a method similar to LCR used to link pairs of probes to a target sequence. A method based on PCR ^TM and oligonucleotide ligase assay (OLA) as disclosed in US Pat. No. 5,912,148 can also be used.

Альтернативные способы амплификации целевых последовательностей нуклеиновых кислот, которые можно использовать для осуществления настоящего изобретения, раскрыты в патентах США 5843650, 5846709, 5,846783, 5849,546, 5849497, 5849547, 5858652, 5866366, 5916776, 5922574, 5928905, 5928906, 5932451, 5935825, 5939291 and 5942391, UK Application No. 2202328 and PCT Application No. PCT/US89/01025, each of which is incorporated herein in its entirety by reference.

The Q-beta replicase described in PCT Application No. PCT/US87/00880 can also be used as an amplification method in the present invention. In this method, a replicative RNA sequence that has a complementary region to the target region is added to the sample in the presence of RNA polymerase. The polymerase copies the replicative sequence, which can then be detected.

The isothermal amplification method, which uses restriction endonucleases and ligases to achieve the amplification of target molecules containing nucleotide 5'-[alpha-thio]-triphosphates on a single strand of the cleavage site, may also be useful for amplifying the nucleic acids of the present invention (Walker et al., 1992). Strand displacement amplification (SDA) as disclosed in US Pat. nick-broadcast.

Other nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence-based amplification (NASBA) and 3SR (Kwoh et al., 1989; PCT application WO 88/10315, herein incorporated by reference in its entirety ). EP 329822 discloses a nucleic acid amplification process involving the cyclic synthesis of single-stranded RNA ("ssRNA"), ssDNA and double-stranded DNA (dsDNA), which can be used in accordance with the present invention.

PCT application WO 89/06700 (incorporated herein by reference in its entirety) discloses a nucleic acid sequence amplification scheme based on hybridization of a promoter region/primer sequence to a single-stranded DNA target ("ssDNA") followed by transcription of multiple RNA copies of the sequence . This scheme is not cyclic; new templates are not created from the resulting RNA transcripts. Other amplification methods include "RACE" and "one way PCR" (Frohman, 1990; Ohara et al., 1989).

After any amplification, it may be necessary to separate the amplification product from the template and/or excess primer. In one embodiment, the amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis by standard methods (Sambrook et al., 2001). The separated amplification products can be excised and eluted from the gel for further manipulation. In the case of low melting agarose gels, the separated band can be removed by heating the gel followed by extraction of the nucleic acid.

Separation of nucleic acids can also be carried out by chromatographic methods known in the art. There are many types of chromatography that can be used to implement the present invention, including adsorption, separation, ion exchange, hydroxyapatite chromatography, molecular sieve chromatography, reversed phase, column, paper, thin layer and gas chromatography, as well as HPLC.

In some embodiments, the amplification products are visualized. A typical imaging technique involves staining the gel with ethidium bromide and visualizing the bands under UV light. Alternatively, if integer labeling of the amplification products is performed with radioactive or fluorometrically labeled nucleotides, then the individual amplification products can be exposed to x-ray film or visualized in the appropriate excitation spectra.

In one embodiment, after separation of the amplification products, the labeled nucleic acid probe is brought into contact with the amplified marker sequence. The probe is preferably conjugated to a chromophore, but radioactive labeling may be performed. In another embodiment, the probe is conjugated to a binding partner, such as an antibody or biotin, or other binding partner carrying a detectable fragment.

In specific embodiments, detection is by Southern blotting and hybridization with a labeled probe. Southern blot techniques are well known to those skilled in the art (see Sambrook et al., 2001). One example of the foregoing is described in US Pat. No. 5,279,721, incorporated herein by reference, which describes an apparatus and method for performing automatic electrophoresis and transfer of nucleic acids. The device allows electrophoresis and blotting without external manipulation of the gel and is ideally suited for carrying out the methods of the present invention.

Различные методы обнаружения нуклеиновых кислот, известные в данной области техники, раскрыты в патентах США 5840873, 5843640, 5843651, 5846708, 5846717, 5846726, 5846729, 5849487, 5853990, 5853992, 5853993, 5856092, 5861244, 5863732, 5863753, 5866331, 5905024, 5910407, 5912124, 5912145, 5919630, 5925517, 5928862, 5928869, 5929227, 5932413 and 5935791, each of which is incorporated herein by reference.

Protease mRNA detection may involve the use of arrays or data generated from an array. An array generally refers to ordered macroarrays or microarrays of nucleic acid molecules (probes) that are completely or nearly complementary or identical to a plurality of mRNA molecules or cDNA molecules and that are spatially separated on a carrier material. Macrochips are typically sheets of nitrocellulose or nylon on which probes are applied. On microarrays, nucleic acid probes are more densely packed such that a region typically 1 to 4 square centimeters in size can contain up to 10,000 nucleic acid molecules. Microarrays can be made by depositing nucleic acid molecules, eg genes, oligonucleotides, etc., on substrates, or by making oligonucleotide sequences in situ on a substrate. Spotted or fabricated nucleic acid molecules can be applied as a high density matrix containing up to 30 non-identical nucleic acid molecules per square centimeter or more, such as up to about 100 or even 1000 per square centimeter. Microchips typically use coated glass as a solid support, as opposed to nitrocellulose-based material for filter matrices. With an ordered array of complementary nucleic acid samples, the position of each sample can be tracked and associated with the original sample. A variety of array devices are known to those skilled in the art in which a plurality of different nucleic acid probes are stably attached to the surface of a solid support. Suitable array substrates include nylon, glass, and silicon. Such matrices can be changed in a variety of ways, including the average length of the probe, the sequence or types of probes, the nature of the bond between the probe and the surface of the matrix, such as covalent or non-covalent, and the like. The use of the labeling and screening methods of the present invention and matrices is not limited in any way, except that the probes must be capable of detecting expression levels; therefore, the methods and compositions can be used with many different types of genes.

Typical methods and devices for making microchips are described in, for example, US Pat. Nos. 5,143,854; 5202231; 5242974; 5288644; 5324633; 5384261; 5405783; 5412087; 5424186; 5429807; 5432049; 5436327; 5445934; 5468613; 5470710; 5472672; 5492806; 5503980; 5510270; 5525464; 5527681; 5529756; 5532128; 5545531; 5547839; 5554501; 5556752; 5561071; 5571639; 5580726; 5580732; 5593839; 5599695; 5599672; 5610287; 5624711; 5631134; 5639603; 5654413; 5658734; 5661028; 5665547; 5667972; 5695940; 5700637; 5744305; 5800992; 5807522; 5830645; 5837196; 5871928; 5847219; 5876932; 5919626; 6004755; 6087102; 6368799; 6383749; 6617112; 6638717; 6720138, as well as WO 93/17126; WO 95/11995; WO 95/21265; WO 95/21944; WO 95/35505; WO 96/31622; WO 97/10365; W097/27317; WO 99/35505; WO 09923256; WO09936760; WO0138580; WO0168255; W003020898; W003040410; WO03053586; WO03087297; W003091426; W003100012; WO 04020085; WO 04027093; EP 373203; EP 785280; EP 799897 and UK 8803000; the disclosure of which is incorporated into the present description by reference.

It is contemplated that the arrays may be high density arrays that may contain 100 or more different probes. It is contemplated that they may contain 1000, 16000, 65000, 250000 or 1000000 or more different probes. Probes can be directed to targets located in one or more different organisms. In some embodiments, the oligonucleotide probes are 5 to 50, 5 to 45, 10 to 40, or 15 to 40 nucleotides in length. In some embodiments, the oligonucleotide probes are 20 to 25 nucleotides in length.

The location and sequence of every other probe sequence in the array is publicly known. In addition, a large number of different probes can occupy a relatively small area, providing a high density array with probe densities typically greater than about 60, 100, 600, 1000, 5000, 10000, 40000, 100000 or 400000 different oligonucleotide probes per ^cm2 . The surface area of the array may be about or less than about 1, 1.6, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cm ² .

In addition, a person skilled in the art can easily analyze the data generated by the array. Such protocols are disclosed above and include information that can be found in patent applications No. WO 9743450; WO03023058; WO03022421; WO03029485; W003067217; WO03066906; W003076928; W003093810; WO 03100448A1, each of which is specifically incorporated herein by reference.

V. Examples

The following examples are included to demonstrate the preferred embodiments. It will be appreciated by those skilled in the art that the methods disclosed in the examples below are methods disclosed by the inventors for effectively practicing embodiments of the invention, and thus may be considered as constituting preferred modes of implementation. However, those skilled in the art, in light of the present disclosure, will appreciate that many changes can be made to the specific disclosed embodiments to achieve the same or similar result without departing from the spirit and scope of the invention.

Example 1

Interferon alpha and beta receptor (IFNAR) IFN prodrug. The inventors have developed an IFN prodrug based on the structure of human immunoglobulin G1 (IgG1) (FIG. 1). The blocking reagent is the extracellular domain (ECD) of native IFNAR1 or IFNAR2 and is linked to the N-terminus of interferon alpha via a cleavable substrate linker. In this form, IFNAR blocks binding to the cell surface IFNAR1/IFNAR2 heterodimer. To increase stability and half-life, the inventors performed a fusion of the Fc domain of IgG1 with the C-terminus of alpha interferon (Fig. 1).

To determine if either IFNAR1 or IFNAR2 ECD is capable of blocking interferon alpha activity, we purified IFNa4-Fc, R1-IFNa4-Fc and R2-IFNa4-Fc. The R1-IFNa4-Fc or R2-IFNa4-Fc linker is a 15 amino acid Gly-Gly-Gly-Gly-Ser ternary peptide that is a flexible analog that allows interaction between the ^23α domains. RAW-Lucia ISG cells were used that express the Lucia luciferase gene under the control of the minimal ISG54 promoter in combination with five IFN-stimulated response elements. RAW-Lucia ISG cells are responsive to mouse IFN-α and IFN-β. Performed a 5-fold serial dilution of 20 nm hIg, IFNa4-Fc, R1-IFNa4-Fc and R2-IFNa4-Fc to analyze the activity of luciferase. Both R1-IFNa4-Fc and R2-IFNa4-Fc showed a significant reduction in IFN activity of at least 125-fold compared to that of IFNa4-Fc (FIG. 2A).

To determine if the activity of the activated IFN prodrug was comparable to that of IFNa4-Fc, purification of R1-NSUB, R1-SUB, R2-NSUB and R2-SUB was performed. The R1-SUB or R2-SUB linker is a 16 amino acid peptide bearing a 6 residue protease cleavable substrate (PVGLIG) ²⁴ that can be cut with MMP-2 or MMP-9, flanked on both sides with flexible peptides Gly-Gly-Gly-Gly-Ser. R1-NSUB and R2-NSUB without protease-cleavable substrate were used as control constructs. R1-NSUB, R1-SUB, R2-NSUB or R2-SUB were incubated with rmMMP-9 at 37°C for 6 hours for activation. Activated R1-SUB showed a 25-fold increase in IFN activity compared to IFNa4-Fc activity, while R1-NSUB activity did not change after enzyme administration (FIG. 2B). A similar result was observed with R2-NSUB and R2-SUB, with activated R2-SUB showing an even greater increase in IFN activity, over 25-fold, compared to IFNa4-Fc (FIG. 2C).

Thus, the biological activity of the protease-activated IFN prodrug was increased in both the IFNA1 and IFNAR2 forms, rather than the expected recovery to the equivalent parental IFNa4-Fc. Moreover, prior to protease cleavage, the IFN prodrug appears to have markedly reduced biological activity, suggesting that the IFN prodrug would be safe in vivo prior to cleavage, but would increase therapeutic activity after cleavage in the local tumor microenvironment.

An IFN prodrug based on IFNAR1 and IFNAR2 shows various antitumor effects in vivo . The possibility of converting local activation of the IFN prodrug into antitumor efficacy in vivo was then studied in a mouse model of B16 melanoma. Without screening for enzyme expression in mouse tumor models, an IFN prodrug carrying a cleavable substrate MMP-2 and MMP-9 was first studied. Mice with established B16 melanoma tumors were treated with R1-SUB, R2-SUB, IFNa4-Fc or control hIg. At a dose of 1 nmol (three injections every three days), R1-SUB, R2-SUB and IFNa4-Fc had different efficacy in suppressing tumor growth in this model. 10 days after treatment, R1-SUB inhibited tumor growth by 24%, R2-SUB by 57% and IFNa4-Fc by 72% relative to the control group treated with hIg (figure 3).

Therefore, the IFNAR2 based IFN prodrug showed superior antitumor effects in vivo than the IFNAR1 based IFN prodrug, which led to the focus of the next study on R2-SUB. However, R2-SUB carrying the cleavable substrate MMP-2 and MMP-9 had a lower efficacy in suppressing tumor growth compared to IFNa4-Fc. It has been suggested that improvement in the efficacy of the IFN prodrug in mouse tumor models can be achieved by selecting an appropriate cleavable substrate linker.

The IFNAR2 based IFN prodrug has improved efficacy and reduced side effects. In order to select a more advantageous cleavable substrate linker, the most studied proteases, which are known to be activated in many different human cancers, were screened in murine tumor models. This substrate selection process involved counter-selection for proteases expressed in normal healthy tissues to reduce the likelihood of systemic (non-specific) activation of the IFN prodrug. Mice with established B16 melanoma tumors or MC38 colon tumors were sacrificed, and tumor tissues and normal tissues, including spleen, heart, liver, lung, and kidney, were used to determine the level of mRNA expression of the following protease genes: uPA, MMP-2, MMP-9 and MMP-14. uPA was only highly expressed in the kidneys, but not in the two tumor tissues (FIG. 4A). MMP-2 was highly expressed in MC38 tumors but not in B16 tumors; however, it had a high background in the heart and lungs (Fig. 4B). The expression level of MMP-9 was very low in all normal and tumor tissues (Fig. 4C). Finally, MC38 and B16 tumors had a high level of expression of MMP-14, while, on the contrary, the expression of MMP-14 in normal tissues was minimal (Fig.4D). Screening data in mouse tumor models indicated that a cleavable linker, which is a MMP-14 sensitive substrate, can be used to generate a potential IFN prodrug in vivo .

The expression level of human proteases between the tumor and adjacent normal tissues was also analyzed. The DiffExp module in TIMER (Tumor Immunity Assessment Resource) provides a comparison of gene expression levels for all samples from the TCGA (Cancer Genome Atlas). Most of the tumor-associated enzymes have been evaluated and it has been found that MMP-1, MMP-3, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13 and MMP-14 are proteases that are highly upregulated in most tumors, but have a relatively low background in normal tissues (FIGS. 6A-H). These proteases are excellent candidates for the development of a human IFN prodrug to achieve maximum antitumor effect and minimal side effects on normal tissues. Interestingly, consistent data on the level of expression of MMP-14 in samples from both mice and humans increase the reliability of the choice of MMP-14 substrate in obtaining IFN prodrug.

Therefore, an IFN prodrug based on IFNAR2 and MMP-14 substrate (SGRSENIRTA) ²⁵ was developed and purified. Potential benefits in terms of safety and antitumor efficacy in vivo have been studied. Mice with established B16-OVA melanoma tumors were treated with R2-SUB, IFNa4-Fc, or control hIg administered as three injections of 1 mM every three days. The relative safety of IFN prodrug and IFNa4-Fc was compared. High levels of inflammatory cytokines in serum, including IL-6, TNF, MCP-1 and IFN-g, were observed in the IFNa4-Fc group the day after the third injection. In contrast, mice treated with R2-SUB showed very low levels of these inflammatory cytokines (FIG. 5A). The level of ALT indicating liver toxicity also showed similar results (FIG. 5B).

Monitoring of tumor growth and body weight continued. At 12 days post-treatment, R2-SUB inhibited tumor growth by 76% and IFNa4-Fc by 67% compared to the hIg-treated control group (FIG. 5C). However, at this point, the mice in the IFN-4-Fc group appeared ill (distracted, inactive, and lean), probably due to severe side effects caused by IFN. Then a comparison was made of the kinetics of body weight. The data showed that mice from the IFNa4-Fc group began to rapidly lose weight after the third injection, however, the body weight of mice from the R2-SUB group did not change significantly after three injections (FIG. 5D). Finally, the toxicity-based survival curve showed that the R2-SUB group had a 100% survival rate, while the IFNa4-Fc group showed a 33% survival rate (FIG. 5E).

Prodrug of human IFN-based interferon receptor alfa and beta (IFNAR).

To determine whether human IFNAR1 or IFNAR2 ECD is capable of blocking human interferon alfa 2 activity, purification of human IFNa2-Fc, R1-IFNa2-Fc and R2-IFNa2-Fc was performed. The R1-IFNa2-Fc or R2-IFNa2-Fc linker was a 15 amino acid Gly-Gly-Gly-Gly-Ser ternary peptide (SEQ ID NO: 13), which is a flexible linker allowing interaction between the ^23α domains. 293T-Dual ^TM hSTING-R232 reporter cells were used expressing the reporter gene SEAP (Secreted Embryonic Alkaline Phosphatase) under the control of the minimal IFN-β promoter fused to the five binding sites of NF-κB and AP-1. Cells 293T-Dual ^TM hSTING-R232 sensitive to human IFN-α and IFN-β. A 10-fold serial dilution of 50 nM human IFNa2-Fc, R1-IFNa2-Fc and R2-IFNa2-Fc was performed to analyze the SEAP reporter gene. Compared to human IFNa2-Fc, R1-IFNa2-Fc showed a 10-fold decrease in IFN activity, while R2-IFNa2-Fc showed a 100-fold decrease in IFN activity (FIG. 7A). Thus, IFNAR2 is a more effective blocking agent for human IFN.

To determine if the activity of the activated human IFN prodrug is comparable to that of IFNa2-Fc, purification of human R2-NSUB and R2-SUB was performed. The R2-SUB linker is a 16 amino acid peptide bearing a 6 residue protease cleavable substrate (PVGLIG) ²⁴ that can be cut with MMP-2 or MMP-9, flanked on both sides by a flexible Gly-Gly- peptide. Gly-Gly-Ser. R2-NSUB without protease-cleavable substrate was used as control constructs. R2-NSUB or R2-SUB was incubated with rmMMP-9 at 37°C for 0, 0.5, 2 or 6 hours. Human R2-SUB was digested with rmMMP-9 in a time dependent manner. After 6 hours, the cleavage efficiency was 100% (FIG. 7C). Activated human R2-SUB showed comparable IFN activity to human IFNa2-Fc, while R2-NSUB activity did not change after enzyme administration (FIG. 7B).

Thus, the biological activity of the protease-activated human IFN prodrug was completely restored in the IFNAR2-based form compared to the parental IFNa4-Fc. In addition, the IFNAR2-based human IFN prodrug appeared to have a markedly reduced biological activity prior to protease cleavage, suggesting that the human IFN prodrug would be safe in vivo prior to cleavage, but would be therapeutic after cleavage in the local human tumor microenvironment. activity will increase.

Thus, the IFN prodrug studied here provides improved targeting of type I interferon to tumor tissue by reducing toxicity in normal tissues. The antitumor efficacy of an IFN prodrug can be tailored to the protease activity in each tumor, making the IFN prodrug a personalized drug in certain situations, with the selected cleavable linker substrate providing the best outcome. In addition, an IFN prodrug can be designed to transfer multiple IFN domains using different linkers that are sensitive to different proteases, thereby greatly improving its therapeutic effect.

All compositions and methods disclosed and claimed in the present description can be made and performed in view of the present description without undue experimentation. While the compositions and methods of the invention are described in terms of the preferred embodiments, it should be apparent to those skilled in the art that changes can be made to the compositions and methods, as well as to the method steps and the sequence of steps, without departing from the concept, spirit, and scope. inventions. More specifically, it will be apparent that certain agents that are both chemically and physiologically related can replace the agents described in this application, with the same or similar results. All such analogous substituents and modifications obvious to those skilled in the art are considered to be within the spirit, scope and concept of the invention as defined in the appended claims.

VII. LITERATURE

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SEQUENCE LISTS

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Glu Asn Ile Asp Val Tyr Ile Ile Asp Asp Asn Tyr Thr Leu Lys Trp

35 40 45

Ser Ser His Gly Glu Ser Met Gly Ser Val Thr Phe Ser Ala Glu Tyr

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Arg Thr Lys Asp Glu Ala Lys Trp Leu Lys Val Pro Glu Cys Gln His

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Thr Thr Thr Thr Lys Cys Glu Phe Ser Leu Leu Asp Thr Asn Val Tyr

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Ile Lys Thr Gln Phe Arg Val Arg Ala Glu Glu Gly Asn Ser Thr Ser

100 105 110

Ser Trp Asn Glu Val Asp Pro Phe Ile Pro Phe Tyr Thr Ala His Met

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Ser Pro Pro Glu Val Arg Leu Glu Ala Glu Asp Lys Ala Ile Leu Val

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His Ile Ser Pro Pro Gly Gln Asp Gly Asn Met Trp Ala Leu Glu Lys

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Pro Ser Phe Ser Tyr Thr Ile Arg Ile Trp Gln Lys Ser Ser Ser Asp

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Lys Lys Thr Ile Asn Ser Thr Tyr Tyr Val Glu Lys Ile Pro Glu Leu

180 185 190

Leu Pro Glu Thr Thr Tyr Cys Leu Glu Val Lys Ala Ile His Pro Ser

195 200 205

Leu Lys Lys His Ser Asn Tyr Ser Thr Val Gln Cys Ile Ser Thr Thr

210 215 220

Val Ala Asn Lys Met Pro Val Pro Gly Asn Leu Gln Val Asp Ala Gln

225 230 235 240

Gly Lys Ser Tyr Val Leu Lys Trp Asp Tyr Ile Ala Ser Ala Asp Val

245 250 255

Leu Phe Arg Ala Gln Trp Leu Pro Gly Tyr Ser Lys Ser Ser Ser Gly

260 265 270

Ser Arg Ser Asp Lys Trp Lys Pro Ile Pro Thr Cys Ala Asn Val Gln

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Thr Thr His Cys Val Phe Ser Gln Asp Thr Val Tyr Thr Gly Thr Phe

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Phe Leu His Val Gln Ala Ser Glu Gly Asn His Thr Ser Phe Trp Ser

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Glu Glu Lys Phe Ile Asp Ser Gln Lys His Ile Leu Pro Pro Pro Pro

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Val Ile Thr Val Thr Ala Met Ser Asp Thr Leu Leu Val Tyr Val Asn

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Cys Gln Asp Ser Thr Cys Asp Gly Leu Asn Tyr Glu Ile Ile Phe Trp

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Glu Asn Thr Ser Asn Thr Lys Ile Ser Met Glu Lys Asp Gly Pro Glu

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Phe Thr Leu Lys Asn Leu Gln Pro Leu Thr Val Tyr Cys Val Gln Ala

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Arg Val Leu Phe Arg Ala Leu Leu Asn Lys Thr Ser Asn Phe Ser Glu

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Asp Lys Trp Leu Glu Gly Met Glu Ser Tyr Val Val Ala Ile Val Ile

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Ser Trp Tyr Glu Val Asp Ser Phe Thr Pro Phe Arg Lys Ala Gln Ile

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Gly Pro Pro Glu Val His Leu Glu Ala Glu Asp Lys Ala Ile Val Ile

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His Ile Ser Pro Gly Thr Lys Asp Ser Val Met Trp Ala Leu Asp Gly

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Leu Ser Phe Thr Tyr Ser Leu Val Ile Trp Lys Asn Ser Ser Gly Val

165 170 175

Glu Glu Arg Ile Glu Asn Ile Tyr Ser Arg His Lys Ile Tyr Lys Leu

180 185 190

Ser Pro Glu Thr Thr Tyr Cys Leu Lys Val Lys Ala Ala Leu Leu Thr

195 200 205

Ser Trp Lys Ile Gly Val Tyr Ser Pro Val His Cys Ile Lys Thr Thr

210 215 220

Val Glu Asn Glu Leu Pro Pro Pro Glu Asn Ile Glu Val Ser Val Gln

225 230 235 240

Asn Gln Asn Tyr Val Leu Lys Trp Asp Tyr Thr Tyr Ala Asn Met Thr

245 250 255

Phe Gln Val Gln Trp Leu His Ala Phe Leu Lys Arg Asn Pro Gly Asn

260 265 270

His Leu Tyr Lys Trp Lys Gln Ile Pro Asp Cys Glu Asn Val Lys Thr

275 280 285

Thr Gln Cys Val Phe Pro Gln Asn Val Phe Gln Lys Gly Ile Tyr Leu

290 295 300

Leu Arg Val Gln Ala Ser Asp Gly Asn Asn Thr Ser Phe Trp Ser Glu

305 310 315 320

Glu Ile Lys Phe Asp Thr Glu Ile Gln Ala Phe Leu Leu Pro Pro Val

325 330 335

Phe Asn Ile Arg Ser Leu Ser Asp Ser Phe His Ile Tyr Ile Gly Ala

340 345 350

Pro Lys Gln Ser Gly Asn Thr Pro Val Ile Gln Asp Tyr Pro Leu Ile

355 360 365

Tyr Glu Ile Ile Phe Trp Glu Asn Thr Ser Asn Ala Glu Arg Lys Ile

370 375 380

Ile Glu Lys Lys Thr Asp Val Thr Val Pro Asn Leu Lys Pro Leu Thr

385 390 395 400

Val Tyr Cys Val Lys Ala Arg Ala His Thr Met Asp Glu Lys Leu Asn

405 410 415

Lys Ser Ser Val Phe Ser Asp Ala Val Cys Glu Lys Thr Lys Pro Gly

420 425 430

Asn Thr Ser Lys

435

<210> 4

<211> 243

<212> Protein

<213> Homo sapiens

<400> 4

Met Leu Leu Ser Gln Asn Ala Phe Ile Phe Arg Ser Leu Asn Leu Val

1 5 10 15

Leu Met Val Tyr Ile Ser Leu Val Phe Gly Ile Ser Tyr Asp Ser Pro

20 25 30

Asp Tyr Thr Asp Glu Ser Cys Thr Phe Lys Ile Ser Leu Arg Asn Phe

35 40 45

Arg Ser Ile Leu Ser Trp Glu Leu Lys Asn His Ser Ile Val Pro Thr

50 55 60

His Tyr Thr Leu Leu Tyr Thr Ile Met Ser Lys Pro Glu Asp Leu Lys

65 70 75 80

Val Val Lys Asn Cys Ala Asn Thr Thr Arg Ser Phe Cys Asp Leu Thr

85 90 95

Asp Glu Trp Arg Ser Thr His Glu Ala Tyr Val Thr Val Leu Glu Gly

100 105 110

Phe Ser Gly Asn Thr Thr Leu Phe Ser Cys Ser His Asn Phe Trp Leu

115 120 125

Ala Ile Asp Met Ser Phe Glu Pro Pro Glu Phe Glu Ile Val Gly Phe

130 135 140

Thr Asn His Ile Asn Val Met Val Lys Phe Pro Ser Ile Val Glu Glu

145 150 155 160

Glu Leu Gln Phe Asp Leu Ser Leu Val Ile Glu Glu Gln Ser Glu Gly

165 170 175

Ile Val Lys Lys His Lys Pro Glu Ile Lys Gly Asn Met Ser Gly Asn

180 185 190

Phe Thr Tyr Ile Ile Asp Lys Leu Ile Pro Asn Thr Asn Tyr Cys Val

195 200 205

Ser Val Tyr Leu Glu His Ser Asp Glu Gln Ala Val Ile Lys Ser Pro

210 215 220

Leu Lys Cys Thr Leu Leu Pro Pro Gly Gln Glu Ser Glu Ser Ala Glu

225 230 235 240

Ser Ala Lys

<210> 5

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 5

Pro Val Gly Leu Ile Gly

15

<210> 6

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 6

Gly Gly Tyr Ala Glu Leu Arg Met Gly Gly

1 5 10

<210> 7

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 7

Gly Pro Arg Pro Phe Asn Tyr Leu

15

<210> 8

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 8

Gly Gly Ala Leu Gly Leu Ser Leu

15

<210> 9

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 9

Gly Pro Met Ser Tyr Asn Ala Leu

15

<210> 10

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 10

Ser Gly Arg Ser Glu Asn Ile Arg Thr Ala

1 5 10

<210> 11

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 11

Thr Ser Gly Pro Asn Gln Glu Gln Lys

15

<210> 12

<211> 4

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 12

Gly Phe Leu Gly

1

<210> 13

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 13

Gly Gly Gly Gly Ser

15

<---

Claims (37)

1. An interferon prodrug for the treatment of cancer, comprising: (a) an interferon alpha and beta receptor 2 (IFNAR2) domain that retains IFN-binding activity; (b) a type 1 interferon (IFN) domain that retains type 1 interferon activity when not bound by said IFNAR2 domain; (c) Fc domain of an immunoglobulin (Ig), (d) a first linker fused at one end to the N-terminus of said IFN and fused at the other end to said IFNAR2, said first linker being cleavable by a protease, said first linker being cleavable by a matrix metalloproteinase, said matrix metalloproteinase being MMP2 or MMP9, said first linker is G4S-PVGLIG-G4S; and (e) a second linker fused at one end to the C-terminus of said IFN and fused at the other end to the N-terminus of said Ig Fc domain. 2. An interferon prodrug according to claim 1, wherein said Ig is an IgG such as IgG1 or IgG2. 3. Prodrug of interferon according to paragraphs. 1, 2, wherein said interferon prodrug contains two copies of said type 1 IFN domain. 4. Prodrug of interferon according to paragraphs. 1-3, wherein said interferon prodrug contains more than two copies of said type 1 IFN domain. 5. Prodrug of interferon according to paragraphs. 1-4, wherein said IFN is IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω, or IFN-ζ. 6. A nucleic acid construct encoding an interferon prodrug containing: (a) an interferon alpha and beta receptor 2 (IFNAR2) domain that retains IFN-binding activity; (b) a type 1 interferon (IFN) domain that retains type 1 interferon activity when not bound by said IFNAR2 domain; (c) Fc domain of an immunoglobulin (Ig), (d) a first linker fused at one end to the N-terminus of said IFN and fused at the other end to said IFNAR2, said first linker being cleavable by a protease, said first linker being cleavable by a matrix metalloproteinase, said matrix metalloproteinase being MMP2 or MMP9, said first linker is G4S-PVGLIG-G4S; (e) a second linker fused at one end to the C-terminus of said IFN and fused at the other end to the N-terminus of said Ig Fc domain; and wherein the construct contains a promoter located 5' to the 5' end of said IFN domain. 7. A nucleic acid construct according to claim 6, wherein said Ig is an IgG such as IgG1 or IgG2. 8. The design of the nucleic acid according to paragraphs. 6, 7, wherein said interferon prodrug contains two copies of said type 1 IFN domain. 9. The design of the nucleic acid according to paragraphs. 6-8, wherein said interferon prodrug contains more than two copies of said type 1 IFN domain. 10. The design of the nucleic acid according to paragraphs. 6-9, wherein said IFN is IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω, or IFN-ζ. 11. Recombinant cell containing the nucleic acid construct according to paragraphs. 6-10, and the cell is designed to express the prodrug of interferon according to paragraphs. 1-5. 12. A method for expressing an interferon prodrug, comprising culturing a cell according to claim 11. 13. The use of an interferon prodrug according to paragraphs. 1-5 in obtaining a drug for the treatment of cancer. 14. The use of an interferon prodrug according to paragraphs. 1-5 for cancer treatment. 15. A method of treating cancer, comprising administering to a patient an interferon prodrug according to paragraphs. 1-5. 16. The method of claim 15, further comprising the step of evaluating protease expression in a cancer cell obtained from said patient. 17. The method of claim 16 wherein said cancer cell is obtained from a biopsy. 18. The method of claim 16 wherein said cancer cell is a circulating tumor cell. 19. The method according to paragraphs. 16-18, wherein said cancer is lung cancer, breast cancer, brain cancer, oral cavity cancer, esophageal cancer, head and neck cancer, skin cancer, stomach cancer, liver cancer, pancreatic cancer, kidney cancer, cancer ovarian cancer, prostate cancer, bladder cancer, colon cancer, testicular cancer, uterine cancer, cervical cancer, lymphoma, or leukemia. 20. The method according to paragraphs. 15-19, wherein said cancer is primary, recurrent, metastatic, or multidrug resistant. 21. The method according to paragraphs. 15-20, wherein said patient is a human or non-human mammal. 22. The method according to paragraphs. 15-21 further comprising administering said interferon prodrug more than once. 23. The method of claim 22, wherein said interferon prodrug is administered daily, every other day, weekly, every other week, or monthly. 24. The method according to paragraphs. 15-23, wherein said interferon prodrug is administered systemically. 25. The method according to paragraphs. 15-23, wherein said interferon prodrug is administered intratumorally, either locally to the tumor or regionally to the tumor. 26. The method according to paragraphs. 15-25, wherein the treatment comprises one or more of: slowing tumor growth, arresting tumor growth, reducing tumor size or volume, increasing survival compared to an untreated patient, inducing cancer remission, inducing tumor cell apoptosis, or inducing tumor necrosis .

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