Fusion protein composition comprising targeted masked type i interferons (ifna and ifnb) and antibody against tumor antigen, for use in treatment of cancer

Abstract

To provide compositions and kits for use in new methods of treating cancer utilizing a masked IFN that inhibits the activity of IFN until it reaches the tumor, in view of current deficiencies associated with delivering IFN to a cancer cell.SOLUTION: A composition comprises a specific polypeptide sequence, wherein the polypeptide sequence masks the activity of a Type-I interferon (IFN), and wherein the composition further comprises a fusion protein in which the polypeptide sequence is fused to an antibody that binds to a tumor associated antigen.SELECTED DRAWING: Figure 26

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 920,140, filed on April 15, 2019, the content of which is hereby incorporated by reference in its entirety.

[0002] Submission of Sequence Listing in ASCII Text File The content of the following submission in ASCII text file is hereby incorporated by reference in its entirety: Sequence Listing in computer - readable format (CRF) (file name: 165802000140.txt, recording date: April 14, 2020, size: 24.7 KB).

[0003] Description of Rights in Inventions Made Under Federally Sponsored Research Not applicable.

[0004] Field of the Invention The present invention described herein relates to the fields of cancer therapies and therapies for other immunological disorders or diseases. Specifically, the present invention relates to masked type I interferon (IFN) compositions that can be fused to tumor - antigen - binding proteins and used as vehicles for targeted cancer therapy in humans. The present invention further relates to the treatment of disorders or diseases such as cancer and other immunological disorders and diseases.

Background Art

[0005] Background of the Invention Cancer is the second most common cause of death globally after coronary artery disease. Millions of people die from cancer every year, and in the United States alone, cancer kills more than half a million people annually, with 1,688,780 new cancer cases diagnosed in 2017 (American Cancer Society). Deaths from heart disease have been significantly declining, while deaths from cancer are generally on the rise. Unless medical advancements change current trends, cancer is predicted to become the leading cause of death early in this century.

[0006] Some cancers stand out as having a high mortality rate. Hematological malignancies, including multiple myeloma, result in 50,000 deaths annually in the United States alone (American Cancer Society, 2018). Additionally, lung cancer (18.4% of all cancer deaths), breast cancer (6.6% of all cancer deaths), colorectal cancer (9.2% of all cancer deaths), liver cancer (8.2% of all cancer deaths), and stomach cancer (8.2% of all cancer deaths) are among the leading causes of cancer death for both genders of all ages worldwide (GLOBOCAN 2018). These and virtually all other cancer tumors metastasize to sites distant from the primary tumor and, with very few exceptions, share the common lethal feature that metastatic disease is fatal. Furthermore, even for cancer patients who initially survive their primary cancer, a common experience of dramatic life changes is shown. Many cancer patients experience intense anxiety stemming from the awareness of the possibility of recurrence or treatment failure. Many cancer patients also experience physical debilitation after treatment. Additionally, many cancer patients experience recurrence of their disease.

[0007] Cancer therapies have improved over the past few decades, and survival rates have been increasing. However, due to the heterogeneous nature of cancer, new treatment strategies that utilize multiple treatment modalities are still needed. This is especially true in the treatment of solid tumors in anatomically difficult locations (e.g., glioblastoma, head and neck squamous cell carcinoma, and lung adenocarcinoma), which are sometimes limited to standard radiotherapy and / or chemotherapy. Nevertheless, the adverse effects of these therapies include severe side effects that reduce the quality of life of patients, as well as chemotherapy resistance and radiotherapy resistance, which promote local recurrence, distant metastasis, and second primary tumors.

[0008] Furthermore, the therapeutic utility of monoclonal antibodies (mAbs) (G. Kohler and C. Milstein, Nature 256:495-497 (1975) (Non-Patent Document 1)) has been realized. Monoclonal antibodies are currently approved as therapies in transplantation, cancer, infectious diseases, cardiovascular diseases, and inflammation. Different isotypes have different effector functions. Such functional differences are reflected in the distinct three-dimensional structures for various immunoglobulin isotypes (P. M. Alzari et al., Annual Rev. Immunol., 6:555-580 (1988) (Non-Patent Document 2)).

[0009] In addition, interferons, including IFNα and IFNβ (type I) and IFNγ (type II), are essential mediators of anti-cancer immunity, having both direct anti-proliferative effects on many cancers and numerous anti-tumor immunotherapy effects. However, although IFNα has shown efficacy against multiple human cancers, its clinical utility has been limited to date because it is unable to achieve effective concentrations of IFN at the tumor site without causing systemic toxicity.

[0010] Due to systemic toxicity, some groups are approaching this problem by using the tumor targeting ability of monoclonal antibodies that deliver IFN directly to the tumor site. See Huang, et al., J. Immunol. 179(10), pp. 6881-6888 (2007) (Non-Patent Document 3) and Vasuthasawat, et. al., J. Immunol. 36(5), pp. 305-318 (2013) (Non-Patent Document 4). It is noted that initial studies have used anti-CD20-IFNα2 protein that targets IFNα to CD20 expressed on lymphoma, and anti-CD138-IFNα2 fusion protein that targets CD138 expressed on multiple myeloma. See Vasuthasawat, et. al., MAbs 8(7), pp. 1386-1397 (2016) (Non-Patent Document 5). These approaches have shown great therapeutic promise and are currently being tested in human clinical trials and commercially developed, but they have some drawbacks.

[0011] It is noted that using the antibody-binding specificity targeting tumor-associated antigens delivers a higher percentage (%) of IFN to the tumor site than is achieved when IFN is injected as is. However, the added interferon is still recognized and bound by interferon receptors expressed throughout the body that are not related to the tumor. Therefore, Mab-fusion IFN can still induce toxicity and / or have increased clearance due to systemic exposure and interaction with interferon receptors throughout the body.

[0012] From the foregoing, it will be readily apparent to those skilled in the art that a new treatment paradigm is needed in the treatment of cancer and immunological diseases.

[0013] In view of the current deficiencies associated with delivering IFN to cancer cells, it is an object of the present invention to provide new and improved methods of treating cancer, immunological disorders, and other diseases by utilizing masked IFN that inhibits the activity of IFN until it reaches the tumor. Compositions, kits, and methods for such uses are provided.

Prior Art Documents

Non-Patent Documents

[0014]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

[0015] The present invention provides antibody, antigen-binding fragment, and fusion protein compositions that bind to the entire range of tumor-associated antigens (TAAs). In a further aspect, the fusion protein comprises type I interferon. In a further aspect, the IFN is masked such that its activity is reduced or ablated until it reaches the tumor cells. In a further aspect, the TAA is shown in Table I. In a preferred aspect, the TAA is associated with solid tumors. In one aspect, the TAA comprises CD138. In a further aspect, the TAA is CD20. In a further aspect, the TAA is mesothelin. In another aspect, the TAA is 5T4. In yet another aspect, the IFN or functionally active variant is shown in Table II. In a preferred aspect, the IFN comprises IFNA2.

[0016] In a further aspect, the present invention comprises a targeted masked IFN. In a preferred aspect, the targeted masked IFN comprises IFNA1.

[0017] In a further aspect, the present invention comprises a targeted masked IFN. In a preferred aspect, the targeted masked IFN comprises IFNA14.

[0018] In a further aspect, the present invention comprises a targeted masked IFN. In a preferred aspect, the targeted masked IFN comprises IFNB1.

[0019] In another aspect, the present disclosure teaches a method of generating a targeted masked IFN.

[0020] In another aspect, the present disclosure teaches a method of treating cancer, immunological disorders, and other diseases in humans.

[0021] In a preferred aspect, the present disclosure teaches a method of treating cancer with a masked IFN fused to a MAb that binds to a TAA.

[0022] In some of any of the aspects, a method for treating cancer comprises administering to a subject, such as a human subject, a therapeutically effective amount of any of the compositions or any of the fusion proteins, such as any of the targeted masked IFNs described herein.

[0023] Also provided is a pharmaceutical composition comprising a therapeutically effective amount of any of the compositions or any of the fusion proteins, such as any of the targeted masked IFNs described herein. In some of any of the aspects, the pharmaceutical composition is for use in a treatment method including the treatment of cancer. In some of any of the aspects, the cancer includes cancer found in solid tumors; or the cancer occurs in the hematopoietic system. In some of any of the aspects, the pharmaceutical composition further comprises one or more anti-neoplastic agents.

[0024] Also provided is a kit, such as a kit comprising any of the compositions or any of the fusion proteins, such as any of the targeted masked IFNs described herein.

Brief Description of the Drawings

[0025]

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Modes for Carrying Out the Invention

[0026] Detailed Description of the Invention Fusion proteins and compositions comprising interferon (IFN) are provided herein. In some aspects, the provided fusion proteins and compositions comprise IFN and an antibody or an antigen-binding fragment thereof, such as an antibody or an antigen-binding fragment thereof specific for a tumor-associated antigen (TAA). In some embodiments, the interferon is a type I IFN. In some aspects, the provided fusion proteins and compositions comprise IFN and a mask, such as a polypeptide sequence that blocks the interaction between IFN and its receptor, such as the IFN-α receptor (IFNAR). In some aspects, the provided fusion protein or composition comprises IFN, an antibody or an antigen-binding fragment thereof, and a mask. In some of any of the provided embodiments, the fusion protein or composition also contains a flexible peptide linker. In some embodiments, the fusion protein or composition also contains a protease cleavage site, such as a tumor-associated protease cleavage site. In some aspects, cleavage of the protease cleavage site, for example, at or near the site of a tumor or in the tumor microenvironment (TME), can result in "unmasking" of the IFN and enable binding of the IFN to its receptor. In some aspects, for example, an antibody or an antigen-binding fragment thereof specific for a TAA can target the fusion protein or composition to a specific site or a tumor or cancer location. Thus, in some aspects, the provided fusion proteins and compositions can be used to treat diseases or disorders such as cancer or tumors. Also provided are methods of making such fusion proteins or compositions, such as methods related to the use of such fusion proteins or compositions in a method of treatment or therapy, and pharmaceutical compositions or kits comprising such fusion proteins or compositions.

[0027] As further described herein, the provided embodiments that include a target-directed masked IFN include a masked IFN whose activity is significantly reduced and / or eliminated until it reaches the tumor site, such that non-specific activity is minimized and the fusion protein is not captured by interferon receptors that are not at the tumor site, and which, for several reasons, provides unique advantages over available approaches. At the tumor site, the mask can be removed and the binding and activity of the IFN reactivated, which can increase the effective concentration of the fusion protein without increasing toxicity and while maximizing efficacy in the tumor. Additionally, the antibody moiety attached to the IFN can be used to target the fusion protein to a specific tumor (e.g., a tumor expressing a TAA to which the antibody specifically binds). Specific targeting allows for a greater chance that the IFN will be directed to the cancer of interest and avoid non-cancerous or non-tumorous tissues.

[0028] Overview of the section I.) Definitions II.) Antibodies III.) Interferons IV.) Methods of Masking IFN a. Discussion of Available Approaches b. Methods of Masking the IFN of the Present Disclosure V.) Treatment of Cancers Expressing Tumor-Associated Antigens (TAAs) VI.) Target-Directed Masked IFN Fusion Protein Cocktails VII.) Combinatorial Therapies VIII.) Kits / Articles of Manufacture

[0029] All publications, including patent documents, scientific papers, and databases, referred to in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were specifically incorporated by reference. In case the definitions set forth herein are contrary to or otherwise inconsistent with the definitions set forth in patents, applications, published applications, and other publications incorporated by reference herein, the definitions set forth herein shall control.

[0030] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0031] I.) Definitions: Unless otherwise defined, all technical field terms, notations, and other scientific terms or terminology used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. In some cases, terms having a commonly understood meaning may be defined herein for clarity and / or ready reference, and the inclusion of such definitions in this specification should not necessarily be construed as representing a substantial difference from what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood by those of ordinary skill in the art and are generally used using conventional methodologies such as, for example, the widely utilized molecular cloning methodology described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Procedures involving the use of commercially available kits and reagents are generally carried out in accordance with the protocols and / or parameters defined by the manufacturer, unless otherwise noted.

[0032] When a trade name is used in this specification, unless otherwise indicated by context, reference to the trade name also refers to the product formulation of the trade name product, generic drugs, and pharmaceutically active ingredients.

[0033] The terms "advanced cancer", "locally advanced cancer", "advanced disease", and "locally advanced disease" mean cancer that has spread through the relevant tissue capsule and are intended to include stage C disease under the American Urological Association (AUA) system, stage C1 - C2 disease under the Whitmore-Jewett system, and stage T3 - T4 and N+ disease under the TNM (tumor, node, metastasis) system. Generally, surgery is not recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes compared to patients with clinically localized (organ-confined) cancer.

[0034] "Modifying the native glycosylation pattern" is intended herein to mean deleting one or more carbohydrate moieties found in the native antibody sequence (either by removing the underlying glycosylation site or by deleting glycosylation by chemical and / or enzymatic means) and / or adding one or more glycosylation sites not present in the native antibody sequence, and "native glycosylation pattern" refers to the native post-translational glycosylation pattern resulting from a particular combination of the antibody sequence used, cell type, and growth conditions. In addition, this phrase includes qualitative changes in the glycosylation of native proteins, including natural changes in the ratio of the various carbohydrate moieties present.

[0035] The term "analog" refers to a molecule that is structurally similar to another molecule (e.g., a protein related to a TAA) or shares similar or corresponding attributes. For example, an analog of a TAA protein can be specifically bound by an antibody that specifically binds to the TAA or a T cell.

[0036] The term "antibody" is used in the broadest sense unless otherwise clearly indicated. Thus, an "antibody" can be naturally occurring or can be artificial, such as a monoclonal antibody produced by conventional hybridoma or transgenic mouse technology. Antibodies include monoclonal and polyclonal antibodies, as well as fragments containing the antigen-binding domains and / or one or more complementarity-determining regions of these antibodies. As used herein, the term "antibody" refers to any form of antibody or fragment thereof that specifically binds to a TAA and / or exhibits a desired biological activity, specifically including monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they specifically bind to a TAA and / or exhibit a desired biological activity. Any specific antibody can be used in the methods and compositions provided herein. Thus, in one aspect, the term "antibody" encompasses a molecule comprising at least one variable region derived from a light chain immunoglobulin molecule and at least one variable region derived from a heavy chain molecule that together form a specific binding site for a target antigen. In one aspect, the antibody is an IgG antibody. For example, the antibody can be an IgG1, IgG2, IgG3, IgG4 antibody, or any known antibody isotype. Antibodies useful in the present methods and compositions can be produced in cell culture, in phage, or in various animals including, but not limited to, cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, and apes. Thus, in one aspect, the antibodies of the present invention are mammalian antibodies. Phage techniques can be used to isolate primary antibodies or to generate variants having altered specificity or binding affinity characteristics. Such techniques are routine and well known in the art. In one aspect, the antibody is produced by recombinant means known in the art. For example, a recombinant antibody can be produced by transfecting a host cell with a vector containing a DNA sequence encoding the antibody.One or more vectors can be used to transfect a host cell with a DNA sequence that expresses at least one VL region and at least one VH region. Exemplary descriptions of recombinant means of antibody generation and production include Delves, ANTIBODY PRODUCTION: ESSENTIAL TECHNIQUES (Wiley, 1997); Shephard, et al., MONOCLONAL ANTIBODIES (Oxford University Press, 2000); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (Academic Press, 1993); and CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons, latest edition). The antibodies of the present invention can be modified by recombinant means to increase the efficacy of the antibody in mediating the desired function. Thus, it is also within the scope of the present invention that the antibody can be modified by substitution using recombinant means. Typically, the substitution will be a conservative substitution. For example, at least one amino acid in the constant region of the antibody can be replaced with a different residue. See, for example, U.S. Patent No. 5,624,821, U.S. Patent No. 6,194,551, Application No. WO 9958572; and Angal, et al., Mol. Immunol. 30: 105-08 (1993). Amino acid modifications include amino acid deletions, additions, and substitutions. In some cases, such changes are made to reduce undesirable activities, such as complement-dependent cytotoxic activity. Frequently, antibodies are labeled by covalently or non-covalently linking a substance that provides a detectable signal. A wide variety of labels and conjugation techniques are known and widely reported in both the chemical and patent literature. These antibodies can be screened for binding to normal or defective TAAs. See, for example, ANTIBODY ENGINEERING: A PRACTICAL APPROACH (Oxford University Press, 1996).Subsequent in vitro assays, including but not limited to proliferation, migration, adhesion, soft agar proliferation, angiogenesis, cell-cell communication, apoptosis, transport, and signal transduction, and subsequent in vivo assays such as inhibition of tumor growth, can be used to identify suitable antibodies having the desired biological activity. The antibodies provided herein may also be useful in diagnostic applications. They can be screened for their ability to bind specifically to the antigen without inhibiting antigen receptor binding or biological activity, as capture antibodies or non-neutralizing antibodies. As neutralizing antibodies, the antibodies can be useful in competitive binding assays. They can also be used to quantify the TAA or its receptor.

[0037] The term "antigen-binding fragment" or "antibody fragment" (or simply "antibody portion") of an antibody, as used herein, refers to one or more fragments of a TAA antibody that retain the ability to specifically bind to a TAA antigen (e.g., CD138, CD20, mesothelin, 5T4, and variants thereof; see also Table I). It has been shown that the antigen-binding function of an antibody can be performed by fragments of the full-length antibody. Examples of binding fragments included within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, which is a monovalent fragment consisting of the V L 、V H 、C L 、and C H1 domains; (ii) an F(ab') 2 fragment, which is a bivalent fragment comprising two Fab fragments linked by disulfide bridges in the hinge region; (iii) an Fd fragment consisting of the V H and C H1 domains; (iv) an Fv fragment consisting of the V L and V H domains of a single arm of the antibody; (v) a dAb fragment consisting of the V H domain (Ward et al., (1989) Nature 341:544-546); and (vi) isolated complementarity-determining regions (CDRs). Further, the two domains of the Fv fragment, V L and V HAlthough encoded by separate genes, they can be linked by a synthetic linker that, using recombinant methods, enables the V L region and the V H region to pair and form a single protein chain that forms a monovalent molecule (known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be included within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0038] The term "Fc", as used herein, refers to a region that includes the hinge region, CH2, and / or CH3 domains.

[0039] As used herein, an "antigen" in any form can be used to generate an antibody specific for a TAA. Thus, an eliciting antigen can be a single epitope, multiple epitopes, or the entire protein, alone or in combination with one or more immunogenic enhancers known in the art. The eliciting antigen can be an isolated full-length protein, a cell surface protein (e.g., immunizing cells transfected with at least a portion of the antigen), or a soluble protein (e.g., immunizing only the extracellular domain portion of the protein). The antigen can be produced in genetically modified cells. The DNA encoding the antigen can be genomic or non-genomic (e.g., cDNA) and can encode at least a portion of an extracellular or intracellular domain. As used herein, the term "portion" in the context of an antigen refers, as appropriate, to the minimum number of amino acids or nucleic acids necessary to constitute an immunogenic epitope of the antigen of interest. Any genetic vector suitable for transformation of cells of interest, including but not limited to adenoviral vectors, plasmids, and non-viral vectors such as cationic lipids, can be used. In one embodiment, the antibodies of the methods and compositions herein specifically bind to at least a portion of the extracellular domain of a TAA of interest.

[0040] The antibodies or antigen-binding fragments thereof provided herein can constitute or be part of a "bioactive agent". As used herein, the term "bioactive agent" refers to any synthetic or naturally occurring compound that binds to an antigen and enhances or mediates the desired biological effect of enhancing and / or potentiating a cell-killing toxin. In one embodiment, the binding fragments useful in the present invention are biologically active fragments. As used herein, the term "biologically active" refers to an antibody or antibody fragment that can bind to a desired antigen epitope and exert a biological effect either directly or indirectly. Direct effects include, but are not limited to, modulation, stimulation, and / or inhibition of growth signals, modulation, stimulation, and / or inhibition of anti-apoptosis signals, modulation, stimulation, and / or inhibition of apoptosis or necrosis signals, modulation, stimulation, and / or inhibition of the ADCC cascade, and modulation, stimulation, and / or inhibition of the CDC cascade.

[0041] As used herein, the term "conservative substitution" is known to those of skill in the art and generally refers to substitutions of amino acids and / or amino acid sequences that can be made without altering the biological activity of the resulting molecule. Those of skill in the art generally recognize that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, e.g., Watson, et al., MOLECULAR BIOLOGY OF THE GENE, The Benjamin / Cummings Pub. Co., p. 224 (4th Edition 1987)). Such exemplary substitutions are preferably made according to the amino acids shown in Table III. For example, such changes include substituting any of these hydrophobic amino acids with any other of isoleucine (I), valine (V), and leucine (L); substituting glutamic acid (E) with aspartic acid (D) and vice versa; substituting asparagine (N) with glutamine (Q) and vice versa; and substituting threonine (T) with serine (S) and vice versa. Other substitutions can also be considered conservative depending on the environment and role of a particular amino acid in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V). The relatively hydrophobic methionine (M) can frequently be exchanged with leucine and isoleucine, and sometimes valine. Lysine (K) and arginine (R) are frequently interchangeable where the significant feature of the amino acid residue is its charge and the different pKs of these two amino acid residues are not significant. Still other changes can be considered "conservative" in a particular environment (see, e.g., Table III herein; pages 13-15 of "Biochemistry" 2nd ED. Lubert Stryer ed. (Stanford University); Henikoff et al., PNAS 1992 Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19; 270(20):11882-6).Other substitutions are also acceptable and can be determined empirically or according to known conservative substitutions.

[0042] As used herein, the term "fusion protein" means a protein of the present invention that is fused to the IFN of the present invention at the C-terminus using linkers and methods known in the art. See, for example, U.S. Patent No. 9,803,021, which is incorporated herein by reference. Exemplary linkers that can be used to fuse IFN to the protein of the present invention include TIFF2025084914000002.tif12162; Landar; Double Landar; 1qo0E_1; IgG3 hinge; IgG3 hingeΔcys; and / or IgG1 hingeΔcys, but are not limited thereto.

[0043] As used herein, the term "inhibit" or "inhibiting" means to reduce by a measurable amount or to block altogether.

[0044] As used herein, the term "interferon" means a group of signaling proteins made and released by host cells in response to the presence of several viruses. In a typical scenario, virus-infected cells are thought to release interferon to cause nearby cells to enhance their antiviral defenses. IFN belongs to a large class of proteins known as cytokines, which are molecules used in cell-to-cell communication to trigger the protective defenses of the immune system that help eradicate pathogens.

[0045] As used herein, the terms "type I interferon" or "IFN-I" refer to a large subgroup of interferon proteins that help regulate the activity of the immune system. All type I IFNs bind to a specific cell surface receptor complex known as the IFN-α receptor (IFNAR), which consists of the IFNAR1 chain and the IFNAR2 chain. An exemplary list of type I interferons of the present disclosure is shown in Table II.

[0046] The term "mammal" refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses, and humans. In one aspect of the invention, the mammal is a mouse. In another aspect of the invention, the mammal is a human.

[0047] The term "mask" when referring to a masked IFN (also denoted as "masked" IFN) means, for the purposes of the present invention, any peptide or protein that blocks the cytokine interaction and / or activation of IFNAR. It is within the scope of the present invention that the "mask" can be modified by substitution using recombinant means. Amino acid modifications include amino acid deletions, additions, and substitutions.

[0048] As used herein, the term "target-directed masked IFN" means a type I interferon in which a polypeptide is added to the carboxy terminus of the IFN, thereby reducing its ability to bind to IFNAR. The masked IFN further includes the addition of a target-directed binding protein (i.e., an antibody) to the carboxy terminus. It is within the scope of the present invention that the "target-directed masked IFN" can be modified by substitution using recombinant means. Amino acid modifications include amino acid deletions, additions, and substitutions.

[0049] The terms "metastatic cancer" and "metastatic disease" mean cancer that has spread to regional lymph nodes or to distant sites, and are intended to include stage D disease under the AUA system and stage T×N×M+ under the TNM system.

[0050] "Molecular recognition" means a chemical event in which a host molecule can form a complex with a second molecule (i.e., a guest). This process occurs through non-covalent chemical bonds including, but not limited to, hydrogen bonds, hydrophobic interactions, and ionic interactions.

[0051] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically contain multiple antibodies that are directed against (or specific for) different epitopes. In one embodiment, a polyclonal antibody contains multiple monoclonal antibodies having different epitope specificities, affinities, or binding strengths within a single antigen containing multiple antigenic epitopes. The modifier "monoclonal" indicates the characteristics of an antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of an antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature 256: 495 (1975), or by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222: 581-597 (1991). These monoclonal antibodies will typically bind with a Kd of at least about 1 μM, more commonly at least about 300 nM, typically at least about 30 nM, preferably at least about 10 nM, more preferably at least about 3 nM, or better, as measured by conventional ELISA.

[0052] "Pharmaceutically acceptable" refers to compositions that are physiologically compatible with humans or other mammals, being non-toxic, inert, and / or the composition.

[0053] As used herein, the terms "single-chain Fv", "scFv", or "single-chain" antibody refer to an antibody fragment that contains the V H domain and the V L domain, and these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further contains a polypeptide linker between the V H domain and the V L domain, which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun, THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

[0054] As used herein, the terms "specific," "specifically binds," and "binds specifically" refer to the selective binding of an antibody to a target antigen epitope. Under a given set of conditions, the specificity of an antibody for binding can be tested by comparing its binding to a relevant antigen to its binding to an irrelevant antigen or mixture of antigens. An antibody is considered specific if it binds at least 2, 5, 7-fold, and preferably 10-fold more to a relevant antigen than to an irrelevant antigen or mixture of antigens. In one embodiment, a specific antibody binds only to a TAA antigen and not to an irrelevant antigen. In another embodiment, a specific antibody binds to a human TAA antigen but does not bind to a non-human TAA antigen having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more amino acid homology to the TAA antigen. In another embodiment, a specific antibody binds to a human TAA antigen but does not bind to a non-human TAA antigen having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more percent identity to the amino acid sequence of the TAA antigen. In another embodiment, a specific antibody binds to a human TAA antigen and to a mouse TAA antigen, but binds more strongly to the human antigen. In another embodiment, a specific antibody binds to a human TAA antigen and to a primate TAA antigen, but binds more strongly to the human antigen. In another embodiment, a specific antibody binds to a human TAA antigen and any non-human TAA antigen, but binds more strongly to the human antigen or any combination thereof.

[0055] As used herein, "treating" or "treatment" and grammatically related terms refer to any improvement in any outcome of a disease, such as long-term survival, lower morbidity, and / or reduction of side effects that are by-products of alternative treatment modalities; although not requiring treatment, complete eradication of the disease is preferred, as will be readily appreciated in the art.

[0056] II.) Antibodies In some embodiments, provided fusion proteins, and compositions, such as a target-directed masked interferon (IFN), comprise an antibody or an antigen-binding fragment thereof. In some of any of the provided embodiments, the antibody binds to, for example specifically binds to, recognizes, and targets an antigen associated with a disease or disorder, such as a cancer or an immunological disorder or disease, such as an antigen that is a tumor-associated antigen (TAA). In some aspects, by virtue of the binding strength to an antigen (e.g., TAA), the provided fusion proteins, such as a target-directed masked IFN, can be targeted to a relevant physical location for treatment, such as a cancer or tumor region. In some aspects, the described antibody or an antigen-binding fragment thereof can be used as a component for any of the provided fusion proteins herein, such as in any of the target-directed IFN, antibody-IFN fusion protein, or target-directed masked IFN provided herein.

[0057] Aspects of the invention provide antibodies that bind to antigens associated with cancer or tumors, such as tumor-associated antigens (TAAs) and proteins associated with TAAs (see Table I). In one embodiment, an antibody that binds to a protein associated with a TAA is an antibody that specifically binds to a TAA protein comprising the amino acids of the protein shown in Table 1. For example, an antibody that binds to a TAA protein comprising one amino acid sequence of the protein shown in Table I can bind to proteins associated with the TAA, such as TAA variants and their homologs or analogs.

[0058] In some aspects, antibodies that bind to a tumor-associated antigen (TAA) or a protein related to a TAA, such as the anti-TAA antibodies of the provided embodiments, are particularly useful in cancer for prognostic assays, imaging, diagnosis, and treatment methodologies. In some aspects, the antibodies of the provided embodiments are therapeutic antibodies that specifically bind to a TAA, such as a TAA shown in Table I. Similarly, such antibodies are useful in the treatment and / or prognosis determination of cancers, such as ovarian cancer, head and neck cancer, multiple myeloma, and other cancers, to the extent that these other cancers also express or overexpress the TAA, for example, when combined with a therapeutic agent, in a fusion protein. Further, the antibodies of the provided embodiments, including antibodies expressed intracellularly (e.g., single-chain antibodies), are therapeutically useful in the treatment of cancers in which TAA expression is involved, such as progressive or metastatic cancer in solid tumors, or other progressive or metastatic cancers. In one aspect, the TAA-binding assays disclosed herein are for use in the detection of cancer, for example, in immunoassays.

[0059] In some embodiments, the provided fusion proteins or compositions include an antibody that binds to a tumor-associated antigen (TAA), such as a TAA selected from the exemplary TAAs shown in Table I. In some embodiments, the TAA is an antigen expressed on the surface of a tumor, such as on the surface of a tumor cell or cancer cell. In some embodiments, the TAA includes any antigen associated with any of the diseases or conditions described herein, such as any cancer described herein. In some embodiments, the TAA is an antigen expressed on the surface of a tumor-related cell, such as a cell present in the tumor microenvironment (TME). In some embodiments, the TAA is an antigen present in the TME.

[0060] In some embodiments, the provided fusion protein or composition comprises an antibody that binds to a tumor - associated antigen associated with tumors that occur in the hematopoietic system, such as hematological malignancies. In some embodiments, the provided fusion protein or composition comprises an antibody that binds to the CD138 antigen. In some embodiments, the antibody binds to, for example specifically binds to, one of the TAAs shown in Table I.

[0061] In some embodiments, the antibody comprises the fusion protein or is included in the fusion protein. In some embodiments, the antibody is included in any of the fusion proteins or compositions provided herein. In some embodiments, the antibody comprises a fusion protein that comprises a type I IFN, such as the type I IFN shown in Table II. In some embodiments, the antibody comprises a fusion protein that further comprises a target - directed IFN - α. In some embodiments, the antibody comprises a fusion protein that further comprises a target - directed masked IFN - α.

[0062] In some aspects, the antibody comprises an antibody fragment, such as an antigen - binding antibody fragment. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab') 2 fragments, single - chain antibody molecules, such as single - chain Fv proteins ("scFv"), disulfide - stabilized Fv proteins ("dsFv"), Fv, Fab'-SH, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0063] A variety of methods for the preparation of antibodies, such as monoclonal antibodies, are well known in the art. For example, antibodies can be prepared by immunizing a suitable mammalian host with a protein, peptide, or fragment related to the TAA, either in isolated form or in an immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). In addition, fusion proteins of TAAs, such as TAA GST fusion proteins, can also be used. In certain embodiments, a GST fusion protein containing all or most of the amino acid sequence of FIG. 1 is produced and then used as an immunogen for generating appropriate antibodies. In another embodiment, a protein related to the TAA is synthesized and used as an immunogen.

[0064] In addition, naked DNA immunization techniques known in the art are used (with or without a protein related to the purified TAA or TAA-expressing cells) to generate an immune response against the encoded immunogen (see, for a review, Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).

[0065] The amino acid sequence of the TAA protein shown in Table I can be analyzed to select specific regions of the TAA protein as, for example, immunogens or epitopes for generating antibodies. For example, hydrophilic regions in the TAA structure can be identified using an analysis of the hydrophobicity and hydrophilicity of the TAA amino acid sequence. Regions of the TAA protein that exhibit immunogenic structures, as well as other regions and domains, can be readily identified using various other methods known in the art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz, or Jameson-Wolf analysis. Hydrophilic profiles can be generated using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydrophobicity profiles can be generated using the method of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol. 157:105-132. The percentage of accessible residues profile can be generated using the method of Janin J., 1979, Nature 277:491-492. The average mobility profile can be generated using the method of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res. 32:242-255. The β-turn profile can be generated using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294. Thus, each region identified by any of these programs or methods is within the scope of the present invention. Preferred methods for generating TAA antibodies are further illustrated by the examples provided herein. Methods for preparing proteins or polypeptides for use as immunogens are well known in the art. Also, methods for preparing immunogenic conjugates of proteins with carriers such as BSA, KLH, or other carrier proteins are well known in the art.In some situations, for example, direct conjugation using a carbodiimide reagent is employed; in other instances, a linking reagent such as those supplied by Pierce Chemical Co., Rockford, Ill. is effective. Administration of the TAA immunogen is often carried out by injection over a suitable period and with the use of a suitable adjuvant, as understood in the art. During the immunization schedule, the antibody titer can be obtained to determine the validity of antibody formation.

[0066] TAA monoclonal antibodies can be produced by various means well known in the art. For example, an immortalized cell line secreting the desired monoclonal antibody is prepared using the standard hybridoma technique of Kohler and Milstein or a modified version that immortalizes antibody-producing B cells, as generally known. The immortalized cell line secreting the desired antibody is screened by an immunoassay in which the antigen is a protein related to TAA. If a suitable immortalized cell culture is identified, the cells can be expanded and the antibody can be produced either from in vitro cultures or from ascites.

[0067] The antibodies or fragments of the present invention can also be produced by recombinant means. Regions that specifically bind to a desired region of a TAA protein can also be produced in the context of chimeric antibodies or complementarity-determining region (CDR)-grafted antibodies of multiple species origin. Humanized TAA antibodies or human TAA antibodies can also be produced and are preferred for use in therapeutic settings. Methods for humanizing mouse antibodies and other non-human antibodies by substituting one or more of the CDRs of the non-human antibody with the corresponding human antibody sequences are well known (see, for example, Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen et al., 1988, Science 239: 1534-1536). See also Carter et al., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151: 2296.

[0068] In one aspect, the human monoclonal antibodies of the present invention can be prepared using VelocImmune mice (Regeneron, Tarrytown, N.Y.) in which genomic sequences having mouse variable segments endogenous to the immunoglobulin heavy chain (VH, DH, and JH segments) and / or kappa light chain (VK and JK) loci are replaced, in whole or in part, with human genomic sequences having unrearranged germline variable segments of the human immunoglobulin heavy chain (VH, DH, and JH) and / or kappa light chain (VK and JK) loci. See, for example, U.S. Patent Nos. 6,586,251, 6,596,541, 7,105,348, 6,528,313, 6,638,768, and 6,528,314.

[0069] In addition, the human antibodies of the present invention can be generated using HuMAb mice (Medarex, Inc.) that contain human immunoglobulin gene miniloci encoding unrearranged human heavy chains (μ and γ) as well as κ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and κ chain loci (see, e.g., Lonberg, et al. (1994) Nature 368(6474): 856-859).

[0070] In another aspect, the fully human antibodies of the present invention can be generated using mice that carry human immunoglobulin sequences on the transgene and the transchromosome, e.g., mice that carry a human heavy chain transgene and a human light chain transchromosome. Such mice are referred to herein as "KM mice" and are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727 and PCT Publication WO 02 / 43478 of Tomizuka, et al.

[0071] The human monoclonal antibodies of the present invention can also be prepared using phage display methods to screen libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies are well established in the art. See, for example, U.S. Patent Nos. 5,223,409; 5,403,484; as well as U.S. Patent Nos. 5,571,698 of Ladner et al.; 5,427,908 and 5,580,717 of Dower et al.; 5,969,108 and 6,172,197 of McCafferty et al.; and 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 of Griffiths et al.

[0072] The human monoclonal antibodies of the present invention can also be prepared using SCID mice in which human immune cells are reconstituted therein so that a human antibody response can be generated upon immunization. Such mice are described, for example, in U.S. Pat. Nos. 5,476,996 and 5,698,767 to Wilson, et al.

[0073] Additionally, the human antibodies of the present invention can be made by a technique using transgenic mice called Xenomouse (Amgen Fremont, Inc., formerly Abgenix, Inc.) that are inactivated for antibody production and engineered at the human heavy and light chain loci. An exemplary description of preparing transgenic mice that produce human antibodies can be found in U.S. Pat. No. 6,657,103. See also U.S. Pat. Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,545,806; as well as Mendez, et. al. Nature Genetics, 15: 146-156 (1998); Kellerman, S. A. & Green, L. L., Curr. Opin. Biotechnol 13, 593-597 (2002).

[0074] Any of the above production methods results in an antibody having a specific ability to bind to a TAA, or a homolog or fragment or polypeptide sequence having 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the TAA. The binding affinity (K D ) of the antibody, its binding fragment, and the antibody-drug conjugate containing it to the TAA can be 1 mM or less, 100 nM or less, 10 nM or less, 2 nM or less, or 1 nM or less. Alternatively, K D can be 5-10 nM; or 1-2 nM. K D can be 1 μM - 500 μM or 500 μM - 1 nM.

[0075] The binding affinity of an antigen-binding protein is determined by the association constant (Ka) and the dissociation constant (Kd) (KD = Kd / Ka). The binding affinity can be measured, for example, by BIACORE by capturing the test antibody on a sensor surface coated with protein A and flowing TAA over this surface. Alternatively, the binding affinity can be measured, for example, by FORTEBIO by capturing the test antibody receptor on a needle coated with protein A and flowing TAA over this surface. A person skilled in the art can identify other suitable assays known in the art for measuring the binding affinity.

[0076] As used herein with respect to TAA antigen binding, the term "specifically binds to" a protein means that the antigen-binding protein binds to TAA, and to discrete domains or discrete amino acid sequences within TAA, and has no binding or has insignificant binding to other (e.g., unrelated) proteins. However, this term does not exclude the fact that an antibody or its binding fragment may also be cross-reactive with closely related molecules. The antibodies and their fragments described herein, and fusion proteins containing them, can specifically bind to TAA with an affinity at least 2, 5, 10, 50, 100, or 1000 times higher than they bind to closely related molecules.

[0077] In one aspect, the invention includes an antibody that binds to a tumor-associated antigen (TAA).

[0078] In another aspect, the invention includes an antibody that binds to a tumor-associated antigen (TAA) associated with a solid cancer tumor.

[0079] In another aspect, the invention includes an antibody that binds to a tumor-associated antigen associated with a tumor that occurs in the hematopoietic system.

[0080] In another aspect, the invention includes an antibody that binds to the CD138 antigen.

[0081] In another aspect, the invention includes an antibody that binds to the CD20 antigen.

[0082] In another aspect, the invention includes an antibody that binds to the mesothelin antigen.

[0083] In another aspect, the invention includes an antibody that binds to the 5T4 antigen.

[0084] In another aspect, the invention includes an antibody that binds to the PSCA antigen.

[0085] In another aspect, the antibody includes a fusion protein.

[0086] In another aspect, the antibody includes a fusion protein that includes type 1 IFN shown in Table II.

[0087] In another aspect, the antibody includes a fusion protein that further includes a target-directed IFN-α.

[0088] In another aspect, the antibody includes a fusion protein that further includes a target-directed masked IFN-α.

[0089] In another aspect, the invention includes an antibody that binds to CD138 and further includes a heavy chain having the following sequence. TIFF2025084914000003.tif56147

[0090] In another aspect, the invention includes an antibody that binds to CD138 and further includes a heavy chain having the following sequence. TIFF2025084914000004.tif55147

[0091] III.) Interferons In some embodiments, provided fusion proteins and compositions that are target-directed interferons (IFNs), such as target-directed masked IFNs, include a component that is an interferon (IFN) or a variant thereof. In some aspects, provided are also target-directed masked IFNs, such as an antibody or a fragment or chain thereof, and a type I IFN fused to an "interferon mask".

[0092] In some embodiments, provided are fusion proteins, such as antibody-IFN fusion proteins or target-directed IFNs, that include an interferon (IFN) or a variant thereof and an antibody or an antigen-binding fragment thereof that specifically binds to a tumor-associated antigen (TAA). In some aspects, the IFN is any type of IFN described herein. In certain aspects, examples of IFNs in the provided embodiments include type I IFNs, including any known type I IFN and any described herein, such as those shown in Table II. Exemplary antibodies in the fusion protein include any described herein, such as in Section II or Table I. In some of any of the embodiments, provided is an interferon (IFN) or a variant thereof that is added to or connected to a "mask", optionally also referred to as an interferon mask, such as a peptide or protein that blocks cytokine interaction and / or activation of the interferon alpha receptor (IFNAR). In some aspects, a masked IFN is provided. In some aspects, examples of IFNs in the provided masked IFNs include type I IFNs, including any known type I IFN and any described herein, such as those shown in Table II. Exemplary masks, and methods for masking interferons, include any described herein, such as in Section IV. In some embodiments, the antibody-IFN fusion protein includes a "masked" IFN that includes an IFN component selected from the IFNs in Table II.

[0093] In some situations, IFN is a protein used for the treatment or management of a disease or disorder. In some situations, the provided fusion proteins and compositions can be effective in the treatment of diseases or disorders such as cancer or tumors, and / or can be used to increase the effectiveness of therapeutic agents such as anti-cancer or anti-neoplastic agents, and contain IFN components.

[0094] IFN is a group of signaling proteins that are made and released by cells of a subject or host, such as host cells, in response to the presence of foreign entities in the body, such as pathogens including some viruses. In a typical scenario, cells infected with a virus are thought to release interferon to cause nearby cells to enhance their antiviral defenses.

[0095] IFN belongs to a large class of proteins known as cytokines, which are molecules used in cell - to - cell communication to trigger the protective defenses of the immune system that help eradicate pathogens. Interferons are named for their ability to "interfere" with viral replication by protecting cells from viral infections. In some situations, IFNs also have various other functions: (i) they activate immune cells such as natural killer cells and macrophages; and (ii) they increase host defense by upregulating antigen presentation by increasing the expression of major histocompatibility complex (MHC) antigens.

[0096] More than 20 distinct IFN genes and proteins have been identified in animals, including humans. They are typically divided into three classes: type I IFN, type II IFN, and type III IFN. IFNs belonging to all three classes are important for fighting viral infections and for the control of the immune system.

[0097] Type I interferons:All type I IFNs bind to a specific cell surface receptor complex known as the IFN-α / β receptor (IFNAR), which consists of the IFNAR1 chain and the IFNAR2 chain. Thirteen types of type I interferons, including but not limited to IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω, exist in humans. Generally, type I interferons are produced when the body recognizes an invading virus. They are produced by fibroblasts and monocytes. However, the production of type I IFN-α is inhibited by another cytokine known as interleukin-10. Once released, type I interferons bind to specific receptors on target cells, which results in the expression of proteins that prevent the virus from producing and replicating its RNA and DNA.

[0098] Type II interferon (IFN-γ in humans) :It is also known as immune interferon and is activated by interleukin-12. Furthermore, type II interferons are released by cytotoxic T cells, and specifically type 1 T helper cells. However, they block the proliferation of type 2 T helper cells. The former results in the inhibition of the T h 2 immune response and the further induction of the T h 1 immune response, which leads to the development of debilitating diseases such as multiple sclerosis. Type II IFNs bind to IFNGR, which consists of the IFNGR1 chain and the IFNGR2 chain.

[0099] Type III interferons :Signals through a receptor complex consisting of IL10R2 (also called CRF2-4) and IFNLR1 (also called CRF2-12). Recent information demonstrates the importance of type III IFNs in several types of viral or fungal infections.

[0100] Generally, type I and type II interferons are responsible for controlling and activating the immune response. The expression of type I and type III IFNs can be induced in substantially all cell types upon recognition of viral components, particularly nucleic acids, by cytoplasmic and endosomal receptors, whereas type II interferon is induced by cytokines such as IL-12 and its expression is restricted to immune cells such as T cells and NK cells.

[0101] In some aspects, interferons and proteins containing or derived from interferons can be used as therapeutic agents. In some of any of the provided aspects, the IFN component of a fusion protein, e.g., a targeted masked IFN, is used as a therapeutic agent for the treatment of diseases or disorders such as cancer.

[0102] In some aspects, interferon therapy is used (in combination with chemotherapy and radiation) as a treatment for some cancers. This treatment can be used in leukemias and lymphomas, including hematological malignancies; hairy cell leukemia, chronic myelogenous leukemia, nodular lymphoma, and cutaneous T cell lymphoma. In some examples, patients with recurrent melanoma receive recombinant IFN-α2b.

[0103] A major limitation in using IFN available in cancer therapies is the inability to achieve an effective concentration of IFN at the tumor site without causing systemic toxicity. To overcome this limitation, several attempts have been made to solve this problem by using the tumor targeting ability of monoclonal antibodies that directly deliver IFN to the tumor site. See Huang, et al., J. Immunol. 179(10), pp. 6881-6888 (2007) and Vasuthasawat, et. al., J. Immunol. 36(5), pp. 305-318 (2013). Initial studies are noted to have used anti-CD20-IFNα2 protein that targets IFNα to CD20 expressed on lymphoma, and anti-CD138-IFNα2 fusion protein that targets CD138 expressed on multiple myeloma. See Vasuthasawat, et. al., MAbs 8(7), pp. 1386-1397 (2016). These approaches have shown great therapeutic promise and are currently being tested in human clinical trials and commercially developed, but there are some drawbacks to these available approaches.

[0104] Using the antibody binding specificity targeting tumor-associated antigens delivers a higher percentage of IFN to the tumor site than is achieved when IFN is injected as is, but the added interferon can still be recognized and bound by interferon receptors expressed by cells throughout the body, such as cells not associated with the tumor or normal cells. This binding can result in both less IFN reaching the tumor and unwanted off-target toxicity. Aspects are provided that overcome such limitations and drawbacks.

[0105] Accordingly, it is an object of the present invention to overcome these limitations by providing a mechanism that "masks" the function or activity of IFN until IFN reaches a location or region associated with the treatment of a disease or disorder such as a tumor. At that time, IFN is "unmasked" and its activity and function are efficiently switched on. In this way, in some embodiments, a fusion protein that is "unmasked" or "activated" only at a location of interest for a therapeutic effect such as a tumor, for example, a target-directed masked IFN, is provided. In some aspects, masking the function or activity of IFN in the rest of the body, for example, generally in the systemic circulation, reduces or prevents the non-specific activity of IFN and also reduces or prevents a therapeutic agent, such as IFN, from being trapped or taken up by non-cancerous or non-tumor cells in the body. In some aspects, masking the function or activity of IFN and targeting IFN to a location of interest, such as a tumor, can efficiently increase the concentration of a therapeutic agent (e.g., IFN) by preventing the binding and / or capture of IFN by the force of an antibody present in the provided embodiment, for example, by specific targeting of the agent by the antibody.

[0106] Accordingly, in some embodiments, the present invention includes an antibody-IFN fusion protein in which IFN selectively binds to an IFN receptor once it reaches a tumor.

[0107] In another embodiment, the antibody-IFN fusion protein includes IFN separated by a peptide linker that is a site for proteolytic cleavage.

[0108] In another embodiment, the antibody-IFN fusion protein includes "masked" IFN.

[0109] In another embodiment, the antibody-IFN fusion protein includes "masked" IFN separated by a peptide linker that is a site for proteolytic cleavage.

[0110] In another aspect, the antibody-IFN fusion protein comprises "masked" IFNA1.

[0111] In another aspect, the antibody-IFN fusion protein comprises "masked" IFNA1 separated by a peptide linker which is a site for proteolytic cleavage.

[0112] In another aspect, the antibody-IFN fusion protein comprises "masked" IFNA2.

[0113] In another aspect, the antibody-IFN fusion protein comprises "masked" IFNA2 separated by a peptide linker which is a site for proteolytic cleavage.

[0114] In another aspect, the antibody-IFN fusion protein comprises "masked" IFNB1.

[0115] In another aspect, the antibody-IFN fusion protein comprises "masked" IFNB1 separated by a peptide linker which is a site for proteolytic cleavage.

[0116] In another aspect, the antibody-IFN fusion protein comprises "masked" IFN selected from the IFN shown in Table II.

[0117] In another aspect, the antibody-IFN fusion protein comprises "masked" IFN separated by a peptide linker which is a site for proteolytic cleavage and selected from the IFN shown in Table II.

[0118] In another aspect, the antibody-IFN fusion protein comprises IFNα2 comprising the following. TIFF2025084914000005.tif19147

[0119] IV.) Methods for masking IFN As previously mentioned, it is an object of the present invention to provide a targeted, masked IFN composition in which the activity of the IFN of the present invention (see Table II) is inhibited until it reaches the tumor and the mask is removed by proteases such as tumor-associated proteases. In some aspects, the provided masked IFN, such as a targeted, masked IFN, is "unmasked" near or at the site of the disease or disorder to be treated, such as a tumor or near a tumor, e.g., in the tumor microenvironment, and can bind to and / or activate an interferon receptor (e.g., IFNAR). In some aspects, the unmasking or activation of the provided fusion protein occurs by the cleavage power of components such as a peptide linker by a protein in the tumor environment such as a tumor-associated protease.

[0120] (a) Discussion of available approaches The available approaches related to this effort are limited. This disclosure presents an explanation of the available approaches to further demonstrate and show the technical advantages of the present invention. Previous approaches to improving the tumor-specific delivery of therapeutic agents have been to create therapeutic agents activated by tumor-related proteases. An example of this approach is to obtain antibodies that recognize tumor-related antigens but also recognize antigens that are present on normal cells, and modify them so that they bind to the antigen only when localized to the target tumor. See U.S. Patent No. 8,563,269 (CytomX Therapeutics, San Francisco, CA). So-called "Probody" has a related peptide that blocks the antibody-binding site, linked by a linker that is cleavable by proteases present in the tumor microenvironment. In one example, cetuximab, an antibody specific for the epidermal growth factor receptor (EGFR) that is activated to bind only when localized to the tumor, was generated. See Desnoyers, et. al., Sci. Transl. Med., 16:5(207) pp.207ra144 (2013). In this "Probody", a mask sequence that binds to the variable region of cetuximab, followed by the GS linker and then the sequence TIFF2025084914000006.tif5128, was added to the amino terminus of the heavy chain of the antibody. In that case, Underlined the sequence is a substrate for urokinase plasminogen activator (UPA) and matriptase, proteases that are known to be upregulated in various human carcinomas and have minimal activity in normal tissues. This probody demonstrated improved safety and increased half-life in non-human primates.

[0121] In addition to generating Probodies with antibody binding that is activated in the tumor microenvironment, it is possible to generate interferon alpha proproteins that are activated by proteases. See U.S. Patent No. 8,399,219 (CytomX Therapeutics, San Francisco, CA). In that case, the peptide mask TIFF2025084914000007.tif5128 was placed at the amino terminus of a single-chain recombinant IFNα that was separated from IFNα by a cleavable sequence. The resulting construct: TIFF2025084914000008.tif12128IFNα contained IFNα that was selectively activated in the tumor microenvironment. VHMPLGFLGP (SEQ ID NO: 11) is taught to be a substrate for MMP-9.

[0122] (b) Methods for masking IFN of the present disclosure The provided fusion proteins, including masked IFN and target-directed masked IFN, can be unmasked at or near the site of the disease or disorder. From the foregoing, the method of masking the IFN of the present disclosure is clearly distinguishable and offers advantages over any available approach.

[0123] For example, as described above, the provided embodiments include a mechanism that "masks" the function or activity of IFN until IFN reaches a location or region associated with the treatment of a disease or disorder such as a tumor, and specific physical targeting of the fusion protein to the tumor location by the force of a TAA-specific antibody fused to IFN. Accordingly, the fusion proteins described herein may provide a number of advantages including, but not limited to, that IFN is "unmasked" or "activated" only at the location of interest for therapeutic effects such as a tumor; that non-specific activity of IFN is reduced; that IFN is prevented from being captured or taken up by non-cancerous or non-tumor cells in the body; and / or that the concentration of a therapeutic agent (e.g., IFN) is efficiently increased without an accompanying increase in toxicity.

[0124] In some of any of the embodiments, the provided fusion protein, e.g., the target-directed masked IFN, is unmasked only at or near the site of a disease or disorder such as a tumor. Accordingly, in some embodiments, the invention includes an antibody-IFN fusion protein in which IFN selectively binds to an IFN receptor once it reaches the location or region of the tumor.

[0125] In some embodiments, the antibody-IFN fusion protein includes IFN separated by a peptide linker that is a site for proteolytic cleavage. In some aspects, proteolytic cleavage of the peptide linker can, for example, "unmask" or activate IFN at or near the tumor.

[0126] As described in the present disclosure, the provided embodiments include antibody-IFNs in which an IFN, such as those described in Section III or Table II herein, is fused to an antibody as described herein, such as in Section II or Table I. The IFN then becomes active only at the site of the tumor and is "masked" to bind to its receptor. In the uncleaved state, an ideal peptide mask inhibits the binding of a protein (e.g., IFN) to its binding partner (e.g., an interferon receptor such as IFNAR), and after cleavage, the peptide mask does not inhibit the binding of the protein to its binding partner. In the embodiments provided herein, for example, cleavage of the "mask" at or near the site of the tumor allows type I IFN to bind to its receptor, such as IFNAR, and exert its therapeutic effect.

[0127] The following describes exemplary embodiments.

[0128] First, using IFNα2, a "mask" that inhibits protease cleavage sites and IFN binding was placed on the 3' end fused to C H 3. At the site of the tumor, it is contemplated by the present disclosure that proteases within the tumor microenvironment cleave the protease cleavage site to release the mask and free the IFN to bind to its receptor.

[0129] Nucleic acids for constructing a recombinant heavy chain having a protease cleavage site and an IFN inhibitory mask were obtained (ATUM, Newark, California) and used to modify the heavy chain (H chain) of anti-CD138-IFNα2 by creating the following fusion at its 3' end. TIFF2025084914000009.tif12156 The single underline indicates the carboxy terminus of IFNα2, the double underline represents the sequence of the protease cleavage site, and the dotted underline represents the IFNα2 mask. The linker sequence is shown in lowercase.

[0130] In another embodiment, the construct is It includes TIFF2025084914000010.tif12158.

[0131] In one embodiment, the "masked" IFN is It includes TIFF2025084914000011.tif5128.

[0132] In one embodiment, the "masked" IFN is It includes TIFF2025084914000012.tif5128 and further includes IFNα1.

[0133] In one embodiment, the "masked" IFN is It includes TIFF2025084914000013.tif5128 and further includes IFNα2.

[0134] In one embodiment, the "masked" IFN is It includes TIFF2025084914000014.tif5128 and further includes IFNα4.

[0135] In one embodiment, the "masked" IFN is It includes TIFF2025084914000015.tif5128 and further includes IFNα5.

[0136] In one embodiment, the "masked" IFN is It includes TIFF2025084914000016.tif5128 and further includes IFNα1 fused to an antibody that binds to CD138.

[0137] In one embodiment, the "masked" IFN is It includes TIFF2025084914000017.tif5128 and further includes IFNα1 fused to an antibody that binds to CD20.

[0138] In one embodiment, the "masked" IFN is It contains TIFF2025084914000018.tif5128 and further contains IFNα1 fused to an antibody that binds to Her2.

[0139] In one embodiment, the "masked" IFN is It contains TIFF2025084914000019.tif5128 and further contains IFNα1 fused to an antibody that binds to CSPG4.

[0140] In one embodiment, the "masked" IFN is It contains TIFF2025084914000020.tif5128 and further contains IFNα1 fused to an antibody that binds to PSCA.

[0141] In one embodiment, the "masked" IFN is It contains TIFF2025084914000021.tif5128 and further contains IFNα1 fused to an antibody that binds to CEA.

[0142] In one embodiment, the "masked" IFN is It contains TIFF2025084914000022.tif5128 and further contains IFNα1 fused to an antibody that binds to RCC.

[0143] In one embodiment, the "masked" IFN is It contains TIFF2025084914000023.tif5128 and further contains IFNα1 fused to an antibody that binds to 5T4.

[0144] In one embodiment, the "masked" IFN is It contains TIFF2025084914000024.tif5128 and further contains IFNα1 fused to an antibody that binds to mesothelin.

[0145] In one embodiment, the "masked" IFN is It includes TIFF2025084914000025.tif5128 and further includes IFNα2 fused to an antibody that binds to CD138.

[0146] In one embodiment, the "masked" IFN is It includes TIFF2025084914000026.tif5128 and further includes IFNα2 fused to an antibody that binds to CD20.

[0147] In one embodiment, the "masked" IFN is It includes TIFF2025084914000027.tif5128 and further includes IFNα2 fused to an antibody that binds to Her2.

[0148] In one embodiment, the "masked" IFN is It includes TIFF2025084914000028.tif5128 and further includes IFNα2 fused to an antibody that binds to CSPG4.

[0149] In one embodiment, the "masked" IFN is It includes TIFF2025084914000029.tif5128 and further includes IFNα2 fused to an antibody that binds to PSCA.

[0150] In one embodiment, the "masked" IFN is It includes TIFF2025084914000030.tif5128 and further includes IFNα2 fused to an antibody that binds to CEA.

[0151] In one embodiment, the "masked" IFN is It includes TIFF2025084914000031.tif5128 and further includes IFNα2 fused to an antibody that binds to RCC.

[0152] In one embodiment, the "masked" IFN is It contains TIFF2025084914000032.tif5128 and further contains IFNα2 fused to an antibody that binds to 5T4.

[0153] In one embodiment, the "masked" IFN is It contains TIFF2025084914000033.tif5128 and further contains IFNα2 fused to an antibody that binds to mesothelin.

[0154] In one embodiment, the "masked" IFN is It contains TIFF2025084914000034.tif5128.

[0155] In one embodiment, the "masked" IFN is It contains TIFF2025084914000035.tif5128 and further contains IFNα1.

[0156] In one embodiment, the "masked" IFN is It contains TIFF2025084914000036.tif5128 and further contains IFNα2.

[0157] In one embodiment, the "masked" IFN is It contains TIFF2025084914000037.tif5128 and further contains IFNα4.

[0158] In one embodiment, the "masked" IFN is It contains TIFF2025084914000038.tif5128 and further contains IFNα5.

[0159] In one embodiment, the "masked" IFN is It contains TIFF2025084914000039.tif5128 and further contains IFNα1 fused to an antibody that binds to CD138.

[0160] In one embodiment, the "masked" IFN is It includes TIFF2025084914000040.tif5128 and further includes IFNα1 fused to an antibody that binds to CD20.

[0161] In one embodiment, the "masked" IFN is It includes TIFF2025084914000041.tif5128 and further includes IFNα1 fused to an antibody that binds to Her2.

[0162] In one embodiment, the "masked" IFN is It includes TIFF2025084914000042.tif5128 and further includes IFNα1 fused to an antibody that binds to CSPG4.

[0163] In one embodiment, the "masked" IFN is It includes TIFF2025084914000043.tif5128 and further includes IFNα1 fused to an antibody that binds to PSCA.

[0164] In one embodiment, the "masked" IFN is It includes TIFF2025084914000044.tif5128 and further includes IFNα1 fused to an antibody that binds to CEA.

[0165] In one embodiment, the "masked" IFN is It includes TIFF2025084914000045.tif5128 and further includes IFNα1 fused to an antibody that binds to RCC.

[0166] In one embodiment, the "masked" IFN is It includes TIFF2025084914000046.tif5128 and further includes IFNα1 fused to an antibody that binds to 5T4.

[0167] In one embodiment, the "masked" IFN is It contains TIFF2025084914000047.tif5128 and further contains IFNα1 fused to an antibody that binds to mesothelin.

[0168] In one embodiment, the "masked" IFN is It contains TIFF2025084914000048.tif5128 and further contains IFNα2 fused to an antibody that binds to CD138.

[0169] In one embodiment, the "masked" IFN is It contains TIFF2025084914000049.tif5128 and further contains IFNα2 fused to an antibody that binds to CD20.

[0170] In one embodiment, the "masked" IFN is It contains TIFF2025084914000050.tif5128 and further contains IFNα2 fused to an antibody that binds to Her2.

[0171] In one embodiment, the "masked" IFN is It contains TIFF2025084914000051.tif5128 and further contains IFNα2 fused to an antibody that binds to CSPG4.

[0172] In one embodiment, the "masked" IFN is It contains TIFF2025084914000052.tif5128 and further contains IFNα2 fused to an antibody that binds to PSCA.

[0173] In one embodiment, the "masked" IFN is It contains TIFF2025084914000053.tif5128 and further contains IFNα2 fused to an antibody that binds to CEA.

[0174] In one embodiment, the "masked" IFN is It includes TIFF2025084914000054.tif5128 and further includes IFNα2 fused to an antibody that binds to RCC.

[0175] In one embodiment, “masked” IFN is It includes TIFF2025084914000055.tif5128 and further includes IFNα2 fused to an antibody that binds to 5T4.

[0176] In one embodiment, “masked” IFN is It includes TIFF2025084914000056.tif5128 and further includes IFNα2 fused to an antibody that binds to mesothelin.

[0177] In one embodiment, “masked” IFN includes a neutralizing scFv that also acts as a mask (as defined within the context of this disclosure) to provide a synergistic ability to mask IFNAR.

[0178] Resulting embodiments such as target-directed masked IFN offer unique advantages over the prior art for several reasons. First, the IFN is masked such that its activity is significantly reduced and / or eliminated until it reaches the tumor. When the mask is removed, the activity is reactivated, which maximizes the efficacy in the tumor. Second, by adding the masked IFN linked to the C-terminus to the C-terminus of an antibody, the masked IFN can be targeted to specific TAAs. Specific targeting allows for a greater chance that the IFN will be directed to the cancer of interest and avoid normal tissues.

[0179] The present disclosure contemplates general embodiments of the resulting target-directed masked IFN, including but not limited to the following: First, compositions as shown herein: (i) antibody-linker-cytokine (e.g., IFN)-linker-protease cleavage (PC) site-linker-mask; and Second, a composition as shown herein: (ii) antibody-linker-PC cleavage site-linker-cytokine (e.g., IFN).

[0180] It is recognized that those skilled in the art will recognize and understand that the composition shown in (ii) has additional properties of the antibody that act as a partial mask through the steric hindrance of the cytokine present in the composition.

[0181] In some embodiments, the protease cleavage site is a tumor-associated protease cleavage site. A "tumor-associated protease cleavage site" as provided herein is an amino acid sequence recognized by a protease whose expression is specific to tumor cells or their tumor cell environment. In some embodiments, exemplary protease cleavage sites include tumor-associated protease cleavage sites such as matrix metalloprotease (MMP) cleavage sites, disintegrin and metalloprotease domain-containing (ADAM) metalloprotease cleavage sites, prostate-specific antigen (PSA) protease cleavage sites, urokinase-type plasminogen activator (uPA) protease cleavage sites, membrane-type serine protease 1 (MT-SP1) protease cleavage sites, matriptase protease cleavage sites (ST14), or legumain protease cleavage sites. In some aspects, the protease cleavage site can be specified by a specific amino acid sequence.

[0182] V.) Treatment of cancers expressing tumor-associated antigens (TAAs) Also provided herein are fusion proteins, such as targeted masked IFNs, or compositions that are useful in various therapeutic, diagnostic, and prophylactic methods and uses. For example, the fusion proteins and compositions are useful in the treatment of various diseases and disorders in a subject, such as cancer or tumors. Such methods and uses include, for example, therapeutic methods and uses that include administration of the fusion protein or composition to a subject having a disease or disorder such as a tumor or cancer. In some embodiments, the fusion protein or composition is administered in an amount effective to effect treatment of the disease or disorder. The use includes the use of the fusion protein or composition in such methods and treatments, as well as in the preparation of a medicament for carrying out such a treatment method. In some embodiments, the fusion protein or composition is for use, for example, in the treatment of various diseases and disorders in a subject according to a treatment method. In some embodiments, the method is carried out by administering the fusion protein or composition to a subject having or suspected of having a disease or disorder such as a tumor or cancer. In some embodiments, the method thereby treats the disease or disorder in the subject.

[0183] In some aspects, the provided fusion proteins, such as targeted masked IFNs, are used in methods or uses for the treatment of diseases or disorders such as tumors or cancers, including those that express tumor-associated antigens (TAAs). Identification of the TAAs of the present disclosure as proteins that are normally expressed in a restricted set of tissues or cells but are also expressed in cancers, such as solid tumor cancers, opens up numerous therapeutic approaches to the treatment of such cancers that utilize the masked fusion proteins disclosed herein.

[0184] Notably, target-directed anti-tumor therapies are useful even when the targeted protein is expressed on normal tissues or cells, and even on normal organ tissues necessary for life support. Organs necessary for life support are those necessary for sustaining life, such as the heart or colon. Organs not necessary for life support are those that can be removed and the individual can still survive. Examples of organs not necessary for life support are the ovaries, breasts, and prostate.

[0185] Expression of the target protein in normal tissues, and even in normal tissues necessary for life support, does not invalidate the usefulness of targeting agents against the protein as a therapeutic agent for certain tumors in which the protein is also overexpressed. For example, expression in organs necessary for life support is not harmful per se. In addition, organs considered not necessary, such as the prostate and ovaries, can be removed without affecting mortality. Finally, some organs necessary for life support are not affected by normal organ expression due to immune privilege. Immune-privileged organs are those protected from the blood by the blood-organ barrier and thus not accessible to immunotherapy. Examples of immune-privileged organs are the brain and testes.

[0186] Accordingly, a therapeutic approach for inhibiting the activity of a TAA protein, comprising the target-directed masked IFN fusion protein of the present invention, is useful for patients suffering from cancers that express TAA (e.g., solid tumor cancers in the lung, kidney, prostate, ovary, breast, and other types of cancers known in the art, etc.). The therapeutic approach includes IFNA-induced killing (e.g., when the "mask" is removed in the tumor of interest and IFN is reactivated), ADCC, CDC, and / or immunomodulation. In addition, antibodies that bind to TAA can also synergistically modulate the functions of cancer cells. Additionally, furthermore, the "unmasked" or activated IFN can regulate the activities of immune cells involved in anti-tumor or anti-cancer immunity by virtue of the binding strength of IFN to the interferon receptor (e.g., IFNAR). The modulation of antibodies that bind to TAA is generally classified into two classes. The first class modulates TAA function such that it is associated with tumor cell proliferation, resulting in inhibition or delay of tumor cell proliferation or induction of its killing. The second class includes various methods for inhibiting the binding or association of the TAA protein with its binding partner or with other proteins.

[0187] Accordingly, cancer patients can preferably be evaluated for the presence and level of TAA expression using immunohistochemical evaluation of tumor tissue, quantitative TAA imaging, or other techniques that reliably indicate the presence and extent of TAA expression. For this purpose, when applicable, immunohistochemical analysis of tumor biopsies or surgical specimens is preferred. Methods for immunohistochemical analysis of tumor tissue are well known in the art.

[0188] VI.) Target-directed masked IFN fusion protein cocktails The treatment methods of the present invention contemplate the administration of a single target-directed masked IFN fusion protein, and a combination or cocktail of different MAbs (i.e., naked MAbs that bind to the same TAA as the masked IFN fusion protein, or MAbs that bind to another protein, or an entire other target-directed masked IFN fusion protein that binds to another TAA). Such MAb cocktails can have certain advantages, just as well as containing MAbs that target different epitopes and utilize different effector mechanisms, or combining directly cytotoxic MAbs with MAbs that rely on immune effector functions. MAbs in such combinations can exhibit a synergistic therapeutic effect. In addition, the target-directed masked IFN fusion protein can be administered in conjunction with other treatment modalities, including but not limited to various chemotherapeutic and biological agents, androgen blockers, immunomodulators (e.g., IL-2, GM-CSF), surgery, or radiation. In a preferred embodiment, the target-directed masked IFN is administered in the form of a fusion protein.

[0189] Target-directed masked IFN fusion protein formulations are administered via any route capable of delivering the antibody to tumor cells. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumoral, intradermal, etc. Treatment generally involves repeated administration of a target-directed masked IFN fusion protein preparation at a dose in the range typically including, but not limited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg / kg body weight via an acceptable route of administration such as intravenous injection (IV). Generally, doses in the range of 10 - 1000 mg of target-directed masked IFN fusion protein per week are effective and show good tolerance.

[0190] Based on clinical experience with Herceptin® (trastuzumab) in the treatment of metastatic breast cancer, an initial IV loading dose of approximately 4 mg / kg patient body weight of the MAb preparation, followed by a subsequent weekly IV dose of approximately 2 mg / kg, corresponds to an acceptable dosing regimen. Preferably, the initial loading dose is administered as an infusion over 90 minutes or more. The periodic maintenance dose is administered as an infusion over 30 minutes or more, provided the initial dose has shown good tolerance. As will be appreciated by those skilled in the art, various factors can affect the ideal dosing regimen in a particular case. Such factors include, for example, the binding affinity and half-life of the TAA MAb used, the degree of TAA expression in the patient, the spread of circulating fragmented TAA antigen, the desired steady-state antibody concentration level, the frequency of treatment, and the effect of chemotherapeutic agents or other agents used in combination with the treatment method of the present invention (i.e., targeted masked IFN), as well as the health status of the particular patient.

[0191] Optionally, the patient should be evaluated for the level of TAA (e.g., the level of circulating TAA and / or TAA-expressing cells) in a given sample to assist in determining, among other things, the most effective dosing regimen. Such evaluation is also used to monitor the goal throughout the course of treatment and is useful in combination with the evaluation of other parameters (e.g., urine cytology and / or ImmunoCyt levels in bladder cancer treatment, or by analogy, serum PSA levels in prostate cancer treatment) to determine treatment success.

[0192] An object of the present invention is to provide a targeted masked IFN fusion protein that inhibits or delays the growth of tumor cells expressing a specific TAA to which the fusion protein binds. A further object of the present invention is to use such a targeted masked IFN-containing fusion protein that binds to a TAA to find a specific TAA, particularly in combination with other drugs or immunologically active treatments. Provided is a method of inhibiting angiogenesis and other biological functions in mammals, preferably humans, using such a targeted masked IFN-containing fusion protein, thereby reducing tumor growth.

[0193] VII.) Combination therapies In some embodiments, also provided are methods and uses comprising combination therapies, including, for example, the use of any of the provided fusion proteins or compositions and additional therapeutic agents such as chemotherapeutic agents or radiation. In some embodiments, the provided fusion protein or composition can be used in combination with additional therapeutic agents for the treatment of diseases or disorders such as anti-cancer or anti-tumor agents.

[0194] In some embodiments, there is a synergistic effect when tumors, including human tumors, are treated with a targeted masked IFN fusion protein that binds to a specific TAA, together with an additional therapeutic agent such as a chemotherapeutic agent, radiation, immunomodulatory therapy, or any combination thereof. In other words, the inhibition of tumor growth by a targeted masked IFN fusion protein that binds to a specific TAA is enhanced to a greater extent than expected when combined with a chemotherapeutic agent or radiation or a combination thereof. The synergistic effect can be demonstrated, for example, by inhibition of tumor growth in combination treatment that is greater than expected from treatment with the targeted masked IFN fusion protein that binds to a specific TAA alone, or from the additive effect of treatment with the targeted masked IFN fusion protein that binds to a specific TAA and a chemotherapeutic agent or radiation. Preferably, the synergistic effect is demonstrated by remission of cancer at a site where remission is not expected from treatment with the targeted masked IFN fusion protein that binds to a specific TAA alone, or from treatment using an additive combination of the targeted masked IFN fusion protein that binds to a specific TAA and a chemotherapeutic agent or radiation.

[0195] A method for inhibiting the growth of tumor cells using a target-directed masked IFN fusion protein that binds to a specific TAA, and a combination of chemotherapy, radiation, or an immunomodulatory agent, or any one, two, or three combinations, comprises administering a target-directed masked IFN fusion protein that binds to a specific TAA before, during, or after (i.e., before and during, before and after, during and after, or before, during, and after) initiating chemotherapy or radiation therapy and any combination thereof. For example, a target-directed masked IFN fusion protein that binds to a specific TAA is typically administered 1 to 60 days, preferably 3 to 40 days, more preferably 5 to 12 days before initiating radiation therapy and / or chemotherapy. However, depending on the treatment protocol and the requirements of the specific patient, the method is carried out in a manner that provides the most effective treatment and ultimately prolongs the patient's life.

[0196] Administration of chemotherapeutic agents can be achieved in a variety of ways, including systemically via parenteral and enteral routes. In one embodiment, a target-directed masked IFN fusion protein that binds to a specific TAA and a chemotherapeutic agent are administered as separate molecules. Specific examples of chemotherapeutic agents or chemotherapies include bortezomib, carfilzomib, lenalidomide, pomalidomide, cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere), aldesleukin, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon α, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, gemcitabine, chlorambucil, taxol, and combinations thereof.

[0197] The source of radiation used in combination with a target-directed masked IFN fusion protein that binds to a specific TAA can be either external or internal to the patient being treated. When the source is external to the patient, the treatment method is known as external beam radiation therapy (EBRT). When the source of radiation is internal to the patient, the treatment is called brachytherapy (BT).

[0198] The above treatment regimen may be further combined with agents and / or regimens for treating additional cancers, such as bortezomib, pomalidomide, and / or additional chemotherapy, cancer vaccines, signal transduction inhibitors, agents useful in treating abnormal cell growth or cancer, antibodies (e.g., anti-CTLA-4 antibodies as described in WO / 2005 / 092380 (Pfizer)), or other ligands that inhibit tumor growth by binding to IGF-1R, and cytokines.

[0199] Examples of immunomodulatory therapeutic agents used in cancer treatment include, but are not limited to, anti-(CTLA-4, PD-1, PD-L1, TIGIT, LAG3, T1B7-H3, B7-H4), and others known in the art.

[0200] When a mammal is subjected to additional chemotherapy, the above chemotherapy agents may be used. Additionally, growth factor inhibitors, biological response modifiers, anti-hormonal therapy, selective estrogen receptor modulators (SERMs), angiogenesis inhibitors, and anti-androgen drugs may be used. For example, anti-hormonal drugs such as anti-estrogen drugs like Nolvadex (tamoxifen), or anti-androgen drugs such as Casodex (4'-cyano-3-(4-fluorophenylsulfonyl)-2-hydroxy-2-methyl-3'-(trifluoromethyl)propionanilide) may be used.

[0201] The above treatment approach can be combined with any one of a variety of surgical, chemotherapy, or radiation therapy regimens. The treatment approach of the present invention enables the use of reduced dosages of chemotherapy (or other treatment methods) and / or less frequent administrations, which is advantageous for all patients, especially those who do not show good tolerance to the toxicity of chemotherapy agents.

[0202] VIII.) Kits / articles of manufacture For use in the laboratories, prognostic determination, prevention, diagnosis, and treatment applications described herein, kits, manufactured articles, systems, and devices are within the scope of the present invention. Such kits can include a carrier, package, or container compartmentalized to receive one or more containers such as vials, tubes, etc., each of the containers containing one of the separate elements used in the method, together with a label or insert containing instructions for use such as those described herein. For example, the container can contain a target-directed masked IFN fusion protein that binds to one specific TAA of the present disclosure or several TAAs. The kit can include a container containing the target-directed masked IFN. The kit can include all or part of the target-directed masked IFN fusion protein that binds to a specific TAA, and / or a diagnostic assay for detecting cancer and / or other immune disorders.

[0203] The kits of the present invention typically include the above containers, as well as one or more other containers associated therewith, including materials desirable from a commercial and user perspective, including buffers, diluents, filters, needles, syringes; labels on the carrier, package, container, vial, and / or tube listing the contents and / or instructions for use, and an accompanying document with instructions for use.

[0204] The label can be present on or with the container such that the composition is indicated for use in a particular therapy or non-therapeutic application, such as prognostic determination, prevention, diagnosis, or laboratory use, and can also indicate instructions for use, such as those described herein, for either in vivo or in vitro use. Instructions and / or other information can also be included on an insert or label included with or on the kit. The label can be on the container or associated with the container. The label can be on the container if the letters, numbers, or other characters forming the label are molded or etched into the container itself; the label can be associated with the container if it is present, for example, as an enclosure that also holds the container or within a carrier. The label can indicate that the composition is used for diagnosing, treating, preventing, or prognosticating a condition such as cancer or other immunological disorders.

[0205] The terms "kit" and "article of manufacture" can be used synonymously.

[0206] In another aspect of the invention, the article of manufacture contains a composition, such as a targeted masked IFN fusion protein that binds to a specific TAA of the present disclosure. The article of manufacture typically includes at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container can be formed of various materials such as glass, metal, or plastic. The container can hold one or several targeted masked IFN fusion proteins that bind to a specific TAA and / or one or more therapeutic doses of the targeted masked IFN.

[0207] The container can alternatively hold a composition that is effective for the treatment, diagnosis, prognosis, or prevention of a condition and can have a sterile access port (e.g., the container can be a vial having a stopper penetrable by an intravenous solution bag or a hypodermic needle). The active agent in the composition can be a targeted masked IFN fusion protein that binds to a specific TAA of the present disclosure.

[0208] The manufactured article can further include a second container containing a pharmaceutically acceptable buffer such as phosphate buffered saline, Ringer's solution, and / or dextrose solution. This can further include other materials desirable from a commercial or user perspective, including other buffers, diluents, filters, stirrers, needles, syringes, and / or accompanying documents with efficacy and / or usage instructions.

[0209] Exemplary embodiments Aspects provided are as follows: 1) Polypeptide sequence A composition comprising TIFF2025084914000057.tif5128, wherein the polypeptide sequence masks the activity of type I interferon (IFN) and the composition further comprises a fusion protein fused to an antibody that binds to a tumor-associated antigen. 2) The composition of aspect 1, further comprising a cleavable polypeptide linker. 3) The composition of aspect 1 or 2, further comprising a tumor-associated protease cleavage site. 4) The composition of any one of aspects 1 to 3, wherein the type I interferon comprises IFNα1. 5) The composition of any one of aspects 1 to 3, wherein the type I interferon comprises IFNα2. 6) The composition of any one of aspects 1 to 3, wherein the type I interferon comprises IFNα4. 7) The composition of any one of aspects 1 to 3, wherein the type I interferon comprises IFNα5. 8) The composition of any one of aspects 1 to 3, wherein the type I interferon comprises IFNα6. 9) The composition according to any one of aspects 1 to 3, wherein the type I interferon comprises IFNα14. 10) The composition according to any one of aspects 1 to 3, wherein the type I interferon comprises IFNβ1. 11) The composition according to any one of aspects 1 to 3, wherein the type I interferon or its functional variant is selected from type I interferons as shown in Table II. 12) The composition according to any one of aspects 1 to 11, wherein the tumor-associated antigen comprises CD138. 13) The composition according to any one of aspects 1 to 11, wherein the tumor-associated antigen comprises CD20. 14) The composition according to any one of aspects 1 to 11, wherein the tumor-associated antigen comprises mesothelin. 15) The composition according to any one of aspects 1 to 11, wherein the tumor-associated antigen comprises 5T4. 16) The composition according to any one of aspects 1 to 11, wherein the tumor-associated antigen is selected from tumor-associated antigens as shown in Table I. 17) a. An antibody comprising a heavy chain and / or a light chain that specifically binds to a tumor-associated antigen; b. A type I interferon in which the N-terminus of the type I interferon is fused to the C-terminus of the antibody heavy chain and / or light chain; and c. An interferon mask (SEQ ID NO: 14) that is added to the C-terminus of the type I interferon. A targeted masked IFN comprising the above. 18) The targeted masked IFN according to aspect 17, further comprising a flexible peptide linker, wherein the N-terminus of the type I interferon is fused to the C-terminus of the antibody heavy chain and / or light chain. 19) The targeted masked IFN according to aspect 17 or 18, further comprising a flexible peptide linker, wherein the interferon mask (SEQ ID NO: 14) is added to the C-terminus of the type I interferon. 20) The targeted masked IFN according to aspect 19, further comprising a tumor-associated protease cleavage site inserted between the antibody and the flexible peptide linker. 21) The target-directed masked IFN of any one of aspects 17 to 19, further comprising a tumor-related protease cleavage site inserted between the type I interferon and the interferon mask. 22) The target-directed masked IFN of any one of aspects 17 to 21, wherein the type I IFN or a functional variant thereof is as shown in Table II. 23) The target-directed masked IFN of any one of aspects 17 to 22, wherein the antibody binds to a tumor-related antigen shown in Table I. 24) The target-directed masked IFN of any one of aspects 17 to 22, wherein the antibody binds to CD138. 25) The target-directed masked IFN of any one of aspects 17 to 22, wherein the antibody binds to CD20. 26) The target-directed masked IFN of any one of aspects 17 to 22, wherein the antibody binds to mesothelin. 27) The target-directed masked IFN of any one of aspects 17 to 22, wherein the antibody binds to 5T4. 28) A method for preparing a composition of any one of aspects 1 to 16 or a target-directed masked IFN of any one of aspects 17 to 27. 29) A pharmaceutical composition comprising a composition of any one of aspects 1 to 16 or a target-directed masked IFN of any one of aspects 17 to 27, (i) Optionally, the pharmaceutical composition is for use in a treatment method including the treatment of cancer, and optionally, (a) the cancer includes a cancer found in a solid tumor; or (b) the cancer occurs in the hematopoietic system and (ii) Optionally, the pharmaceutical composition further comprises one or more anti-cancer agents, The pharmaceutical composition. 30) A kit comprising a composition of any one of aspects 1 to 16 or a target-directed masked IFN of any one of aspects 17 to 27. 31) A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a composition of any one of aspects 1-16 or a targeted masked IFN of any one of aspects 17-27, optionally wherein the subject is a human subject, said method.

Example

[0210] Various aspects of the invention are further described and illustrated by several of the following examples, none of which are intended to limit the scope of the invention.

[0211] Example 1: Characterization of a target-directed masked IFNα2 fused to anti-CD138 (anti-CD138-IFNα2) In this example, it is shown that the IFN mask can be cleaved from the H chain using matriptase ST 14. Briefly, anti-CD138-IFNα2 and anti-CD138-IFNα2-mask were generated using the procedure shown above. Methods for masking IFN of the present disclosure See. The modified heavy chain was then transiently expressed in 293T cells with the appropriate L chain to yield anti-CD138-IFNα2-mask. Confirmation by SDS-PAGE analysis showed that the fusion protein had H chains and L chains of the appropriate size and was correctly assembled into H 2 L 2 molecules. Then, FACS analysis showed that the modified fusion protein bound to CD138-expressing cells.

[0212] Analysis resulting via Western blot showed that matriptase ST 14 could cleave the IFN mask from the H chain. Anti-CD138-IFNα2 without a mask was used as a control (see Figure 1).

[0213] Example 2: Characterization of a target-directed masked IFNα2 fused to non-glycosylated anti-CD138 (N297Q) (anti-CD138-IFNα2 N297Q) In this example, it is shown that the IFN mask can be cleaved from the H chain using matriptase ST14. Briefly, for samples treated with MST14 (R&D Systems), 50 μg of antibody was incubated with 0.5 μg of MST14 for 1 hour at 37°C. Then, 1 μg of each purified antibody was denatured by heating to 95°C, reduced with approximately 2% β-mercaptoethanol (Thermofisher), and electrophoresed on a 4-12% Bis-Tris SDS-PAGE gel (Invitrogen). 4 μg of each non-reduced antibody was denatured by heating to 95°C and electrophoresed on a 5% PO4 SDS-PAGE gel. The resulting analysis shows that matriptase ST14 efficiently cleaves the IFN mask on the non-glycosylated fusion antibody. Anti-CD138-IFNα2 without a mask was used as a control (see Figure 2).

[0214] Example 3: Characterization of target-directed masked IFNα2 fusion antibodies fused to anti-5T4 and anti-mesothelin In this example, it is shown that the IFN mask can be cleaved from the H chain of the fusion antibody against multiple targets using matriptase ST14. Briefly, for samples treated with MST14 (R&D Systems), 50 μg of antibody was incubated with 0.5 μg of MST14 for 1 hour at 37°C. Then, 1 μg of each purified antibody was denatured by heating to 95°C, reduced with approximately 2% β-mercaptoethanol (Thermofisher), and electrophoresed on a 4-12% Bis-Tris SDS-PAGE gel (Invitrogen). 4 μg of each non-reduced antibody was denatured by heating to 95°C and electrophoresed on a 5% PO4 SDS-PAGE gel. The resulting analysis shows that matriptase ST14 efficiently cleaves the IFN mask on the 5T4 and mesothelin fusion antibodies. Anti-CD138-IFNα2 without a mask was used as a control (see Figure 3).

[0215] Example 4: Binding of a masked fusion antibody to the IFNα receptor In this example, it is shown that multiple masked fusion antibodies of the present disclosure can bind to the IFNα2 receptor. Briefly, Immulon 2 HB plates (Thermofisher) were coated overnight at 4°C with 10 ug / mL of IFNαR2 (R&D Systems) and blocked with 2% BSA (Fisher) for at least 2 hours at room temperature. The wells were then washed three times with PBS + 0.05% Tween (Sigma). The surface was covered overnight at 4°C with the indicated antibody concentrations. The wells were then washed three times with PBS + 0.05% Tween. Bound antibodies were detected with anti-human κ-AP (Southern Biotech) diluted 1:3000 in PBS + 1% BSA. The change in absorbance after addition of the AP substrate (Sigma) was assayed at 410 nm using a Biotek EPOCH ELISA reader. The results show that the masked 5T4, mesothelin, CD20, and CD138 antibodies bind to IFNαR2 with lower affinity compared to the unmasked 5T4, mesothelin, CD20, and CD138 fusion antibodies (see FIGS. 4 and 5).

[0216] Example 5: Methods for reducing and restoring masked IFNα activity In this example, it is shown that the masked IFN fusion protein (anti-CD138-IFNα2) does not activate the type I IFN signaling pathway. Activation could then be restored after matriptase ST14 treatment. Briefly, HEK-Blue™ IFN-α / β cells (Invivogen, San Diego, CA) were used to detect activation of the type I IFN signaling pathway using a reporter gene that expresses secreted embryonic alkaline phosphatase (SEAP), and gene activation was monitored by quantifying alkaline phosphatase activity in the supernatant of the treated cells. HEK-Blue™ IFN-α / β cells were incubated for 24 hours with hIFNα2, anti-CD138 masked IFNα2 without MST14, anti-CD138 masked IFNα2 with MST14; and anti-CD138-IFNα2. The results showed that the IFNα activity in the masked anti-CD138-IFNα2 was reduced to less than 10% of that seen in the unmasked protein; and that the IFNα2 activity was shown to be almost 100% restored after matriptase / ST14 treatment (see Figure 6).

[0217] Example 6: Methods for reducing and restoring masked IFNα activity In this example, it is shown that multiple masked fusion antibodies of the present disclosure can reduce and recover IFNα activity. Briefly, HEK Blue IFNa / b cells (Invivogen) were seeded in a 96-well tissue culture plate (Fisher) at a density of 1×10e4 cells / well (50 uL / well). Recombinant IFNa (Novus Biologicals) or the indicated antibodies (5T4 or mesothelin) at 50 uL / well were incubated with the cells overnight at 37°C at the indicated concentrations. Antibodies cleaved with MST14 were prepared by incubating 50 ug of the antibody with 0.5 ug of MST14 (R&D Systems) for 1 hour at 37°C. Then, 10 uL of the supernatant was added to plates containing 90 uL / well of Quanti-Blue substrate (Invivogen). Changes in absorbance were read at 630 nm using a Biotek EPOCH ELISA reader. The results show that IFNα activity in masked anti-5T4-IFNα and masked anti-mesothelin-IFNα was reduced by approximately 1 to 2 logs compared to when the mask was cleaved (see Figures 7(A) and 7(B)).

[0218] Example 7: Method for reducing IP-10 induction in PBMC In this example, it is shown that multiple masked fusion antibodies of the present disclosure can reduce IP-10 induction. Briefly, freshly thawed human PBMCs (Human Cells Biosciences) were washed once with cold RPMI + 10% FBS (Invitrogen) and seeded into a 12-well plate (Themofisher) at a density of approximately 1×10e6 cells / well (1 mL / well). Prior to proceeding with the described experiments, any Fc receptor and / or CD138 antigen expression was blocked / reduced by adding 300 nM of non-fused anti-CD138 IgG1 to the cells for 1 hour. Recombinant human IFNα (Novus Biologicals) or the indicated antibodies (5T4 or mesothelin) were then added to the cells and incubated at 37°C for an additional 7 hours. Antibodies cleaved with MST14 were prepared by incubating 50 μg of the antibody with 0.5 μg of MST14 (R&D Systems) at 37°C for 1 hour. After 7 hours of incubation, the cells were spun down at 500×g for 3 minutes, and 20 μL of the supernatant from each sample was assayed for IP-10 (Abcam) by ELISA according to the manufacturer's protocol. The results show that masking reduces IP-10 induction in both the anti-5T4 fusion antibody and the anti-mesothelin fusion antibody (see Figure 8).

[0219] Example 8: Method for producing a masked fusion protein In this example, the construction and characterization of the QXL138AM are shown. Briefly, anti-CD138 IgG1 was prepared using standard methods in the art. The heavy chain isotype is human γ1 and the light chain isotype is human κ. The mask was generated as described herein. Methods for masking IFN of the present disclosure See. The resulting construct designated QXL138AM (anti-CD138-IFNα2-cleavable linker-mask) consists of an IgG heavy chain (shown in yellow) (SEQ ID NO: 6), a fusion protein linker (SGGGGS) (SEQ ID NO: 3), IFNα2 (shown in green) (SEQ ID NO: 7), a cleavable linker TIFF2025084914000058.tif4128 (shown in cyan) (SEQ ID NO: 16), and the mask of the present disclosure (shown in purple) (SEQ ID NO: 15) (see Figure 9; the entire sequence is shown in SEQ ID NO: 17, and the entire sequence without a signal peptide is shown in SEQ ID NO: 18).

[0220] The construct shown in Figure 9 was transiently expressed in TunaCHO for 14 days. The titer reached 179 mg / L and was purified by Protein A chromatography using standard methods (Lake Pharma, Belmont, CA) (Figure 10). Further analysis of the heavy and light chains was evaluated by mass spectrometry using standard methods (Lake Pharma, Belmont, CA) (Figure 11).

[0221] Example 9: Determination of the structural integrity of antibody-cytokine-mask In this example, the structural integrity of the antibody-cytokine-mask construct of QXL138AM was determined. Briefly, two lots of QXL138AM were evaluated by reducing SDS-PAGE. For samples treated with MST14 (R&D Systems), 8 μg of antibody was incubated with approximately 125 ng of MST14 for 15 minutes at 37°C. Then, 2 μg of each purified antibody was denatured by heating to 95°C, reduced with approximately 2% β-mercaptoethanol (Sigma), and electrophoresed on a 4-12% Bis-Tris SDS-PAGE gel (Invitrogen). Lot 2 was evaluated with pretreatment with matriptase (MST14) + / - The results indicate that the antibody-cytokine-mask structure appears to be intact when purified. Furthermore, the mask is released by MST14 without accompanying destruction of the antibody-cytokine protein (see Figure 12).

[0222] Example 10: Determination of the structural integrity and cleavage specificity of the fusion protein In this example, the structural integrity of the multiple fusion antibodies and the ability to cleave the mask by MST14 were determined. Briefly, for samples treated with MST14 (R&D Systems), 50 μg of antibody was incubated with 0.5 μg of MST14 for 1 hour at 37 °C. 1 μg of each purified antibody was denatured by heating to 95 °C, reduced with approximately 2% β-mercaptoethanol (Thermofisher), and electrophoresed on a 4-12% Bis-Tris SDS-PAGE gel (Invitrogen). 4 μg of each non-reduced antibody was denatured by heating to 95 °C and electrophoresed on a 5% PO4 SDS-PAGE gel. 10 μg of the indicated masked antibody was incubated with 1×10e6 RPMI 8226S cells for 4 hours in 100 μL of serum-free RPMI (Invitrogen). After 4 hours, the cells were spun down at 500×g for 3 minutes, and 10 μL of the supernatant was denatured by heating to 95 °C and reduced with approximately 2% β-mercaptoethanol (Thermofisher). The remaining cells were then resuspended and incubated overnight at 37 °C. Then, approximately 24 hours later, the cells were gently spun down and processed as described above. The results show that the masked fusion antibody appears to be of the correct size and is specifically cleaved by MST14 (see Figure 13(A)).

[0223] In a parallel setting, 10 μg of the indicated masked antibody was incubated with 1×10e6 RPMI 8226S cells for 24 hours at 37 °C in 1 mL of serum-free RPMI (Invitrogen). Approximately 24 hours later, the cells were spun down as mentioned in the above paragraph, and the supernatant was immunoprecipitated with protein A agarose (Sigma). The samples were denatured and reduced as shown above and electrophoresed on a 4-12% Bis-Tris SDS-PAGE gel. The results show that targeting the masked anti-CD138 fusion antibody to 8226S cells does not affect mask cleavage in vitro (see Figure 13(B)).

[0224] Example 11: Method for binding a fusion antibody to a masking peptide In this example, it is shown that multiple fusion antibodies specifically bind to some of the peptide masks of the present disclosure. For reference, the following two peptide masks were tested. Peptide 1 (Mask 1): TIFF2025084914000059.tif5128 Peptide 2 (Mask 2): TIFF2025084914000060.tif5128

[0225] Briefly, plates (Pierce) coated with streptavidin were overlaid with the surface at room temperature for at least 2 hours with each of the indicated peptides (Thermofisher) at 50 μM. The wells were then washed three times with PBS + 0.05% Tween. Antibodies at the indicated concentrations were then bound overnight at 4°C. The wells were washed three times with PBS + 0.05% Tween (Sigma). Bound antibodies were detected with anti-human κ-AP (Southern Biotech) diluted 1:3000 in PBS + 1% BSA. The change in absorbance after addition of the AP substrate (Sigma) was assayed at 410 nm using a Biotek EPOCH ELISA reader. The results show that Peptide 1 binds to all of the IFN fusion antibodies tested (see Figure 14(A)). Additionally, Peptide 2 binds to all of the IFN fusion antibodies tested to varying degrees compared to Peptide 1 (see Figure 14(B)).

[0226] Example 12: Characterization of target-directed fusion antibodies versus non-target-directed fusion antibodies The fusion antibodies of the present disclosure were tested in several cell lines to compare the efficacy of the target-directed fusion antibodies versus the non-target-directed fusion antibodies. Briefly, 1×10e4 cells / well (50 μL / well) of each cell line were seeded in a 96-well tissue culture plate (Becton Dickinson). The next day, the cells were treated with each of the indicated antibodies at the indicated concentrations at 50 μL / well. Six days later, 20 μL / well of MTS reagent (Promega) was added to the plate. The change in absorbance was read at 490 nm with a Biotek EPOCH reader. The results show that the target-directed fusion antibodies are more effective than the non-target-directed fusion antibodies (see FIGS. 15(A) and 15(B)).

[0227] In another experiment, 1.5×10e4 U266 cells / well (50 μL / well) or 1×10e4 CAPAN-2 cells / well (50 μL / well) were seeded in a 96-well tissue culture plate (Becton Dickinson). The U266 cells were treated with each of the indicated antibodies at the indicated concentrations at 50 μL / well on the same day, while the CAPAN-2 cells were treated the next day. Six days after treatment, 20 μL / well of MTS reagent (Promega) was added to the plate. The change in absorbance was read at 490 nm with a Biotek EPOCH reader. The results show that the target-directed fusion antibodies are more effective than the non-target-directed fusion antibodies. In addition, the masked fusion antibodies are less effective than the cleaved version (see FIGS. 16(A) and 16(B)).

[0228] Example 13: Method for inhibiting cell proliferation using anti-CD138-IFNα2 In this example, it is shown that the IFN fusion protein (anti-CD138-IFNα) inhibits cell proliferation in cancer cells (i.e., Daudi cells). The inhibition of cancer cell proliferation was significantly decreased while the IFN mask was in a predetermined position. After removing the IFN mask, the inhibition of cell proliferation was restored. Briefly, Daudi cells were incubated with hIFNα2, anti-CD138 masked IFNα2 without MST14, anti-CD138 masked IFNα2 with MST14; and anti-CD138-IFNα2 for 3 days, and proliferation was measured using the MTS assay. It is noted that proliferation is expressed as a percentage (%) compared to non-treated cells. The results show that removal of the IFN mask restores the inhibition of cell proliferation (see Figure 17).

[0229] Example 14: Method for reducing off-target IFNα-induced cytotoxic activity In this experiment, the ability of the mask was shown to reduce off-target IFNα-induced cytotoxic activity. Briefly, each cell line at 1×10e4 cells / well (50 μL / well) was seeded into a 96-well tissue culture plate (Becton Dickinson). Each indicated antibody at the indicated concentration of 50 μL / well was added to the plate. Three days later, 20 μL / well of MTS reagent (Promega) was added to the plate. The change in absorbance was read at 490 nm with a Biotek EPOCH reader. For samples treated with MST14 (R&D Systems), 50 μg of antibody was incubated with 0.5 μg of MST14 for 1 hour at 37 °C. The results show that the CD138 fusion protein and the cleaved CD138 fusion protein are approximately 50-fold less potent than free IFNα against CD138 cells, and the masked CD138 fusion protein is approximately 1000-fold less potent than IFNα against CD138 cells (see Figure 18(A)). Additionally, masking reduces on-target or off-target activity by approximately 10-fold in cell culture. Antibody targeting to the cell surface enhances activity by approximately 100-fold. The targeted unmasked fusion protein is approximately 10,000-fold more potent than the non-targeted masked fusion protein (see Figure 18(B)).

[0230] Example 15: Tumor cell line cytotoxic activities of masked fusion proteins versus unmasked fusion proteins, and target-directed fusion proteins versus non-target-directed fusion proteins A study comparing the cytotoxic activities of various cell lines using masked fusion proteins versus unmasked fusion proteins and target-directed fusion proteins versus non-target-directed fusion proteins of the present disclosure was conducted using the following protocol. Briefly, 1.5 × 10e4 U266, H929, or OCI-My5.5 cells / well (50 μL / well) were seeded into 96-well tissue culture plates (Becton Dickinson). Additionally, 1 × 10e4 OVCAR3 or BCMW1 cells / well (50 μL / well) were seeded into 96-well tissue culture plates (Becton Dickinson). U266, H929, or OCI-My5.5 cells were treated with each of the indicated antibodies at the indicated concentrations at 50 μL / well on the same day, while OVCAR3 or BCMW1 cells were treated the next day. U266, H929, or OCI-My5.5 were assayed 4 days after treatment by adding 20 μL / well of MTS reagent (Promega) to the plates and measuring the change in absorbance at 490 nm with a Biotek EPOCH reader. OVCAR3 or BCMW1 cells were assayed similarly 6 days after treatment. The results indicate that antigen targeting increases the anti-proliferative effects of masked and unmasked fusion antibodies compared to their non-target-directed counterparts. Additionally, masking of the interferon portion reduces the anti-proliferative effects of the fusion antibodies compared to their unmasked counterparts in the cell lines assayed (see FIGS. 19 and 20).

[0231] Example 16: Method for reducing the IFNγ activation of PBMCs In this experiment, freshly thawed human PBMC (Human Cells Biosciences) were washed once with cold RPMI + 10% FBS (Invitrogen) and seeded into 12-well plates (Themofisher) at a density of approximately 1×10e6 cells / well (1 mL / well). Prior to proceeding with the experiment, any Fc receptor expression was blocked / reduced by the addition of 167 nM of human Fc Block (Becton Dickinson) to the cells for 1 hour. Recombinant human IFNa (Novus Biologicals) or the indicated antibodies were added to the cells and incubated for an additional 7 hours at 37°C. Antibodies cleaved with MST14 were prepared by incubating 50 ug of the antibody with 0.5 ug of MST14 (R&D Systems) for 1 hour at 37°C. After 7 hours of incubation, the cells were spun down at 500×g for 3 minutes, and 20 uL of the supernatant from each sample was assayed for IP-10 (Abcam) by ELISA according to the manufacturer's protocol.

[0232] Additionally, Western blot was performed on cell pellets from the same experiment. Cells were lysed in 100 uL NDET (1% Nonidet P-40, 0.4% deoxycholate, 66 mM EDTA, and 10 mM Tris, pH 7.4). 10 uL / sample was denatured by heating to 95 °C, reduced with approximately 2% β-mercaptoethanol (Thermofisher), and run on a 4-12% Bis-Tris SDS-PAGE gel (Invitrogen). Proteins were transferred to a 0.45 um PVDF membrane (GE Healthcare) and blocked overnight at 4 °C with PBS+3% BSA (Thermofisher). The membrane was then blotted overnight at 4 °C with 1:3000 rabbit anti-pSTAT1 (Cell Signaling) in PBS+3% BSA. After three 10-minute washes with PBS+0.1% Tween (Sigma), the blot was incubated for 1 hour at RT with anti-rabbit IgG-HRP diluted 1:10000 in PBS+3% BSA. Following another three 10-minute washes with PBS+0.1% Tween, the blot was incubated with SuperSignal West Pico HRP substrate (Pierce). Blot images were captured using Azure 280 (Azure Biosystems). Immediately after blot image capture, the blot was washed three times with PBS+0.1% Tween and incubated overnight at 4 °C with rabbit anti-GAPDH diluted 1:20000 in PBS+3% BSA.

[0233] Detection was achieved as previously described. Finally, the blot was stripped by heating to 65 °C for 15 minutes in 10 mM Tris pH 6.8+2% SDS+0.7% β-mercaptoethanol. After four 10-minute washes with PBS+0.1% Tween, the blot was re-blocked with PBS+3% BSA and blotted overnight at 4 °C with 1:1000 diluted rabbit anti-STAT1 in PBS+3% BSA. Detection was carried out as previously described.

[0234] The results show that there is dose-dependent STAT1 activation, and the masked fusion protein activates STAT1 with lower efficiency compared to the equimolar concentration of the unmasked fusion protein (see Figure 21(A)). In addition, Figure 21(B) shows that there is dose-dependent induction of IP-10, and the masked fusion protein induces IP-10 with lower efficiency compared to the equimolar concentration of the unmasked fusion protein.

[0235] In another experiment, freshly thawed human PBMCs (Human Cells Biosciences) were washed once with cold RPMI + 10% FBS (Invitrogen) and seeded into a 12-well plate (Themofisher) at a density of approximately 1×10e6 cells / well (1 mL / well). Prior to proceeding with the experiment, any Fc receptor expression was blocked / reduced by adding 167 nM of human Fc Block (Becton Dickinson) to the cells for 1 hour. Recombinant human IFNα (Novus Biologicals) or the indicated antibodies were added to the cells and further incubated at 37°C for 7 hours. Antibodies cleaved with MST14 were prepared by incubating 50 μg of the antibody with 0.5 μg of MST14 (R&D Systems) at 37°C for 1 hour. After 7 hours of incubation, the cells were spun down at 500×g for 3 minutes, and 20 μL of the supernatant from each sample was assayed for IP-10 (Abcam) by ELISA according to the manufacturer's protocol. The results show that masking of IFNα significantly reduces (by more than 10-fold) the potency of IP-10 induction (see Figure 22).

[0236] Example 17: In vitro functional study of QXL138AM The study on the functional characterization of QXL138AM was conducted using the following protocol. Briefly, HEK Blue IFNa / b cells (Invivogen) were seeded in a 96-well tissue culture plate (Fisher) at a density of 5×10e4 cells / well (50 uL / well). Then, 50 uL / well of recombinant IFNa (Novus Biologicals) or the indicated antibodies were incubated with the cells overnight at 37°C at the indicated concentrations. Antibodies cleaved with MST14 were prepared by incubating 50 ug of the antibody with 0.5 ug of MST14 (R&D Systems) for 1 hour at 37°C. Next, 10 uL of the supernatant was added to a plate containing 90 uL / well of Quanti-Blue substrate (Invivogen). The change in absorbance was read at 630 nm using a Biotek EPOCH ELISA reader. The results show that (i) the fusion antibody-targeted IFNα has the same potency as wild-type IFNα, (ii) the non-targeted antibody-IFNα is 100-fold less potent than wild-type IFNα, (iii) the mask is effective in blocking non-targeted IFNα, and (iv) the mask reduces IFNα activity even when targeted to transformed (non-tumorigenic) HEK cells expressing CD138 (see Figures 23(A) and 23(B)).

[0237] Example 18: In vivo potency study of QXL138AM The potency study of QXL138AM was conducted using the following protocol. Briefly, 1×10 6Individual H929 cells were s.c. injected onto the back of NSG mice together with matrigel (BD). Mice were treated i.v. with 100 μg (5 mg / kg, left graph) and 300 μg (15 mg / kg, right graph) on days 14, 16, and 18 post-infection. Tumor size areas were measured three times a week until they grew larger than 1.4 cm and were sacrificed at that time point. The target-directed fusion protein was compared with the non-target-directed fusion protein. Each masked version was also compared. The results show that (i) QXL138AM has the same efficacy as QXL138A, and (ii) single-agent QXL138A and QXL138AM can achieve durable CR that persists for several months after treatment (see Figures 24 and 25).

[0238] In another experiment, synergy with standard treatment (bortezomib) in multiple myeloma is shown. Briefly, 5×10 6 Individual MM1-144 cells were s.c. injected onto the back of NSG mice together with matrigel (BD). Mice were treated i.v. with 100 μg (5 mg / kg) of the fusion protein alone on days 14, 16, and 18 post-infection, and the target-directed masked and unmasked fusion proteins were treated in combination with 0.38 mg / kg bortezomib i.v. or with 0.38 mg / kg bortezomib i.v. alone. Tumor size areas were measured three times a week until they grew larger than 1.4 cm and were sacrificed at that time point. The results show that both masked IFNα and unmasked IFNα are synergistic and show similar efficacy when used with bortezomib (see Figure 26).

[0239] In another experiment, 1×10 6Individual H929 cells were s.c. injected onto the backs of NSG mice together with matrigel (BD). The mice were treated on days 14, 16, and 18 after injection with 100 μg (5 mg / kg) of the fusion protein alone i.v., 0.38 mg / kg of bortezomib alone i.v., or 0.15 mg / kg of bortezomib alone i.v. The target-directed fusion protein (QXL138A) was treated in combination with 0.38 mg / kg of bortezomib i.v., or 0.15 mg / kg of bortezomib i.v. The tumor size area was measured three times a week until they grew larger than 1.4 cm and then sacrificed. The results show a synergistic effect between QXL138A and bortezomib in H929 cells in vivo (see Figure 27).

[0240] In another experiment, a synergistic effect with the standard treatment (pomalidomide) in multiple myeloma is shown. Briefly, 1×10 6 Individual H929 cells were s.c. injected onto the backs of NSG mice together with matrigel (BD). The mice were treated on days 14, 16, and 18 after injection with 100 μg (5 mg / kg) of the fusion protein alone i.v., or 500 μg (25 mg / kg) of pomalidomide alone i.v. The unmasked fusion protein (QXL138A) was treated in combination with 25 mg / kg of pomalidomide i.v., and the tumor size area was measured three times a week until they grew larger than 1.4 cm and then sacrificed. The results show a synergistic effect between QXL138A M and pomalidomide (see Figure 28).

[0241] In another experiment, 5×10 6Individual MM1-144 cells were s.c. injected onto the backs of NSG mice together with matrigel (BD). The mice were treated on days 14, 16, and 18 after injection with 100 ug (5 mg / kg) of the fusion protein (QXL138A) i.v. alone, or a non-targeted fusion protein i.v. alone, or 25 mg / kg pomalidomide i.p. alone. 25 mg / kg pomalidomide i.p. was treated in combination with 5 mg / kg of the targeted fusion protein (QXL138A) i.v., or 5 mg / kg of the non-targeted fusion protein i.v. The tumor size area was measured three times a week until they grew larger than 1.4 cm, and then sacrificed at that time point. The results show a synergistic effect between QXL138A and pomalidomide (see Figure 29).

[0242] Example 19: Characterization of a target-directed masked IFNα2 fused to anti-CD138 using a second mask (anti-CD138-IFNα2-mask 2) In this example, it is shown that the second IFN mask (mask 2) can be cleaved from the heavy chain using matriptase ST 14. Briefly, for samples treated with MST14 (R&D Systems), 50 ug of antibody was incubated with 0.5 ug of MST14 for 1 hour at 37 °C. Then, 1 ug of each purified antibody was denatured by heating to 95 °C, reduced with approximately 2% β-mercaptoethanol (Thermofisher), and electrophoresed on a 4-12% Bis-Tris SDS-PAGE gel (Invitrogen). The resulting analysis shows that matriptase ST 14 efficiently cleaves the IFN mask (mask 2) on the anti-CD138 fusion antibody (see Figure 30).

[0243] Example 20: Binding of a masked fusion antibody (utilizing mask 2) to the IFNα2 receptor In this example, it is shown that the masked fusion antibody (using mask 2) of the present disclosure can bind to the IFNα receptor. Briefly, Immulon 2 HB plates (Thermofisher) were coated overnight at 4°C with 10 ug / mL of IFNαR2 (R&D Systems) and blocked with 2% BSA (Fisher) for at least 2 hours at room temperature. The wells were then washed three times with PBS + 0.05% Tween (Sigma). The surface was covered overnight at 4°C with the indicated antibody concentrations. The wells were then washed three times with PBS + 0.05% Tween. Bound antibody was detected with anti-human κ-AP (Southern Biotech) diluted 1:3000 in PBS + 1% BSA. The change in absorbance after addition of the AP substrate (Sigma) was assayed at 410 nm using a Biotek EPOCH ELISA reader. The results show that both mask 1 and mask 2 can inhibit the binding of the fusion antibody to IFNαR2 (see Figure 31).

[0244] Example 21: Methods for reducing and restoring masked IFNα activity In this example, it is shown that both Mask 1 and Mask 2 of the present disclosure can reduce and recover IFNα activity. Briefly, HEK Blue IFNa / b cells (Invivogen) were seeded in a 96-well tissue culture plate (Fisher) at a density of 1×10e4 cells / well (50 uL / well). 50 uL / well of recombinant IFNa (Novus Biologicals) or the indicated antibodies (5T4 or mesothelin) were incubated with the cells overnight at 37°C at the indicated concentrations. Antibodies cleaved with MST14 were prepared by incubating 50 ug of the antibody with 0.5 ug of MST14 (R&D Systems) for 1 hour at 37°C. Then, 10 uL of the supernatant was added to a plate containing 90 uL / well of Quanti-Blue substrate (Invivogen). The change in absorbance was read at 630 nm using a Biotek EPOCH ELISA reader. The results show that the IFNα activity in masked anti-CD138-IFNα (Mask 1) and masked anti-CD138-IFNα (Mask 2) was reduced compared to when the mask was cleaved (see FIGS. 32(A) and 32(B)).

[0245] Example 22: Characterization of a target-directed masked IFNα1 fused to anti-PSCA (anti-PSCA-IFNα1) In this example, it is shown that the IFN mask can be cleaved from the H chain using matriptase ST 14. Briefly, anti-PSCA-IFNα1 and anti-PSCA-IFNα1 + mask are generated using the procedure shown above. Method for masking the IFN of the present disclosure See. The modified heavy chain is transiently expressed in 293T cells together with the appropriate L chain, resulting in anti-PSCA-IFNα1 + mask in 293T cells. Confirmation by SDS-PAGE analysis shows that the fusion protein has H chains and L chains of the appropriate size and is correctly assembled into H 2 L 2 molecules. FACS analysis shows that the modified fusion protein binds to PSCA-expressing cells.

[0246] The resulting analysis shows that matriptase ST 14 can cleave the IFN mask from the H chain. Anti-PSCA-IFNα1 without the mask is used as a control.

[0247] Example 23: Characterization of a target-directed masked IFNα2 fused to anti-PSCA (anti-PSCA-IFNα2) In this example, it is shown that the IFN mask can be cleaved from the H chain using matriptase ST 14. Briefly, anti-PSCA-IFNα2 and anti-PSCA-IFNα2 + mask are generated using the procedure shown above. Method for masking the IFN of the present disclosure See. The modified heavy chain is transiently expressed in 293T cells together with the appropriate L chain, resulting in anti-PSCA-IFNα2 + mask in 293T cells. Confirmation by SDS-PAGE analysis shows that the fusion protein has H chains and L chains of the appropriate size and is accurately assembled into H 2 L 2 L molecules. FACS analysis shows that the modified fusion protein binds to PSCA-expressing cells.

[0248] The resulting analysis shows that matriptase ST 14 can cleave the IFN mask from the H chain. Anti-PSCA-IFNα2 without the mask is used as a control.

[0249] Example 24: Method for inhibiting tumor growth using a target-directed masked IFN fusion protein in vivo Significant expression of TAAs in tumor cells, along with restricted expression in normal cells, makes the TAAs of the present disclosure, and preferably TAAs expressed in solid tumor cancers, good targets for targeted masked IFN fusion protein therapy. Accordingly, the therapeutic efficacy of a targeted masked IFN fusion protein that binds to TAAs expressed in a human cancer xenograft mouse model is evaluated.

[0250] The efficacy of the masked IFN fusion protein against tumor growth and metastasis formation is studied in a mouse cancer xenograft model (eg, subcutaneous and orthotopic).

[0251] 5×10 mixed with Matrigel at a 1:1 dilution 4 ~10 6 Subcutaneous (s.c.) tumors are generated (co - research) by injecting 5×10 to 10 cancer cells mixed with Matrigel at a 1:1 dilution into the right flank of SCID mice. To test the efficacy of the masked IFN fusion protein against tumor formation, injection of the masked IFN fusion protein is started on the same day as the tumor cell injection. As a control, mice are injected with either purified human IgG or PBS; or a purified MAb that recognizes an unrelated antigen not expressed in human cells. In preliminary studies, no difference is found between control IgG or PBS with respect to tumor growth. Tumor size is determined by vernier caliper measurement, and tumor volume is calculated as width 2 × length / 2, where width is the minimum dimension and length is the maximum dimension. Mice with subcutaneous tumors larger than 1.4 cm in diameter are sacrificed.

[0252] The advantage of the xenograft cancer model is that it allows for the study of angiogenesis and neovascularization. Tumor growth depends in part on the development of new blood vessels. The capillary system and the developing blood network are of host origin, but the initiation and organization of the neovascular system are controlled by the xenograft tumor (Davidoff et al., Clin Cancer Res. (2001) 7:2870; Solesvik et al., Eur J Cancer Clin Oncol. (1984) 20:1295). The effects of antibodies and small molecules on angiogenesis are studied according to procedures known in the art, such as by IHC analysis of tumor tissue and the surrounding microenvironment.

[0253] Masked IFN fusion proteins that bind to TAAs expressed in human cancers are shown to inhibit tumor growth in vivo.

[0254] Example 25: Human clinical trial for the treatment of human cancer tumors through the use of a masked IFN fusion protein that binds to a specific TAA Masked IFN fusion proteins that specifically accumulate in tumor cells and bind to specific TAAs are synthesized according to the present invention and used in the treatment of certain tumors and other immunological disorders, and / or other diseases. In connection with each of these indications, two clinical approaches are pursued successfully.

[0255] I.) Adjuvant therapy : In adjuvant therapy, patients are treated with a masked IFN fusion protein that binds to a specific TAA in combination with a chemotherapeutic agent or drug or biologic or combination thereof. Protocol design addresses efficacy as evaluated by examples including, but not limited to, reduction of tumor burden in primary or metastatic lesions, increased progression-free survival, overall survival, improvement in patient health, disease stabilization, and the ability to reduce the normal dosages of standard chemotherapies and other biologics. These dosage reductions enable additional and / or prolonged therapy by reducing the dose-related toxicities of chemotherapeutic or biologic agents.

[0256] II.) Monotherapy : In connection with the use of a masked IFN fusion protein that binds to a specific TAA in monotherapy of tumors, the masked IFN fusion protein that binds to a specific TAA is administered to patients without a chemotherapeutic agent or drug or biologic. In one embodiment, the monotherapy is conducted clinically in end-stage cancer patients with a wide range of metastatic disease. Protocol design addresses efficacy as evaluated by examples including, but not limited to, reduction of tumor burden in primary or metastatic lesions, increased progression-free survival, overall survival, improvement in patient health, disease stabilization, and the ability to reduce the normal dosages of standard chemotherapies and other biologics.

[0257] Dosage The dosing regimen may be adjusted to provide an optimal desirable response. For example, an injection of a single masked IFN fusion protein that binds to a specific TAA may be administered, several divided doses may be administered over a period of time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the treatment situation. As used herein, "unit dosage form" refers to physically discrete units suitable as unit dosages for the mammalian subject to be treated; each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect, with the required pharmaceutical carrier. The specifications for the unit dosage forms of the present invention are defined by and directly depend on (a) the unique characteristics of the masked IFN fusion protein that binds to a specific TAA and the specific therapeutic or prophylactic effect to be achieved, and (b) the inherent limitations in the art of compounding such compounds for the treatment of sensitivity in an individual.

[0258] Clinical Development Plan (CDP) CDP is developed in accordance with and for the treatment of cancer and / or immunological disorders using the masked IFN fusion proteins that bind to the specific TAAs of the present disclosure, in connection with adjuvant and monotherapy. Clinical trials first demonstrate safety and then confirm efficacy at repeated doses. The clinical trial is an open-label comparison of standard chemotherapy with standard therapy + masked IFN fusion protein that binds to a specific TAA. As will be appreciated, one non-limiting criterion that may be utilized in connection with patient enrollment is the concentration of the masked IFN fusion protein that binds to a specific TAA in the tumor, as determined by standard detection methods known in the art.

[0259] The present invention is not intended to be limited by the embodiments disclosed herein as a single illustration of the individual aspects of the present invention, and anything that is functionally equivalent is within the scope of the present invention. Various modifications to the models, methods, and life cycle methodologies of the present invention will become apparent to those skilled in the art from the foregoing description and teachings in addition to those described herein, and are similarly intended to fall within the scope of the present invention. Such modifications or other aspects can be implemented without departing from the true scope and spirit of the present invention.

[0260] (Table I) Selected tumor-associated antigens TIFF2025084914000061.tif157128

[0261] (Table II) List of interferons and functional variants TIFF2025084914000062.tif20590

[0262] (Table III) Abbreviations of amino acids TIFF2025084914000063.tif133128

[0263] Array information SEQUENCE LISTING <110> Qwixel Therapeutics LLC <120> Fusion Protein Composition(s) Comprising Targeted Masked Type I Interferons (IFNa and IFNb) And An Antibody Against Tumor Antigen, For Use In The Treatment of Cancer <150> US 62 / 920,140 <151> 2019-04-15 <160> 21 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Unknown <400> 1 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Unknown <400> 2 Gly Gly Gly Gly Ser 1 5 <210> 3 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Unknown <400> 3 Ser Gly Gly Gly Gly Ser 1 5 <210> 4 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Unknown <400> 4 Ala Gly Ala Ala Ala Lys Gly Ala Ala Ala Lys Ala Gly 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Unknown <400> 5 Ser Gly Gly Ala Gly Gly Ser 1 5 <210> 6 <211> 475 <212> PRT <213> Artificial Sequence <220> <223> Unknown <400> 6 Met Asp Pro Lys Gly Ser Leu Ser Trp Arg Ile Leu Leu Phe Leu Ser 1 5 10 15 Leu Ala Phe Glu Leu Ser Tyr Gly Gln Val Gln Leu Gln Gln Ser Gly 20 25 30 Ser Glu Leu Met Met Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala 35 40 45 Thr Gly Tyr Thr Phe Ser Asn Tyr Trp Ile Glu Trp Val Lys Gln Arg 50 55 60 Pro Gly His Gly Leu Glu Trp Ile Gly Glu Ile Leu Pro Gly Thr Gly 65 70 75 80 Arg Thr Ile Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Phe Thr Ala 85 90 95 Asp Ile Ser Ser Asn Thr Val Gln Met Gln Leu Ser Ser Leu Thr Ser 100 105 110 Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Arg Asp Tyr Tyr Gly Asn 115 120 125 Phe Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val 130 135 140 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 145 150 155 160 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 165 170 175 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 180 185 190 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 195 200 205 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 210 215 220 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 225 230 235 240 Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 245 250 255 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 260 265 270 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 275 280 285 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 290 295 300 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 305 310 315 320 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 325 330 335 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 340 345 350 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 355 360 365 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 370 375 380 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 385 390 395 400 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 405 410 415 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 420 425 430 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 435 440 445 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 450 455 460 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 465 470 475 <210> 7 <211> 165 <212> PRT <213> Artificial Sequence <220> <223> Unknown <400> 7 Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu 65 70 75 80 Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys 100 105 110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu 115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser 145 150 155 160 Leu Arg Ser Lys Glu 165 <210> 8 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Unknown <400> 8 Leu Ser Gly Arg Ser Asp Asn His Gly Ser Ser Gly Thr 1 5 10 <210> 9 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Unknown <400> 9 Thr Asp Val Asp Tyr Tyr Arg Glu Trp Ser Trp Thr Gln Val Ser 1 5 10 15 <210> 10 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Unknown <400> 10 Gly Gln Ser Gly Gln Thr Asp Val Asp Tyr Tyr Arg Glu Trp Ser Glu 1 5 10 15 Thr Gln Val Ser Gly Ser Ser Gly Gly Ser Val His Met Pro Leu Gly 20 25 30 Phe Leu Gly Pro Gly Gly Ser 35 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Unknown <400> 11 Val His Met Pro Leu Gly Phe Leu Gly Pro 1 5 10 <210> 12 <211> 66 <212> PRT <213> Artificial Sequence <220> <223> Unknown <400> 12 Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu 1 5 10 15 Gln Glu Ser Leu Arg Ser Lys Glu Gly Ser Ser Gly Leu Ser Gly Arg 20 25 30 Ser Asp Asn His Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser 35 40 45 Gly Thr Asp Val Asp Tyr Tyr Arg Glu Trp Ser Trp Thr Gln Val Ser 50 55 60 Gly Gly 65 <210> 13 <211> 65 <212> PRT <213> Artificial Sequence <220> <223> Unknown <400> 13 Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu 1 5 10 15 Gln Glu Ser Leu Arg Ser Lys Glu Gly Ser Ser Gly Leu Ser Gly Arg 20 25 30 Ser Asp Asn His Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser 35 40 45 Gly Thr Asp Val Asp Tyr Tyr Arg Glu Trp Ser Trp Thr Gln Val Gly 50 55 60 Gly 65 <210> 14 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Unknown <400> 14 Thr Asp Val Asp Tyr Tyr Arg Glu Trp Ser Trp Thr Gln Val 1 5 10 <210> 15 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Unknown <400> 15 Thr Asp Val Asp Tyr Tyr Arg Glu Trp Ser Trp Thr Gln Val Ser 1 5 10 15 <210> 16 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Unknown <400> 16 Gly Ser Ser Gly Leu Ser Gly Arg Ser Asp Asn His Gly Ser Ser Gly 1 5 10 15 Gly Ser Gly Gly Ser Gly Gly Ser Gly 20 25 <210> 17 <211> 688 <212> PRT <213> Artificial Sequence <220> <223> Unknown <400> 17 Met Asp Pro Lys Gly Ser Leu Ser Trp Arg Ile Leu Leu Phe Leu Ser 1 5 10 15 Leu Ala Phe Glu Leu Ser Tyr Gly Gln Val Gln Leu Gln Gln Ser Gly 20 25 30 Ser Glu Leu Met Met Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala 35 40 45 Thr Gly Tyr Thr Phe Ser Asn Tyr Trp Ile Glu Trp Val Lys Gln Arg 50 55 60 Pro Gly His Gly Leu Glu Trp Ile Gly Glu Ile Leu Pro Gly Thr Gly 65 70 75 80 Arg Thr Ile Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Phe Thr Ala 85 90 95 Asp Ile Ser Ser Asn Thr Val Gln Met Gln Leu Ser Ser Leu Thr Ser 100 105 110 Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Arg Asp Tyr Tyr Gly Asn 115 120 125 Phe Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val 130 135 140 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 145 150 155 160 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 165 170 175 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 180 185 190 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 195 200 205 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 210 215 220 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 225 230 235 240 Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 245 250 255 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 260 265 270 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 275 280 285 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 290 295 300 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 305 310 315 320 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 325 330 335 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 340 345 350 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 355 360 365 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 370 375 380 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 385 390 395 400 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 405 410 415 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 420 425 430 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 435 440 445 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 450 455 460 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Gly 465 470 475 480 Ser Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu 485 490 495 Met Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys 500 505 510 Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe 515 520 525 Gln Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile 530 535 540 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 545 550 555 560 Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu 565 570 575 Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met 580 585 590 Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 595 600 605 Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 610 615 620 Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 625 630 635 640 Ser Leu Arg Ser Lys Glu Gly Ser Ser Gly Leu Ser Gly Arg Ser Asp 645 650 655 Asn His Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Thr 660 665 670 Asp Val Asp Tyr Tyr Arg Glu Trp Ser Trp Thr Gln Val Ser Gly Gly 675 680 685 <210> 18 <211> 664 <212> PRT <213> Artificial Sequence <220> <223> Unknown <400> 18 Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Met Met Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Asn Tyr 20 25 30 Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Leu Pro Gly Thr Gly Arg Thr Ile Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Phe Thr Ala Asp Ile Ser Ser Asn Thr Val Gln 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Tyr Gly Asn Phe Tyr Tyr Ala Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Ser Gly Gly Gly Gly Ser Cys Asp Leu Pro Gln Thr His 450 455 460 Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg 465 470 475 480 Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro 485 490 495 Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val 500 505 510 Leu His Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp 515 520 525 Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu 530 535 540 Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val 545 550 555 560 Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val 565 570 575 Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr 580 585 590 Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe 595 600 605 Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu Gly Ser 610 615 620 Ser Gly Leu Ser Gly Arg Ser Asp Asn His Gly Ser Ser Gly Gly Ser 625 630 635 640 Gly Gly Ser Gly Gly Ser Gly Thr Asp Val Asp Tyr Tyr Arg Glu Trp 645 650 655 Ser Trp Thr Gln Val Ser Gly Gly 660 <210> 19 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Unknown <400> 19 Gly Ser Gly Thr Asp Val Asp Tyr Tyr Arg Glu Trp Ser Trp Thr Gln 1 5 10 15 Val Ser <210> 20 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Unknown <400> 20 Gly Ser Gly Thr Asp Val Asp Tyr Tyr Arg Glu Trp Ser Trp Thr Gln 1 5 10 15 Val <210> 21 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> Unknown <400> 21 Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Met Met Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Asn Tyr 20 25 30 Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Leu Pro Gly Thr Gly Arg Thr Ile Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Phe Thr Ala Asp Ile Ser Ser Asn Thr Val Gln 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Tyr Gly Asn Phe Tyr Tyr Ala Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly 450

Claims

1. Polypeptide sequence 1. A composition comprising: wherein the polypeptide sequence masks the activity of a type I interferon (IFN); and the composition further comprises a fusion protein fused to an antibody that binds to a tumor-associated antigen.

2. The composition of claim 1, further comprising a flexible peptide linker.

3. 3. The composition of claim 1 or 2, further comprising a tumor-associated protease cleavage site.

4. The composition of any one of claims 1 to 3, wherein the type I interferon comprises IFNα1.

5. The composition of any one of claims 1 to 3, wherein the type I interferon comprises IFNα2.

6. The composition of any one of claims 1 to 3, wherein the type I interferon comprises IFNα4.

7. The composition of any one of claims 1 to 3, wherein the type I interferon comprises IFNα5.

8. The composition of any one of claims 1 to 3, wherein the type I interferon comprises IFNα6.

9. The composition of any one of claims 1 to 3, wherein the type I interferon comprises IFNα14.

10. The composition of any one of claims 1 to 3, wherein the type I interferon comprises IFNβ1.

11. The composition of any one of claims 1 to 3, wherein the type I interferon or functional variant thereof is selected from the type I interferons as shown in Table II.

12. The composition of any one of claims 1 to 11, wherein the tumor-associated antigen comprises CD138.

13. The composition of any one of claims 1 to 11, wherein the tumor-associated antigen comprises CD20.

14. The composition of any one of claims 1 to 11, wherein the tumor-associated antigen comprises mesothelin.

15. The composition of any one of claims 1 to 11, wherein the tumor-associated antigen comprises 5T4.

16. The composition of any one of claims 1 to 11, wherein the tumor associated antigen is selected from the tumor associated antigens as shown in Table I.

17. a. an antibody comprising a heavy chain and / or a light chain that specifically binds to a tumor-associated antigen; b. a type I interferon, wherein the N-terminus of the type I interferon is fused to the C-terminus of the antibody heavy and / or light chain; and c. An interferon mask comprising (SEQ ID NO: 14), thereby added to the C-terminus of the type I interferon. A targeted masked IFN comprising:

18. 18. The targeted masked IFN of claim 17, further comprising a flexible peptide linker, wherein the N-terminus of the type I interferon is fused to the C-terminus of the antibody heavy and / or light chain.

19. 19. The targeted masked IFN of claim 17 or 18, further comprising a flexible peptide linker, wherein an interferon mask (SEQ ID NO:14) is added to the C-terminus of the type I interferon.

20. 20. The targeted masked IFN of claim 19, further comprising a tumor-associated protease cleavage site inserted between the antibody and the flexible peptide linker.

21. The targeted masked IFN of any one of claims 17 to 19, further comprising a tumor-associated protease cleavage site inserted between said type I interferon and said interferon mask.

22. The targeted masked IFN of any one of claims 17 to 21, wherein said type I IFN or a functional variant thereof is shown in Table II.

23. The targeted masked IFN of any one of claims 17 to 22, wherein said antibody binds to a tumor-associated antigen as shown in Table I.

24. The targeted masked IFN of any one of claims 17 to 22, wherein said antibody binds to CD138.

25. The targeted masked IFN of any one of claims 17 to 22, wherein the antibody binds to CD20.

26. The targeted masked IFN of any one of claims 17 to 22, wherein the antibody binds to mesothelin.

27. The targeted masked IFN of any one of claims 17 to 22, wherein said antibody binds to 5T4.

28. A method of producing the composition of any one of claims 1 to 16 or the targeted masked IFN of any one of claims 17 to 27.

29. A pharmaceutical composition comprising a composition according to any one of claims 1 to 16 or a targeted masked IFN according to any one of claims 17 to 27, (i) optionally, the pharmaceutical composition is for use in therapy, including the treatment of cancer, and optionally (a) the cancer comprises a cancer found in a solid tumor; or (b) the cancer arises in the hematopoietic system; and (ii) optionally, the pharmaceutical composition further comprises one or more anti-neoplastic agents; The pharmaceutical composition.

30. A kit comprising the composition of any one of claims 1 to 16 or the targeted masked IFN of any one of claims 17 to 27.

31. A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a composition according to any one of claims 1 to 16 or a targeted masked IFN according to any one of claims 17 to 27, optionally wherein the subject is a human subject.

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