Fusion protein compositions comprising masked type I interferons (IFNα and IFNβ) for use in and methods of treating cancer - Patents.com

Abstract

Disclosed herein are fusion protein compositions comprising masked IFN and methods for making masked IFN. As a result, masked IFN can be fused to a monoclonal antibody or binding fragment thereof and administered to a patient as a therapeutic modality, providing a method for treating cancer, immune disorders, and other diseases. In view of the current deficiencies associated with delivering IFN to cancer cells, one object of the present invention is to provide a new and improved method for treating cancer, immune disorders, and other diseases utilizing masked IFN, in which the activity of IFN is inhibited until the IFN reaches the tumor. Compositions, kits, and methods for use that meet such needs are provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 259,105, filed June 18, 2021, the contents of which are incorporated by reference in their entirety herein.

[0002] Submission of paper copy and ASCII file of sequence listing The contents of the following submissions are incorporated herein by reference in their entirety: a paper copy of the Sequence Listing recorded on June 17, 2022. Additionally, the contents of the Sequence Listing in computer readable form (CRF) in an ASCII text file entitled COPY 1 REPLACEMENT (Filename: 1441-20003.40-SEQ LIST-As-Filed XX-Jun-2022.txt, recorded on June XX, 2022, size: XXX KB), and the contents of the Sequence Listing in computer readable form (CRF) in an ASCII text file entitled COPY 2 REPLACEMENT (Filename: 1441-20003.40-SEQ LIST-As-Filed XX-Jun-2022.txt, recorded on June XX, 2022, size: XXX KB).

[0003] STATEMENT AS TO RIGHTS FOR INVETIONS MADE UNDER FEDERALLY SPONSORED RESEARCH Not applicable

[0004] FIELD OF THEINVENTION The invention described herein relates to the field of cancer therapy and other immune disorders or diseases.Specifically, the invention relates to a masked type I interferon (IFN) composition that can be fused with tumor antigen binding protein and used as a vehicle for targeted cancer therapy in humans.The invention further relates to the treatment of disorders or diseases, such as cancer and other immune disorders and diseases. [Background technology]

[0005] 2. Background of the Invention Cancer is the second leading cause of death worldwide after coronary artery disease. Millions of people die from cancer each year, with well over half a million deaths annually in the United States alone, with 1,688,780 new cancer cases diagnosed in 2017 (American Cancer Society). Deaths from heart disease have declined significantly, while deaths attributable to cancer are generally increasing. Unless medical advances change current trends, cancer is projected to become the leading cause of death in the early part of this century.

[0006] Mortality from some cancers is particularly high. Hematological malignancies, including myeloma, cause 50,000 deaths annually in the United States alone (American Cancer Society, 2018). Furthermore, 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 gastric cancer (8.2% of all cancer deaths) are the leading causes of cancer deaths in both sexes and of all ages worldwide (GLOBOCAN 2018). These and virtually all other carcinomas share the common fatal feature of metastasizing from the primary tumor to sites distal to the tumor, and with very few exceptions, metastatic disease is fatal. Furthermore, even cancer patients who initially survive their primary cancer have a common experience of having their lives dramatically altered. Many cancer patients experience significant anxiety driven by the realization of the possibility of recurrence or that the treatment may not work. Many cancer patients also experience physical debilitation following treatment. Furthermore, many cancer patients experience a recurrence of the disease.

[0007] Although cancer treatments have improved over the past decades and survival rates have increased, the heterogeneity of cancers still demands new therapeutic strategies that utilize multiple treatment modalities. This is especially true for the treatment of solid tumors in critical anatomical sites (e.g., glioblastoma, squamous cell carcinoma of the head and neck, and lung adenocarcinoma), which are sometimes limited to standard radiation therapy and / or chemotherapy. Nevertheless, these treatments have the deleterious effects of chemoresistance and radioresistance that promote locoregional recurrence, distant metastases, and second primary tumors, in addition to severe side effects that reduce the patient's quality of life.

[0008] Furthermore, the therapeutic utility of monoclonal antibodies (mAbs) is being recognized (G. Kohler and C. Milstein, Nature 256: 495-497 (1975)). Monoclonal antibodies are currently approved for the treatment of transplantation, cancer, infectious diseases, cardiovascular diseases, and inflammation. Different isotypes have distinct effector functions. Such functional differences are reflected in the distinct three-dimensional structures of various immunoglobulin isotypes (PM ALZARI, et. al., Annual Rev. Immunol., 6: 555-580 (1988)).

[0009] In addition, interferons, including IFNα and IFNβ (type I) and IFNγ (type II), are essential mediators of anticancer immunity and have both direct antiproliferative and numerous antitumor immunotherapeutic effects against many cancers. However, although the efficacy of IFNα has been demonstrated against several human cancers, its clinical utility has, to date, been limited by the inability to achieve effective concentrations of IFN at the tumor site without causing systemic toxicity.

[0010] Due to systemic toxicity, several groups have addressed this issue by using the tumor targeting ability of monoclonal antibodies to deliver IFN directly 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). Note that initial studies used anti-CD20-IFNα2 proteins to target IFNα to CD20 expressed in lymphomas and anti-CD138-IFNα2 fusion proteins to target CD138 expressed in multiple myelomas. See Vasuthasawat, et. al., MAbs 8 (7), pp. 1386-1397 (2016). Although these approaches have shown great therapeutic promise and are currently being tested in human clinical trials and under commercial development, they suffer from several deficiencies.

[0011] Although it has been found that by using antibody binding specificity to target tumor-associated antigens, a greater percentage (%) of IFN is delivered to the tumor site than would be achieved if IFN were injected by itself, the bound interferon is still recognized by and binds to interferon receptors expressed throughout the body that are not tumor associated. Thus, IFN fused to monoclonal antibodies may still induce toxicity and / or have increased clearance due to systemic exposure and interaction with systemic IFN receptors. From the above, it will be readily apparent to one of skill in the art that new treatment paradigms are needed for the treatment of cancer and immunological diseases. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] G. Kohler and C. Milstein, Nature 256: 495-497 (1975) [Non-Patent Document 2] PM ALZARI, et. al., Annual Rev. Immunol., 6: 555-580 (1988) Summary of the Invention [Means for solving the problem]

[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, immune disorders, and other diseases that utilize masked IFN, in which the activity of the IFN is inhibited until the IFN reaches the tumor. Compositions, kits and methods for use that meet such needs are provided.

[0014] Summary of the Invention The present invention provides antibodies, antigen-binding fragments, and fusion protein compositions that bind to a wide range of tumor-associated antigens (TAA). In a further embodiment, the fusion protein composition comprises a type I interferon. In a further embodiment, the IFN is masked such that its activity is reduced or absent until it reaches the tumor cells. In a further embodiment, the TAA is listed in Table I. In a preferred embodiment, the TAA is associated with a solid tumor. In one embodiment, the TAA comprises CD138. In a further embodiment, the TAA is CD20. In a further embodiment, the TAA is mesothelin. In another embodiment, the TAA is 5T4. In another embodiment, the TAA is a FAP. In yet another embodiment, the IFN or functionally active variant is listed in Table II. In a preferred embodiment, the IFN comprises IFNA2.

[0015] In a further embodiment, the invention comprises a masked targeted IFN. In a preferred embodiment, the masked targeted IFN comprises IFNA1.

[0016] In a further embodiment, the invention comprises a masked targeted IFN. In a preferred embodiment, the masked targeted IFN comprises IFNA14.

[0017] In a further embodiment, the invention comprises a masked targeted IFN. In a preferred embodiment, the masked targeted IFN comprises IFNB1.

[0018] In another embodiment, the present disclosure teaches a method for producing a masked targeted IFN.

[0019] In another embodiment, the disclosure teaches methods of treating cancer, immune disorders, and other diseases in humans.

[0020] In a preferred embodiment, the present disclosure teaches a method of treating cancer with a masked IFN fused to a monoclonal antibody that binds to a TAA.

[0021] In some of any of the embodiments, methods for treating cancer involve administering to a subject, such as a human subject, a therapeutically effective amount of any of the compositions or any of the fusion proteins described herein, e.g., a masked targeted IFN.

[0022] Also provided is a pharmaceutical composition comprising a therapeutically effective amount of any of the compositions or any of the fusion proteins described herein, such as masked targeted IFN.In some of the embodiments, the pharmaceutical composition is for use in therapy, including the treatment of cancer.In some of the embodiments, the cancer comprises cancer found in solid tumors, or the cancer originates in the hematopoietic system.In some of the embodiments, the pharmaceutical composition further comprises one or more anti-cancer agents.

[0023] Kits are also provided, eg, kits that include any of the compositions or any of the fusion proteins, eg, masked targeted IFN, described herein. [Brief description of the drawings]

[0024] [Figure 1] Amino acid sequence of IFNAR2. The signal sequence is shaded in gray. Peptides selected as masking peptides based on crystal structure data showing that they are regions that interact with type I interferons are underlined.

[0025] [Diagram 2] IFNα masking peptide.

[0026] [Diagram 3] Description of fusion proteins and masking peptides.

[0027] [Figure 4] Description of fusion proteins and masking peptides.

[0028] [Diagram 5] Matriptase ST14 cleaves multiple IFNα masks from the heavy chain of an anti-CD138 fusion antibody.

[0029] [Figure 6] The masked anti-CD138 (mask 1) fusion antibody and the masked anti-CD138 (mask 2) fusion antibody bind to the IFNα2 receptor with lower affinity compared to the unmasked fusion protein.

[0030] [Figure 7] Masked anti-CD138 (mask 1) fusion antibody and masked anti-CD138 (mask 2) and masked anti-CD138 (mask 3) fusion antibodies bind to the IFNα2 receptor with lower affinity compared to the unmasked fusion protein.

[0031] [Figure 8]Masked anti-CD138 (Mask 1) fusion antibody, masked anti-CD138 (Mask 2), masked anti-CD138 (Mask 2.2), and masked anti-CD138 (Mask 3) fusion antibody bind to the IFNα2 receptor with lower affinity compared to the unmasked fusion proteins.

[0032] [Figure 9] Binding of the fusion antibody to the mask by ELISA. Figure 9 shows the binding to the mask (peptide 6).

[0033] [Figure 10] Masked anti-5T4 (mask1) and masked anti-mesothelin (mask1) fusion antibodies bind to the IFNα2 receptor with lower affinity compared to unmasked anti-CD138 fusion proteins.

[0034] [Figure 11] Methods for reducing and restoring masked IFNα activity. Figure 11(A) shows anti-CD138 fusion antibody (mask 1 and mask 2) without MST. Figure 11(B) shows anti-CD138 fusion antibody (mask 1 and mask 2) with MST.

[0035] [Figure 12] Methods for reducing and restoring masked IFNα activity. Figure 12(A) shows anti-CD138 fusion antibody and anti-5T4 mask 1 with and without MST. Figure 12(B) shows anti-CD138 fusion antibody and anti-mesothelin mask 1 with and without MST.

[0036] [Figure 13] Methods for reducing and restoring masked IFNα activity. Figure 13(A) shows anti-CD138 fusion antibodies (mask 1, mask 2, and mask 3) without MST. Figure 13(B) shows anti-CD138 fusion antibodies (mask 1, mask 2, and mask 3) with MST.

[0037] [Figure 14] Methods for reducing and restoring masked IFNα activity. Figure 14(A) shows anti-CD138 fusion antibodies (mask 1, mask 2, mask 2.2, and mask 3) without MST. Figure 14(B) shows anti-CD138 fusion antibodies (mask 1, mask 2, mask 2.2, and mask 3) with MST.

[0038] [Figure 15] Methods for reducing and restoring masked IFNα activity. Figure 15(A) shows anti-CD138 fusion antibodies (mask 1, mask 1 N297Q, mask 1 w / MST, and mask 1 N297Q w / MST). Figure 15(B) shows anti-CD138 fusion antibodies (mask 2.2, mask 2.2 N297Q, mask 2.2 w / MST, and mask 2.2 N297Q w / MST). Figure 15(C) shows anti-CD138 fusion antibodies (mask 3, mask 3.2 N297Q, mask 3 w / MST, and mask 3.2 N297Q w / MST).

[0039] [Figure 16] Methods for reducing and restoring masked IFNα activity. Figure 16(A) shows anti-CD138 fusion antibodies (Mask1 N297Q, Mask1 N297Q w / MST, Mask3.2 N297Q, and Mask3.2 N297Q w / MST).

[0040] [Figure 17] Induction of IP-10 in human peripheral blood mononuclear cells ("PMBCs") using ELISA.

[0041] [Figure 18] Induction of IP-10 in human peripheral blood mononuclear cells ("PMBCs") using ELISA.

[0042] [Figure 19] Comparative analysis of glycosylated and non-glycosylated MASK in human peripheral blood mononuclear cells ("PMBC") using ELISA.

[0043] [Figure 20] Dose-dependent induction of IP-10 in human peripheral blood mononuclear cells ("PMBCs") using ELISA.

[0044] [Figure 21] Dose-dependent comparative analysis of glycosylated vs. non-glycosylated MASK in human peripheral blood mononuclear cells ("PMBCs") using ELISA.

[0045] [Figure 22] Dose-dependent comparative analysis of glycosylated vs. non-glycosylated MASK in human peripheral blood mononuclear cells ("PMBCs") using ELISA.

[0046] [Figure 23] Dose-dependent comparative analysis of glycosylated vs. non-glycosylated masked MCP-1 induction in human peripheral blood mononuclear cells ("PMBCs") using ELISA.

[0047] [Figure 24] QXL138AM2.2 light chain characteristics and sequence information.

[0048] [Diagram 25] Non-glycosylated QXL138AM2.2 heavy chain amino acid sequence information.

[0049] [Figure 26] Sequence information for non-glycosylated QXL138AM2.2 heavy chain nucleic acid.

[0050] [Figure 27] Methods for reducing and restoring masked IFNα activity.

[0051] [Figure 28] Dose-dependent induction of IP-10 in human PBMCs using ELISA.

[0052] [Figure 29] The masked, non-glycosylated 2.2 fusion protein binds to the IFNα2 receptor with lower affinity compared to the unmasked, non-glycosylated 2.2 fusion protein.

[0053] [Diagram 30] Dose-dependent induction of IP-10 in human PBMCs using ELISA.

[0054] [Diagram 31] Binding of QXL138AM2.2-N297Q to soluble CD138.

[0055] [Diagram 32] Comparison of binding of multiple manufacturing lots of QXL138AM2.2-N297Q (Lot 2 and Lot 3) to soluble CD138.

[0056] [Diagram 33] Methods for reducing and restoring masked IFNα activity.

[0057] [Diagram 34] Methods for reducing and restoring masked IFNα activity of QXL138AM2.2-N297Q.

[0058] [Diagram 35] Reduction and restoration of induction of IP-10 in PBMCs.

[0059] [Diagram 36] Tumor inhibition by QXL138AM in OVCAR3 cells in vivo.

[0060] [Figure 37] Tumor inhibition by QXL138AM in H929 cells in vivo.

[0061] [Figure 38]Tumor inhibition by QXL138AM in vivo in Capan-2 cells.

[0062] [Figure 39] Additional IFNα masking peptides.

[0063] [Diagram 40] Description of additional fusion proteins and masking peptides.

[0064] [Diagram 41] Description of additional fusion proteins and masking peptides.

[0065] [Diagram 42] The masked IFNα YNS mutant is cleaved from the heavy chain of the anti-CD138-IFNα fusion antibody by matriptase ST14.

[0066] [Diagram 43] Binding of fusion antibodies to the mask by ELISA.

[0067] [Diagram 44] Binding of fusion antibodies to the mask by ELISA.

[0068] [Diagram 45] Binding of fusion protein to mask by ELISA.

[0069] [Figure 46] Methods for reducing and restoring masked IFNα activity.

[0070] [Figure 47] Methods for reducing and restoring masked IFNα activity.

[0071] [Figure 48] Methods for reducing and restoring masked IFNα activity.

[0072] [Figure 49] Methods for reducing and restoring masked IFNα activity.

[0073] [Figure 50] Methods for reducing and restoring masked IFNα activity.

[0074] [Figure 51] Reduction and restoration of induction of IP-10 in PBMCs.

[0075] [Figure 52] Methods for reducing and restoring masked IFNα activity.

[0076] [Diagram 53] Matriptase ST 14 cleaves mask 4 of QXL138AM4.2-N297Q from the heavy chain of the anti-CD138-IFNα fusion antibody.

[0077] [Figure 54] Methods for reducing and restoring masked IFNα activity.

[0078] [Figure 55] Methods for reducing and restoring masked IFNα activity.

[0079] [Figure 56] Methods for reducing and restoring masked IFNα activity. Figure 56(A) shows QXL138A-N297Q, QXL138AM2.2-N297Q (lot 10), and QXL138AM4.2-N297Q. Figure 56(B) shows QXL138A-N297Q, QXL138AM2.2-N297Q (lot 10) + MST, and QXL138AM4.2-N297Q + MST.

[0080] [Figure 57] Methods for reducing and restoring masked IFNα activity.

[0081] [Figure 58] Reduction and restoration of induction of IP-10 in PBMCs.

[0082] [Figure 59] QXL138AM4.2 heavy chain amino acid characteristics and sequence information.

[0083] [Figure 60] QXL138AM4.2 heavy chain nucleic acid characteristics and sequence information.

[0084] [Figure 61] QXL138YNS4.2-N297Q heavy chain amino acid characteristics and sequence information.

[0085] [Figure 62] QXL138YNS4.2-N297Q heavy chain nucleic acid characteristics and sequence information. [Figure 63] Figure 63. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0086] Detailed Description of the Invention Provided herein are fusion proteins and compositions comprising interferon (IFN). In some aspects, the presented fusion proteins and compositions comprise IFN and an antibody or antigen-binding fragment thereof, e.g., an antibody or 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 presented fusion proteins and compositions comprise IFN and a mask, e.g., a polypeptide sequence that blocks the interaction of IFN with its receptor, e.g., IFN-alpha receptor (IFNAR). In some aspects, the presented fusion proteins or compositions comprise IFN, an antibody or antigen-binding fragment thereof, and a mask. In some of any of the presented embodiments, the fusion proteins or compositions also contain a flexible peptide linker. In some embodiments, the fusion proteins or compositions also contain a protease cleavage site, such as a tumor-associated protease cleavage site. In some aspects, cleavage of the protease cleavage site, e.g., at or near the tumor site or within the tumor microenvironment (TME), can result in "unmasking" of the IFN, allowing binding of the IFN to its receptor. In some embodiments, the fusion protein or composition can be targeted to a specific site or location of tumor or cancer, for example, by an antibody or its antigen-binding fragment specific to TAA.Thus, in some embodiments, the presented fusion protein and composition can be used to treat disease or disorder, such as cancer or tumor.Methods of making such fusion protein or composition, methods related to the use of such fusion protein or composition, for example, in treatment or therapeutic methods, and pharmaceutical compositions or kits comprising such fusion protein or composition are also provided.

[0087] As further described herein, the presented embodiments including masked targeted IFN provide unique advantages over available approaches for several reasons, including that the activity of the masked IFN is significantly reduced and / or eliminated until it reaches the tumor site, thus minimizing non-specific activity, and the fusion protein is not trapped by interferon receptors that are not present at the tumor site. At the tumor site, the mask can be removed and the binding and activity of IFN is reactivated, thereby maximizing efficacy and increasing the effective concentration of the fusion protein in the tumor without increasing toxicity. Furthermore, an antibody conjugated to IFN can target the fusion protein to a specific tumor (e.g., a tumor that expresses a TAA that the antibody specifically binds). Specific targeting allows for a higher probability that IFN is directed to the cancer of interest and avoids non-cancer or non-tumor tissues.

[0088] Section Overview I.) Definition II.) Antibodies III.) Interferon IV.) IFN Masking Methods Consideration of available methods b. IFN masking methods of the present disclosure i. IFNα Masking Peptides and Masking Constructs V.) Treatment of Cancers Expressing Tumor-Associated Antigens (TAA) VI.) Masked Targeted IFN Fusion Protein Cocktails VII.) Combination Therapy VIII.) Kits / Manufactured Products All publications referenced in this application, including patent documents, scientific articles, and databases, are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. To the extent that a definition set forth herein contradicts or is otherwise inconsistent with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein shall take precedence over the definition incorporated herein by reference.

[0089] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0090] I.) Definition: Unless otherwise defined, all technical terms, notations and other scientific terms or terminology used herein shall have the meanings commonly understood by those skilled in the art to which the present invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ready reference, and the incorporation of such definitions into this specification should not necessarily be interpreted as representing a substantial difference from what is commonly understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly used by those skilled in the art using conventional methodologies, such as the widely used molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where necessary, procedures involving the use of commercially available kits and reagents are generally carried out according to the protocols and / or parameters defined by the manufacturer, unless otherwise noted.

[0091] When trade names are used herein, reference to the trade name also refers to that product formulation, generic drugs, and the active pharmaceutical ingredients of the trade name product, unless the context indicates otherwise.

[0092] The terms "advanced cancer", "locally advanced cancer", "advanced disease" and "locally advanced disease" refer to cancer that has spread to the relevant tissue capsule and are intended to include stage C disease under the American Urological Association (AUA) system, stages C1-C2 disease under the Whitmore-Jewett system, and stages T3-T4 and N+ disease under the TNM (tumor, node, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease, which has a substantially less favorable outcome compared with patients with clinically localized (organ-confined) cancer.

[0093] "Altering the native glycosylation pattern," for purposes herein, shall mean deleting one or more carbohydrate moieties found in the native antibody sequence (by removing the underlying glycosylation site or by deleting glycosylation by chemical and / or enzymatic means) and / or adding one or more glycosylation sites that are not present in the native antibody sequence, where "native glycosylation pattern" refers to the natural post-translational glycosylation pattern that results from the particular combination of antibody sequence, cell type, and growth conditions used. Furthermore, the phrase encompasses qualitative alterations in the glycosylation of the native protein, involving changes in the nature and proportions of the various carbohydrate moieties present.

[0094] The term "analog" refers to a molecule that is structurally similar or shares similar or corresponding characteristics with another molecule (e.g., a TAA-associated protein). For example, an analog of a TAA protein may be specifically bound by an antibody or T cell that specifically binds to the TAA.

[0095] The term "antibody" is used in the broadest sense unless expressly indicated otherwise. Thus, an "antibody" may be naturally occurring or synthetic, e.g., 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 domain 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 the desired biological activity, and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they specifically bind to a TAA and / or exhibit the desired biological activity. Any particular antibody can be used in the methods and compositions presented herein. Thus, in one embodiment, the term "antibody" encompasses molecules that include at least one variable region from a light chain immunoglobulin molecule and at least one variable region from a heavy chain molecule that combine to form a specific binding site for a target antigen. In one embodiment, the antibody is an IgG antibody. For example, the antibody is an IgG1, IgG2, IgG3, IgG4 antibody or any known antibody isotype. The 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 embodiment, the antibody of the present invention is a mammalian antibody. Phage technology can be used to isolate the original antibody or to produce variants with altered specificity or avidity characteristics. Such techniques are routine and well known in the art. In one embodiment, the antibody is produced by recombinant means known in the art. For example, recombinant antibodies can be produced by transfecting a vector containing a DNA sequence encoding the antibody into a host cell.One or more vectors can be used to transfect the DNA sequence that expresses at least one VL region and at least one VH region in a host cell. 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, most recent edition). The antibody of the present invention can be modified by recombinant means to increase the effectiveness of the antibody in mediating the desired function. Thus, it is within the scope of the present invention that the antibody can be modified by substitution using recombinant means. Generally, the substitution is 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. WO9958572; 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 cytotoxicity. Frequently, antibodies are labeled by either covalent or non-covalent attachment with a substance that produces a detectable signal. A wide variety of labeling and conjugation techniques are known and have been extensively reported in both scientific 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). Suitable antibodies with desired biologic activity can be identified using in vitro assays including, but not limited to, proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, trafficking, signal transduction, and in vivo assays such as inhibition of tumor growth. The antibodies presented herein can also be useful in diagnostic applications. The antibodies presented herein can be screened as capture or non-neutralizing antibodies for their ability to bind to a particular antigen without inhibiting the binding or biological activity of that antigen's receptor. As neutralizing antibodies, the antibodies can be useful in competitive binding assays. The antibodies can also be used to quantify TAAs or their receptors.

[0096] The term "antigen-binding fragment" or "antibody fragment" of an antibody (or simply "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 a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include (i) Fab fragments, V L , V H , C L and C H1 (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and C H1 (iv) a single arm V of an antibody; L and V H (v) an Fv fragment consisting of a V H(vi) isolated complementarity determining regions (CDRs). In addition, Fv fragments, V L and V H The two domains are encoded by separate genes, but they can be synthesized using recombinant methods to L Area and V H The pair of domains can be joined by a synthetic linker that allows them to be made into a single protein chain forming a monovalent molecule (known as single-chain Fv (scFv); see, e.g., 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 encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the utility of these fragments is screened in the same manner as intact antibodies.

[0097] The term "Fc," as used herein, refers to the region comprising the hinge region, CH2 and / or CH3 domain.

[0098] As used herein, any form of "antigen" can be used to generate antibodies specific for a TAA. Thus, the inducing antigen can be a single epitope, multiple epitopes, or the entire protein alone or in combination with one or more immunogenicity enhancing agents known in the art. The inducing antigen can be an isolated full-length protein, a cell surface protein (e.g., immunization with cells transfected with at least a portion of the antigen), or a soluble protein (e.g., immunization with only the extracellular domain portion of the protein). The antigen can be produced in a genetically modified cell. The DNA encoding the antigen can be genomic or non-genomic DNA (e.g., cDNA) and encodes at least a portion of the extracellular or intracellular domain. As used herein, the term "portion," in reference to an antigen, refers to the minimum number of amino acids or nucleic acids, as appropriate, to constitute an immunogenic epitope of the antigen of interest. Any genetic vector suitable for transformation of the cells of interest can be used, including, but not limited to, non-viral vectors such as adenoviral vectors, plasmids, and cationic lipids. In one embodiment, an antibody of the methods and compositions herein specifically binds to at least a portion of the extracellular domain of a TAA of interest.

[0099] The antibodies or antigen-binding fragments thereof presented herein may 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 / or enhances or mediates a desired biological effect to enhance a toxin that kills cells. 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 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-apoptotic signals, modulation, stimulation, and / or inhibition of apoptotic or necrotic signals, modulation, stimulation, and / or inhibition of the ADCC cascade, and modulation, stimulation, and / or inhibition of the CDC cascade.

[0100] As used herein, the term "conservative substitution" is known to those skilled in the art and generally refers to the substitution of amino acids and / or amino acid sequences that can be made without changing the biological activity of the resulting molecule. In general, those skilled in the art understand that a single amino acid substitution in a non-essential region of a polypeptide will not substantially alter biological activity (see, for example, 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 listed in Table III. For example, such changes include the substitution of any of these hydrophobic amino acids with any of isoleucine (I), valine (V), and leucine (L); the substitution of glutamic acid (E) with aspartic acid (D) and vice versa; the substitution of asparagine (N) with glutamine (Q) and vice versa; and the substitution of threonine (T) with serine (S) and vice versa. Other substitutions can also be considered conservative, depending on the particular amino acid's environment and its role in the protein's three-dimensional structure. For example, glycine (G) and alanine (A) are frequently interchangeable, as are alanine (A) and valine (V). Methionine (M) is relatively hydrophobic and can be frequently interchanged with leucine and isoleucine, and occasionally with valine. Lysine (K) and arginine (R) are frequently interchangeable in positions where the important feature of the amino acid residue is the charge and it is not important that the pK of these two amino acid residues differ. Still other changes may be considered "conservative" in certain circumstances (see, e.g., Table III herein; pages 13-15 "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 permissible and can be determined empirically or in accord with known conservative substitutions.

[0101] The term "fusion protein" as used herein refers to a protein of the present invention fused at the C-terminus to an IFN of the present invention using linkers and methods known in the art. See, for example, US 9,803,021, which is incorporated herein by reference. Exemplary linkers that can be used to fuse an IFN to a protein of the present invention include, but are not limited to, (i) GGGGSGGGSGGGGS (SEQ ID NO: 1); (ii) GGGGS (SEQ ID NO: 2); (iii) SGGGGS (SEQ ID NO: 3); AGAAAKGAAAKAG (SEQ ID NO: 4); SGGAGGS (SEQ ID NO: 5); Landar; Double Landar; 1qo0E_1; IgG3 hinge; IgG3 hingeΔcys; and / or IgG1 hingeΔcys.

[0102] The terms "inhibit" or "inhibition of," as used herein, mean to reduce by a measurable amount or to prevent completely.

[0103] The term "interferon" as used herein refers to a group of signaling proteins made and released by host cells in response to the presence of some viruses. In a typical scenario, a virus-infected cell releases interferons that cause nearby cells to mount antiviral defenses. IFNs belong to a large class of proteins known as cytokines, molecules used for communication between cells that trigger protective defenses by the immune system to help eradicate pathogens.

[0104] The term "type 1 interferon" or "type I interferon" as used herein refers 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 and IFNAR2 chains. An exemplary list of type I interferons of the present disclosure is set forth in Table II.

[0105] The term "mammal" refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses, and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human.

[0106] The term "mask", when referring to masked IFN (also referred to as "masked" IFN), means, for the purposes of the present invention, any peptide or protein that blocks 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.

[0107] The term "masked targeted IFN" as used herein refers to a type I interferon that has a polypeptide attached to the carboxy terminus of the IFN, thereby reducing its ability to bind to IFNAR. The masked IFN further comprises a targeting binding protein (i.e., an antibody) attached to the carboxy terminus. It is within the scope of the present invention that the "masked targeted IFN" can be modified by substitution using recombinant means. Amino acid modifications include amino acid deletions, additions, and substitutions.

[0108] The terms "metastatic cancer" and "metastatic disease" mean cancer that has spread to regional lymph nodes or distant sites and are intended to include stage D disease under the AUA system and stage TxNxM+ under the TNM system.

[0109] "Molecular recognition" refers to a chemical event by 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.

[0110] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., a population in which the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations generally contain a large number of antibodies directed against (or specific for) different epitopes. In one embodiment, a polyclonal antibody contains a plurality of monoclonal antibodies with different epitopic specificities, affinities, or avidities within a single antigen that contains multiple antigenic epitopes. The modifier "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present invention may be produced by the hybridoma method first described in Kohler et al., Nature 256: 495 (1975), or may be produced by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222: 581-597 (1991). These monoclonal antibodies generally bind with a Kd of at least about 1 μM, more generally at least about 300 nM, typically at least about 30 nM, preferably at least about 10 nM, and more preferably at least about 3 nM or better, usually as determined by ELISA.

[0111] "Pharmaceutically acceptable" refers to a composition that is non-toxic, inert, and / or physiologically compatible with humans or other mammals.

[0112] As used herein, the term "single chain Fv" or "scFv" or "single chain" antibody refers to the V H Domain and V L Fv refers to an antibody fragment comprising a V domain, where these domains are present as a single polypeptide chain. Generally, an Fv polypeptide is H Domain and V L It further comprises a polypeptide linker between the domains which enables the sFv to form the desired structure for antigen binding. For a general review of scFvs, see Pluckthun, THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

[0113] As used herein, the terms "specific", "specifically binds" and "binds specifically" refer to the selective binding of an antibody to a target antigen epitope. An antibody can be tested for specificity of binding by comparing binding to the appropriate antigen with binding to an unrelated antigen or antigen mixture under a given set of conditions. An antibody is considered specific if it binds to the appropriate antigen at least 2-fold, 5-fold, 7-fold, and preferably 10-fold more than binding to an unrelated antigen or antigen mixture. In one embodiment, a specific antibody is an antibody that binds only to a TAA antigen and not to unrelated antigens. In another embodiment, a specific antibody is an antibody that binds to a human TAA antigen but not to a non-human TAA antigen that has 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, the specific antibody is an antibody that binds to a human TAA antigen but does not bind to a non-human TAA antigen that has 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, the specific antibody is an antibody that binds to a human TAA antigen and also binds to a mouse TAA antigen, but binds to a greater extent to the human antigen. In another embodiment, the specific antibody is an antibody that binds to a human TAA antigen and also binds to a primate TAA antigen, but binds to a greater extent to the human antigen. In another embodiment, the specific antibody binds to a human TAA antigen and any non-human TAA antigen, but binds to a greater extent to the human antigen, or any combination thereof.

[0114] As used herein, "to treat" or "therapeutic" and grammatically related terms refer to any improvement in any outcome of a disease, e.g., prolonging survival, reducing morbidity, and / or mitigating side effects that are a by-product of alternative treatment modalities; complete eradication of the disease is preferred, but is not a requirement for the treatment effect, as is readily understood in the art.

[0115] II.) Antibodies In some embodiments, the presented fusion proteins, such as masked targeted interferon (IFN), and compositions include an antibody or antigen-binding fragment thereof. In some of the presented embodiments, the antibody binds to, e.g., specifically binds to, recognizes, targets, an antigen that is an antigen associated with a disease or disorder, such as cancer or an immunological disorder or disease, e.g., a tumor-associated antigen (TAA). In some aspects, by binding to an antigen (e.g., a TAA), the presented fusion protein, such as masked targeted IFN, can be targeted to a relevant physical location for treatment, e.g., the area of ​​a cancer or tumor. In some aspects, the antibody or antigen-binding fragment thereof described herein can be used as a component of any of the fusion proteins presented herein, e.g., any of the targeted IFN, antibody-IFN fusion proteins, or masked targeted IFN presented herein.

[0116] Aspects of the invention provide antibodies that bind to antigens associated with cancer or tumors, such as tumor-associated antigens (TAA) and TAA-associated proteins (see Table I). In one embodiment, an antibody that binds to a TAA-associated protein is an antibody that specifically binds to a TAA protein that includes amino acids of a protein listed in Table I. For example, an antibody that binds to a TAA protein that includes the amino acid sequence of one of the proteins listed in Table I may bind to a TAA-associated protein, such as a TAA variant and its homologs or analogs.

[0117] In some aspects, antibodies that bind to TAAs or TAA-associated proteins, such as the anti-TAA antibodies of the embodiments presented, are particularly useful for prognostic assays, imaging, diagnostics, and therapeutic methodologies with respect to cancer. In some aspects, the antibodies of the embodiments presented are therapeutic antibodies, such as therapeutic antibodies that specifically bind to TAAs, such as the TAAs listed in Table I. Similarly, such antibodies are useful (e.g., when combined with therapeutic agents, as fusion proteins, in the treatment and / or prognosis of cancers, such as ovarian cancer, head and neck cancer, multiple myeloma, and other cancers, as long as TAAs are also expressed or overexpressed in these cancers). Furthermore, the antibodies of the embodiments presented, including intracellularly expressed antibodies (e.g., single chain antibodies), are therapeutically useful in the treatment of cancers associated with expression of TAAs, such as solid tumor progression or metastatic cancers or other progression or metastatic cancers. One embodiment is the TAA binding assay disclosed herein for use in, for example, cancer detection in an immunoassay.

[0118] In some embodiments, the presented fusion protein or composition comprises an antibody that binds to a tumor-associated antigen (TAA), e.g., a TAA selected from the exemplary TAAs set forth in Table I. In some embodiments, the TAA is an antigen expressed on the surface of a tumor, e.g., on the surface of a tumor cell or a cancer cell. In some embodiments, the TAA comprises any antigen associated with any of the diseases or conditions described herein, such as any of the cancers described herein. In some embodiments, the TAA is an antigen expressed on the surface of a cell associated with a tumor, e.g., a cell present within the tumor microenvironment (TME). In some embodiments, the TAA is an antigen present within the TME.

[0119] In some embodiments, the presented fusion protein or composition comprises an antibody that binds to a tumor-associated antigen associated with a tumor occurring in the hematopoietic system, e.g., a hematological malignancy. In some embodiments, the presented fusion protein or composition comprises an antibody that binds to the CD138 antigen. In some embodiments, the antibody binds, e.g., specifically binds, to one of the TAAs listed in Table I.

[0120] In some embodiments, the antibody comprises or is comprised in a fusion protein. In some embodiments, the antibody comprises any of the fusion proteins or compositions presented herein. In some embodiments, the antibody comprises a fusion protein comprising a type 1 IFN, such as a type I IFN listed in Table II. In some embodiments, the antibody comprises a fusion protein further comprising a targeted IFN-alpha. In some embodiments, the antibody comprises a fusion protein further comprising a masked targeted IFN-alpha.

[0121] In some embodiments, antibodies include antibody fragments, such as antigen-binding antibody fragments. 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.

[0122] Various methods for preparing 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 TAA-related protein, peptide, or fragment, in isolated or 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 TAA, such as TAA GST-fusion proteins, can be used. In certain embodiments, a GST fusion protein is produced that contains all or most of the amino acid sequence of Figure 1, which is then used as an immunogen to generate suitable antibodies. In another embodiment, a TAA-related protein is synthesized and used as an immunogen.

[0123] Additionally, naked DNA immunization techniques known in the art can be used (with or without purified TAA-associated proteins or TAA-expressing cells) to generate an immune response against the encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).

[0124] The amino acid sequences of the TAA proteins listed in Table I can be analyzed to select specific regions of the TAA proteins, for example, as immunogens or epitopes for generating antibodies. For example, hydrophobicity and hydrophilicity analysis of the TAA amino acid sequences can be used to identify hydrophilic regions within the TAA structures. Regions of the TAA proteins that exhibit immunogenic structures, as well as other regions and domains, can be readily identified using a variety of other methods known in the art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles can be generated using the methods of Hopp, TP and Woods, KR, 1981, Proc. Natl. Acad. Sci. USA 78: 3824-3828. Hydropathicity Index profiles can be generated using the methods of Kyte, J. and Doolittle, RF, 1982, J. Mol. Biol. 157: 105-132. Percent (%) accessible residue profiles can be generated using the method of Janin J., 1979, Nature 277: 491-492. Average flexibility profiles can be generated using the method of Bhaskaran R., Ponnuswamy PK, 1988, Int. J. Pept. Protein Res. 32: 242-255. Beta turn profiles 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 falls within the scope of the present invention. A preferred method for generating TAA antibodies is further illustrated by the examples presented herein. Methods for preparing proteins or polypeptides for use as immunogens are well known in the art. Methods for preparing immunogenic conjugates of proteins with carriers such as BSA, KLH or other carrier proteins are also well known in the art.In some cases, direct conjugation is used, for example using carbodiimide reagents. In other cases, linking reagents such as those supplied by Pierce Chemical Co., Rockford, Ill. are effective. Administration of the TAA immunogen is often by injection over a suitable period of time and with a suitable adjuvant as understood in the art. During the immunization schedule, antibody titers can be obtained to determine the adequacy of antibody formation.

[0125] TAA monoclonal antibodies can be produced by various means well known in the art.For example, immortalized cell lines secreting desired monoclonal antibodies are prepared using standard hybridoma techniques or modifications of Kohler and Milstein that immortalize antibody-producing B cells, as is generally known.Immortalized cell lines secreting desired antibodies are screened by immunoassay with TAA-associated protein as antigen.Once a suitable immortalized cell culture is identified, the cells can be expanded and antibody can be produced from either in vitro culture or ascites.

[0126] The antibody or fragment of the present invention can also be produced by recombinant means. For chimeric antibodies of multiple species origin or antibodies with grafted complementarity determining regions (CDRs), the region that specifically binds to the desired region of the TAA protein can also be produced. Humanized or human TAA antibodies can also be produced for use in therapeutic situations, which is preferred. Methods for humanizing mouse and other non-human antibodies by replacing one or more of the non-human antibody CDRs with the corresponding human antibody sequence 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.

[0127] In one embodiment, the human monoclonal antibodies of the present invention can be prepared using VelocImmune mice, in which the genomic sequence with endogenous mouse variable segments in immunoglobulin heavy chain (VH, DH, and JH segments) and / or kappa light chain (VK and JK) loci is replaced in whole or in part by the human genomic sequence with unrearranged germline variable segments in human immunoglobulin heavy chain (VH, DH, and JH) and / or kappa light chain (VK and JK) loci (Regeneron, Tarrytown, NY).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.

[0128] Additionally, human antibodies of the invention can be generated using HuMAb mice (Medarex, Inc.) that contain human immunoglobulin gene miniloci encoding unrearranged human heavy (mu and gamma) and kappa light chain immunoglobulin sequences with targeted mutations that inactivate the endogenous mu and kappa chain loci (see, e.g., Lonberg, et al. (1994) Nature 368 (6474): 856-859).

[0129] In another embodiment, mice carrying human immunoglobulin sequences on a transgene and transchromosome, e.g., a mouse carrying a human heavy chain transgene and a human light chain transchromosome, can be used to generate fully human antibodies of the invention. Such mice are referred to herein as "KM mice," and such mice are described in Tomizuka, et al. (2000) Proc. Natl. Acad. Sci. USA 97: 722-727 and PCT Publication No. WO02 / 43478 to Tomizuka et al.

[0130] The human monoclonal antibody of the present invention can also be prepared by using phage display method for screening the library of human immunoglobulin genes.Such phage display method for isolating human antibody has been established in the art.See, for example, U.S. Patent No. 5,223,409; U.S. Patent No. 5,403,484; and U.S. Patent No. 5,571,698 to Ladner et al.; U.S. Patent No. 5,427,908 and U.S. Patent No. 5,580,717 to Dower et al.; U.S. Patent No. 5,969,108 and U.S. Patent No. 6,172,197 to McCafferty et al.; and U.S. Patent No. 5,885,793; U.S. Patent No. 6,521,404; U.S. Patent No. 6,544,731; U.S. Patent No. 6,555,313; U.S. Patent No. 6,582,915 and U.S. Patent No. 6,593,081 to Griffiths et al.

[0131] Human monoclonal antibodies of the invention can also be prepared using SCID mice reconstituted with human immune cells such that a human antibody response can be generated upon immunization. Such mice are described, for example, in U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al.

[0132] Furthermore, the human antibodies of the present invention can be produced using a technique that uses transgenic mice with engineered human heavy and light chain loci that are inactivated for antibody production, called Xenomouse (Amgen Fremont, Inc., formerly Abgenix, Inc.). An exemplary description of the preparation of transgenic mice that produce human antibodies can be found in U.S. Patent No. 6,657,103. See also U.S. Patent Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,545,806; and Mendez, et. al. Nature Genetics, 15: 146-156 (1998); Kellerman, SA & Green, LL, Curr. Opin. Biotechnol 13, 593-597 (2002).

[0133] Any of the above production methods will result in an antibody having a certain ability to bind to a TAA or a homolog or fragment or polypeptide sequence having 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9%, 98%, or 99% sequence identity to the TAA. The binding affinity (K D ) may 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 between 5 nM and 10 nM; or between 1 nM and 2 nM. Dcan be between 1 micromolar and 500 micromolar or between 500 micromolar and 1 nM.

[0134] The binding affinity of an antigen-binding protein is determined by the association constant (Ka) and dissociation constant (Kd) (KD=Kd / Ka). Binding affinity can be measured by BIACORE, for example, by capturing the test antibody on a protein A-coated sensor surface and flowing TAA over the surface. Alternatively, binding affinity can be measured by FORTEBIO, for example, by capturing the test antibody receptor on a protein A-coated needle and flowing TAA over the surface. Those skilled in the art can identify other suitable assays known in the art for measuring binding affinity.

[0135] The term "specifically binds" as used herein in the context of a TAA antigen binding protein means that the antigen binding protein binds to the TAA as well as to a discrete domain or discrete amino acid sequence within the TAA, and does not or only weakly binds to other (e.g., unrelated) proteins. However, this term does not exclude the fact that an antibody or binding fragment thereof may also cross-react with closely related molecules. The antibodies and fragments thereof and fusion proteins comprising them described herein may specifically bind to a TAA with an affinity at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, or 1000-fold greater than that of the binding to closely related molecules.

[0136] In one aspect, the invention includes an antibody that binds to a tumor-associated antigen (TAA).

[0137] In another aspect, the invention includes antibodies that bind to tumor-associated antigens (TAA) associated with solid cancer tumors.

[0138] In another aspect, the invention includes antibodies that bind to tumor-associated antigens associated with tumors arising in the hematopoietic system.

[0139] In another aspect, the invention includes an antibody that binds to the CD138 antigen.

[0140] In another aspect, the invention includes an antibody that binds to the CD20 antigen.

[0141] In another aspect, the invention includes antibodies that bind to a mesothelin antigen.

[0142] In another aspect, the invention includes antibodies that bind to the 5T4 antigen.

[0143] In another aspect, the invention includes an antibody that binds to a FAP antigen.

[0144] In another aspect, the invention includes antibodies that bind to the PSCA antigen.

[0145] In another embodiment, the antibody comprises a fusion protein.

[0146] In another embodiment, the antibody constitutes a fusion protein containing a type 1 IFN as described in Table II.

[0147] In another embodiment, the antibody constitutes a fusion protein further comprising the targeting IFN-alpha.

[0148] In another embodiment, the antibody constitutes a fusion protein further comprising a masked targeted IFN-alpha.

[0149] In another embodiment, the invention includes an antibody that binds CD138, further comprising a heavy chain as depicted in FIG.

[0150] In another aspect, the present invention provides a method for the production of a medicament comprising the following sequence: [ka] The antibody that binds to CD138 further comprises a heavy chain having the following structure:

[0151] In another aspect, the present invention provides a method for the production of a medicament comprising the following sequence: [ka] The antibody includes an antibody that binds to CD138, further comprising a heavy chain variable region having the following structure:

[0152] In another aspect, the invention includes an antibody that binds CD138, further comprising a heavy chain with an amino acid substitution at position 297 (N297Q).

[0153] In another aspect, the invention includes an antibody that binds CD138, further comprising a heavy chain as set forth in SEQ ID NO:6 having an amino acid substitution at position 297 (N297Q).

[0154] In another aspect, the invention includes an antibody that binds CD138, further comprising a heavy chain variable region as set forth in SEQ ID NO:7 having an amino acid substitution at position 297 (N297Q).

[0155] In another aspect, the invention includes an antibody that binds CD138, further comprising a heavy chain variable region set forth in (SEQ ID NO:7), further comprising a flexible linker set forth in (SEQ ID NO:5), further comprising an IFNa2 set forth in (SEQ ID NO:10), further comprising a protease-cleavable linker set forth in (SEQ ID NO:38), further comprising a mask sequence set forth in (SEQ ID NO:34), and further comprising an amino acid substitution at position 297 (N297Q). See Figure 25 (SEQ ID NO:41).

[0156] In another embodiment, the invention includes an antibody that binds CD138, further comprising a light chain as depicted in FIG.

[0157] In another aspect, the present invention provides a method for the production of a medicament comprising the following sequence: [ka] The antibody binds to CD138 and further comprises a light chain having the following structure:

[0158] In another aspect, the present invention provides a method for the production of a medicament comprising the following sequence: [ka] The antibody includes an antibody that binds to CD138, further comprising a light chain variable region having the following structure:

[0159] In another embodiment, the invention includes an antibody that binds CD138, further comprising a light chain variable region (SEQ ID NO:9) and a heavy chain variable region (SEQ ID NO:7).

[0160] In another aspect, the invention includes an antibody that binds CD138, further comprising a light chain variable region (SEQ ID NO:9) and a heavy chain variable region (SEQ ID NO:7), further comprising an amino acid substitution at position 297 (N297Q).

[0161] In another embodiment, the invention includes an antibody that binds CD138, further comprising a light chain (SEQ ID NO:8) and a heavy chain (SEQ ID NO:6), further comprising an amino acid substitution at position 297 (N297Q).

[0162] III.) Interferon In some embodiments, the presented fusion proteins and compositions, such as targeted interferon (IFN), e.g., masked targeted IFN, comprise a component that is an interferon (IFN) or a variant thereof. In some aspects, masked targeted IFN, e.g., type I IFN, fused to an antibody, fragment or chain thereof, and an "interferon mask" are also provided.

[0163] In some embodiments, a fusion protein is provided, such as an antibody-IFN fusion protein or targeted IFN, comprising an interferon (IFN) or variant thereof and an antibody or 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, exemplary IFNs in the presented embodiments include any known type I IFNs, and type I IFNs including any described herein, e.g., in Table II. Exemplary antibodies for the fusion protein include any described herein, e.g., in Section II or Table I. In some of the embodiments, an interferon (IFN) or variant thereof is provided that is bound or connected to a "mask," such as a peptide or protein that blocks cytokine interactions and / or activation of the interferon alpha receptor (IFNAR), also referred to in some cases as an interferon mask. In some aspects, a masked IFN is provided. In some aspects, exemplary IFNs for the presented masked IFNs include any known type I IFNs, and type I IFNs including any described herein, e.g., in Table II. Exemplary masks and methods for masking interferon include any described herein, e.g., in Section IV. In some embodiments, the antibody-IFN fusion protein comprises a "masked" IFN that includes an IFN component selected from the IFNs in Table II.

[0164] In some embodiments, IFN is a protein that is used to treat or treat disease or disorder.In some embodiments, the presented fusion protein and composition contain IFN components that can be effective in treating disease or disorder such as cancer or tumor, and / or can be used to increase the effect of therapeutic agents such as anti-cancer or anti-neoplastic agents.

[0165] IFNs are a group of signaling proteins that are made and released by the cells of a subject or host, e.g., host cells, in response to the presence of foreign entities in the body, such as pathogens, including some viruses. In a typical scenario, a virus-infected cell releases interferons that cause nearby cells to mount an antiviral defense.

[0166] IFN belongs to a large class of proteins known as cytokines, molecules used for cell-cell communication that triggers protective defenses by the immune system to help eradicate pathogens. The name interferon comes from its ability to "interfere" with viral replication by protecting cells from viral infection. In some embodiments, IFN also has various other functions: (i) activates immune cells such as natural killer cells and macrophages; and (ii) increases host defense by upregulating antigen presentation by increasing the expression of major histocompatibility complex (MHC) antigens.

[0167] More than 20 distinct IFN genes and proteins have been identified in animals, including humans. These are generally divided into three classes: type I IFN, type II IFN, and type III IFN. IFNs from all three classes are important for fighting viral infections and regulating the immune system.

[0168] Interferon Type I: All type I IFNs bind to a specific cell surface receptor complex known as the IFN-α / β receptor (IFNAR), which consists of IFNAR1 and IFNAR2 chains. In humans, there are 13 types of type I interferons, including but not limited to IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω. Generally, type I interferons are produced when the body recognizes an invading virus. Type I interferons are produced by fibroblasts and monocytes. However, the production of type I IFN-α is prevented by another cytokine known as interleukin-10. Once released, type I interferons bind to specific receptors on target cells, which leads to the expression of proteins that prevent the virus from producing and replicating RNA and DNA.

[0169] Interferon type II (IFN-γ in humans): Interferon type II is also known as immune interferon and is activated by interleukin-12. Furthermore, type II interferons are released by cytotoxic T cells and T helper cells in a type I specific manner. However, type II interferons block the proliferation of type 2 T helper cells. The former inhibits the proliferation of T h 2. Inhibition of immune responses and T h 1 immune response, which leads to the development of debilitating diseases such as multiple sclerosis. IFN type II binds to IFNGR, which is composed of IFNGR1 and IFNGR2 chains.

[0170] Interferon type III: 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.

[0171] In general, type I and type II interferons are involved in the regulation and activation of immune responses. The expression of type I and type III IFNs can be induced in virtually all cell types upon recognition of viral components, especially nucleic acids, by cytoplasmic and endosomal receptors, whereas type II interferons are induced by cytokines such as IL-12 and their expression is restricted to immune cells such as T cells and NK cells.

[0172] In some aspects, IFN and IFN-containing or IFN-derived proteins can be used as therapeutic agents. In some of the embodiments presented, the IFN component of the fusion protein, such as masked targeted IFN, is used as a therapeutic agent to treat diseases or disorders, such as cancer.

[0173] In some embodiments, interferon therapy is used as a treatment for some cancers (combined with chemotherapy and radiation).This treatment can be used for hematological malignancies; leukemia and lymphoma, including hairy cell leukemia, chronic myelogenous leukemia, nodal lymphoma, and cutaneous T-cell lymphoma.In some cases, patients with recurrent melanoma receive recombinant IFN-α2b.

[0174] The main limitation with the use of available IFN in cancer therapy has been the inability to achieve effective concentrations 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 to deliver IFN directly 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). Note that in the first study, anti-CD20-IFNα2 protein was used to target IFNα to CD20 expressed in lymphoma, and anti-CD138-IFNα2 fusion protein was used to target CD138 expressed in multiple myeloma. See Vasuthasawat, et. al., MAbs 8 (7), pp. 1386-1397 (2016). Although these approaches show great therapeutic promise and are currently being tested in human clinical trials and in commercial development, there are several deficiencies in these available approaches.

[0175] By using antibody binding specificity for target tumor-associated antigen, a larger percentage of IFN is delivered to the tumor site than would be achieved if IFN were injected by itself, but the bound interferon can still be recognized and bound by interferon receptors expressed by cells throughout the body, such as non-tumor-associated or normal cells. This binding can result in both a reduction in IFN reaching the tumor and undesirable off-target toxicity. An embodiment is provided that overcomes such limitations and deficiencies.

[0176] Thus, it is an object of the present invention to overcome these limitations by providing a mechanism to "mask" the function or activity of IFN until it reaches a location or area of ​​relevance for the treatment of a disease or disorder, e.g., a tumor, at which point the IFN is "unmasked" and effectively switched back on to be active and functional. Thus, in some embodiments, a fusion protein is provided, e.g., a masked targeted IFN, which is "unmasked" or "activated" only in the desired location of therapeutic effect, e.g., a tumor. In some aspects, masking the function or activity of IFN in the rest of the body, e.g., the systemic circulation, can generally reduce or prevent non-specific activity of IFN, and can also reduce or prevent the therapeutic agent, e.g., IFN, from being trapped or taken up by non-cancer or non-tumor cells in the body. In some aspects, by masking the function or activity of IFN and targeting it to a location of interest, e.g., a tumor, by an antibody present in the embodiment in which IFN is presented, the concentration of a therapeutic agent (e.g., IFN) can be effectively increased, e.g., by preventing IFN from binding and / or being trapped by specific targeting of the agent by the antibody.

[0177] Thus, in some embodiments, the invention comprises an antibody-IFN fusion protein that selectively binds to an IFN receptor once the IFN reaches the tumor.

[0178] In another embodiment, the antibody-IFN fusion protein comprises the IFN separated by a peptide linker that is a proteolytic cleavage site.

[0179] In another embodiment, the antibody-IFN fusion protein comprises a "masked" IFN.

[0180] In another embodiment, the antibody-IFN fusion protein comprises a "masked" IFN separated by a peptide linker that is a proteolytic cleavage site.

[0181] In another embodiment, the antibody-IFN fusion protein comprises a "masked" IFNA1.

[0182] In another embodiment, the antibody-IFN fusion protein contains a "masked" IFNA1 separated by a peptide linker that is a proteolytic cleavage site.

[0183] In another embodiment, the antibody-IFN fusion protein comprises a "masked" IFNA2.

[0184] In another embodiment, the antibody-IFN fusion protein contains a "masked" IFNA2 separated by a peptide linker that is a proteolytic cleavage site.

[0185] In another embodiment, the antibody-IFN fusion protein comprises a "masked" IFNB1.

[0186] In another embodiment, the antibody-IFN fusion protein contains a "masked" IFNB1 separated by a peptide linker that is a proteolytic cleavage site.

[0187] In another embodiment, the antibody-IFN fusion protein comprises a "masked" IFN selected from the IFNs listed in Table II.

[0188] In another embodiment, the antibody-IFN fusion protein comprises a "masked" IFN separated by a peptide linker that is a proteolytic cleavage site and selected from the IFNs listed in Table II.

[0189] In another embodiment, the antibody-IFN fusion protein comprises: [ka] Contains IFNα.

[0190] In another embodiment of the present disclosure, it is understood that the increased binding affinity particularly increases the anti-proliferative efficacy of IFNα.See KALLE, et. al., J. Bio. Chem., vol. 282, no. 15 (April 13, 2007) and URIN, et. al., Plos One, DOI: 10.1371 / journal.pone.01030797 (July 9, 2015).Thus, in one embodiment of the present disclosure, the antibody-IFN fusion protein comprises IFNα (SEQ ID NO: 10) further comprising YNS mutations (H57Y, E58N, and Q61S).

[0191] In another embodiment, the antibody-IFN fusion protein comprises: [ka] See, for example, Figure 62 (QXL138YNS4.2-N297Q)

[0192] IV.) Methods for masking IFN As mentioned above, it is an object of the present invention to provide a masked targeted IFN composition in which the IFN of the present invention (see Table II) reaches the tumor and the activity of the IFN is inhibited until such time as the IFN is unmasked by a protease, such as a tumor-associated protease. In some embodiments, the presented masked IFN, e.g., masked targeted IFN, is "unmasked" at or near the location of the disease or disorder to be treated, e.g., at or near the tumor, such as the tumor microenvironment, and is able to bind to and / or activate an interferon receptor (e.g., IFNAR). In some embodiments, the presented fusion protein is unmasked or activated by a component, such as a peptide linker, being cleaved by a protein in the tumor, e.g., the environment of a tumor-associated protease.

[0193] (a) Discussion of available methods. The available approaches related to this endeavor are limited. In this disclosure, a description of the available approaches is presented to further demonstrate and illustrate the technical advantages of the present invention. A previous approach to improve tumor-specific delivery of therapeutics has been to create therapeutics that are activated by tumor-associated proteases. An example of this approach was to take an antibody that recognizes a tumor-associated antigen, but has limited effectiveness because it also recognizes antigens present on normal cells, and engineer it to bind to the antigen only when localized to the target tumor. See US8,563,269 (CytomX Therapeutics, San Francisco, CA). So-called "probodies" are accompanied by peptides that block the antibody binding site, joined by a linker that is cleavable by proteases present in the tumor microenvironment. In one case, CETUXIMAB, an antibody specific for epidermal growth factor receptor (EGFR), was created that is activated and binds only when localized to the tumor. See Desnoyers, et. al., Sci. Transl. Med., 16 :5 (207) pp.207ra144 (2013). This "Probody" contains a mask sequence that binds to the variable region of CETUXIMAB, followed by a GS-linker, followed by the sequence [ka] to the amino terminus of the heavy chain of an antibody, where the underlined sequences are substrates for UPA and matriptase, proteases that have been shown to be upregulated in a variety of human carcinomas and have minimal activity in normal tissues. This probody has demonstrated improved safety and increased half-life in non-human primates.

[0194] In addition to creating probodies with antibody binding properties that are activated in the tumor microenvironment, it has also been possible to create interferon alpha proproteins that are activated by proteases. See US8,399,219 (CytomX Therapeutics, San Francisco, CA). In this case, a peptide mask for IFN-α, TDVDYYREWSWTQVS (SEQ ID NO: 12), was placed at the amino terminus of a single-chain recombinant IFNα, separated from the IFNα by a cleavable sequence. The resulting construct: GQSGQ TDVDYYREWSETQVS GSSGGS VHMPLGFLGP GGS (SEQ ID NO: 13) IFNα contained IFNα that is selectively activated in the tumor microenvironment. VHMPLGFLGP (SEQ ID NO: 14) is taught to be a substrate for MMP-9.

[0195] (b) A method of masking IFN of the present disclosure. Previously, the inventors have disclosed alternative approaches to generate masked IFN that can be fused to an antibody that binds to a TAA. See, for example, US Patent Application Publication No. US2020 / 0331966 (Qwixel Therapeutics, Inc., Los Angeles, CA.), published October 22, 2020. The mask(s) disclosed herein are derived from peptides identified by phage display and are not native human sequences. The subject matter of the present disclosure teaches novel alternatives to generate masked IFN. The peptide masks disclosed herein are capable of being unmasked at or near the location of the disease or disorder. From the above, the disclosed method of masking IFN is clearly distinct and offers advantages over all available approaches. Some advantages include, but are not limited to, (i) the ability to substantially mask type I interferons, (ii) superior masking properties based on high affinity, and (iii) reduced risk of immunogenicity.

[0196] Furthermore, as noted above, the embodiments presented include a mechanism for "masking" the function or activity of IFN until the IFN reaches a location or region of relevance for the treatment of a disease or disorder, e.g., a tumor, and specifically physically targeting the fusion protein to the tumor location by a TAA-specific antibody fused to the IFN. Thus, the fusion proteins described herein provide a number of advantages, including, but not limited to, IFN being "unmasked" or "activated" only in the location of interest for therapeutic effect, e.g., a tumor; reduced non-specific activity of IFN; prevention of IFN being trapped or internalized by non-cancer or non-tumor cells in the body; and / or effective increase in concentration of therapeutic agent (e.g., IFN) without increased toxicity.

[0197] In some of the embodiments, the presented fusion protein, e.g., the masked targeted IFN, is unmasked only at or near the site of the disease or disorder, e.g., the tumor. Thus, in some embodiments, the present invention includes an antibody-IFN fusion protein that selectively binds to the IFN receptor once the IFN reaches the location or region of the tumor.

[0198] In some embodiments, the antibody-IFN fusion protein comprises an IFN separated by a peptide linker that is a proteolytic cleavage site. In some aspects, the IFN can be "unmasked" or activated by proteolytic cleavage of the peptide linker, for example, at or near a tumor.

[0199] As described in this disclosure, the embodiments presented include antibody-IFN, in which an IFN, such as those described in Section III or Table II herein, is fused to an antibody, for example, as described in Section II or Table I herein. The IFN is then "masked" to be active and bind to its receptor only at the tumor site. An ideal peptide mask, in its uncleaved state, inhibits 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 binding of the protein to its binding partner. In the embodiments presented herein, cleavage of the "mask," for example, at or near the tumor site, allows type I IFN to bind to its receptor (e.g., IFNAR) and exert a therapeutic effect.

[0200] Exemplary embodiments are described below.

[0201] (i) IFNα Masking Peptides and Masking Constructs Disclosed herein are novel peptide masks designed based on a crystal structure model of IFNα binding to IFNAR2. As a result of these studies, regions of IFNAR2 that interact with IFNα were synthesized as peptides and tested for binding to IFNα as well as for competitive inhibition of IFNα-IFNAR2.

[0202] The development of the peptides disclosed herein is based on the rationale that all type I IFNs share the same receptor binding mode and form structurally highly similar signaling complexes. See PIEHLER, et. at., Immunol Rev., 250 (1): 317-334 (Nov. 2012). Furthermore, it has been shown that the receptor-ligand cross-reactivity of IFNα2 and IFNAR2 is enabled by conserved receptor-ligand "anchor points" interspersed among the ligand-specific interactions that "tune" the relative IFN binding affinity in an apparent extracellular "ligand proofreading" mechanism that modulates biological activity. Furthermore, functional differences between IFNs are linked to their respective receptor recognition chemistries, which, in concert with ligand-induced conformational changes in IFNAR1, collectively control signal initiation and complex stability, ultimately modulating differential STAT phosphorylation profiles, receptor internalization rates, and downstream gene expression patterns. See THOMAS, et. al., Cell, 146 (4): 621-632 (Aug. 2011). Furthermore, a comparison of the reciprocal binding sites of IFNα2 and IFNAR2 suggests that IFNα2 interacts with both domains of IFNAR2, exposing several "hot spot" residues. See ROISMAN, et. al., PNAS, vol. 98, no. 23, 13231-13236 (Nov. 2001) and FIG. 1. Based on the above, several peptides were generated, which are described in FIG. 2 and FIG. 39.

[0203] IFNα2 is used with a protease cleavage site (e.g., LSGRSDNH (SEQ ID NO: 15) or KQSRVVNH (SEQ ID NO: 16)) and a peptide "mask" that inhibits IFN binding (Figures 2 and 39) is placed at the 3' end, H 3. It is contemplated in the present disclosure that at the tumor site, the protease cleavage site is cleaved by a protease in the tumor microenvironment, thereby unmasking and liberating the IFN, such that the IFN can bind to its receptor.

[0204] Nucleic acid for constructing a recombinant heavy chain with a protease cleavage site and an IFN inhibitory mask was obtained (ATUM, Newark, California) and used to modify the heavy chain (H chain) of anti-TAA-IFNα2 by making the following fusion at the 3′ end: [ka]

[0205] The single underline indicates the carboxy terminus of IFNα2, the double underline represents the sequence of the protease cleavage site, and the dashed underline represents the IFNα mask (peptide 4). The linker sequence is shown in lower case.

[0206] In another embodiment, the construct comprises: [ka] Includes.

[0207] The single underline indicates the carboxy terminus of IFNα2, the double underline represents the sequence of the protease cleavage site, and the dashed underline represents the IFNα mask (peptide 5). The linker sequence is shown in lower case.

[0208] In another embodiment, the construct comprises: [ka] Includes.

[0209] The single underline indicates the carboxy terminus of IFNα2, the double underline represents the sequence of the protease cleavage site, and the dashed underline represents the IFNα mask (peptide 6). The linker sequence is shown in lower case.

[0210] In another embodiment, the construct comprises: [ka] Includes.

[0211] The single underline indicates the carboxy terminus of IFNα2, the double underline represents the sequence of the protease cleavage site, and the dashed underline represents the IFNα mask (peptide 7). The linker sequence is shown in lower case.

[0212] In another embodiment, the construct comprises: [ka] Includes.

[0213] The single underline indicates the carboxy terminus of IFNα2, the double underline represents the sequence of the protease cleavage site, and the dashed underline represents the IFNα mask (peptide 1). The linker sequence is shown in lower case.

[0214] In another embodiment, the construct comprises: [ka] Includes.

[0215] The single underline indicates the carboxy terminus of IFNα2, the double underline represents the sequence of the protease cleavage site, and the dashed underline represents the IFNα mask (peptide 21). The linker sequence is shown in lower case.

[0216] In another embodiment, the construct comprises: [ka] Includes.

[0217] The single underline indicates the carboxy terminus of IFNα2, the double underline represents the sequence of the protease cleavage site, and the dashed underline represents the IFNα mask (peptide 22). The linker sequence is shown in lower case.

[0218] In another embodiment, the construct comprises: [ka] Includes.

[0219] The single underline indicates the carboxy terminus of IFNα2, the double underline represents the sequence of the protease cleavage site, and the dashed underline represents the IFNα mask (peptide 23). The linker sequence is shown in lower case.

[0220] In another embodiment, the construct comprises: [ka] Includes.

[0221] The single underline indicates the carboxy terminus of IFNα2, the double underline represents the sequence of the protease cleavage site, and the dashed underline represents the IFNα mask (peptide 24). The linker sequence is shown in lower case.

[0222] Nucleic acid for constructing a recombinant heavy chain with a protease cleavage site and an IFN inhibitory mask was obtained (ATUM, Newark, California) and used to modify the heavy chain (H chain) of anti-TAA-IFNα2 by making the following fusion at the 3′ end: [ka]

[0223] The single underline indicates the carboxy terminus of IFNα2, the double underline represents the sequence of the protease cleavage site, and the dashed underline represents the IFNα mask (peptide 4). The linker sequence is shown in lower case.

[0224] In another embodiment, the construct comprises: [ka] Includes.

[0225] The single underline indicates the carboxy terminus of IFNα2, the double underline represents the sequence of the protease cleavage site, and the dashed underline represents the IFNα mask (peptide 5). The linker sequence is shown in lower case.

[0226] In another embodiment, the construct comprises: [ka] Includes.

[0227] The single underline indicates the carboxy terminus of IFNα2, the double underline represents the sequence of the protease cleavage site, and the dashed underline represents the IFNα mask (peptide 6). The linker sequence is shown in lower case.

[0228] In another embodiment, the construct comprises: [ka] Includes.

[0229] The single underline indicates the carboxy terminus of IFNα2, the double underline represents the sequence of the protease cleavage site, and the dashed underline represents the IFNα mask (peptide 7). The linker sequence is shown in lower case.

[0230] In another embodiment, the construct comprises: [ka] Includes.

[0231] The single underline indicates the carboxy terminus of IFNα2, the double underline represents the sequence of the protease cleavage site, and the dashed underline represents the IFNα mask (peptide 1). The linker sequence is shown in lower case.

[0232] In another embodiment, the construct comprises: [ka] Includes.

[0233] The single underline indicates the carboxy terminus of IFNα2, the double underline represents the sequence of the protease cleavage site, and the dashed underline represents the IFNα mask (peptide 21). The linker sequence is shown in lower case.

[0234] In another embodiment, the construct comprises: [ka] Includes.

[0235] The single underline indicates the carboxy terminus of IFNα2, the double underline represents the sequence of the protease cleavage site, and the dashed underline represents the IFNα mask (peptide 22). The linker sequence is shown in lower case.

[0236] In another embodiment, the construct comprises: [ka] Includes.

[0237] The single underline indicates the carboxy terminus of IFNα2, the double underline represents the sequence of the protease cleavage site, and the dashed underline represents the IFNα mask (peptide 23). The linker sequence is shown in lower case.

[0238] In another embodiment, the construct comprises: [ka] Includes.

[0239] The single underline indicates the carboxy terminus of IFNα2, the double underline represents the sequence of the protease cleavage site, and the dashed underline represents the IFNα mask (peptide 24). The linker sequence is shown in lower case.

[0240] In one embodiment, the "masked" IFN is [ka] Includes.

[0241] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1.

[0242] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2. In one embodiment, the "masked" IFN is [ka] and further comprising IFNα4.

[0243] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα5.

[0244] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds CD138.

[0245] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds CD20.

[0246] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds Her2.

[0247] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds CSPG4.

[0248] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds PSCA.

[0249] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds CEA.

[0250] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds to RCC.

[0251] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds to 5T4.

[0252] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds mesothelin.

[0253] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds CD138.

[0254] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds CD20.

[0255] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds Her2.

[0256] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds CSPG4.

[0257] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds PSCA.

[0258] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds CEA.

[0259] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds to RCC.

[0260] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds to 5T4.

[0261] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds mesothelin.

[0262] In one embodiment, the "masked" IFN is [ka] Includes.

[0263] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1.

[0264] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2. In one embodiment, the "masked" IFN is [ka] and further comprising IFNα4.

[0265] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα5.

[0266] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds CD138.

[0267] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds CD20.

[0268] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds Her2.

[0269] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds CSPG4.

[0270] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds PSCA.

[0271] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds CEA.

[0272] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds to RCC.

[0273] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds to 5T4.

[0274] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds mesothelin.

[0275] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds CD138.

[0276] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds CD20.

[0277] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds Her2.

[0278] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds CSPG4.

[0279] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds PSCA.

[0280] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds CEA.

[0281] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds to RCC.

[0282] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds to 5T4.

[0283] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds mesothelin.

[0284] In one embodiment, the "masked" IFN is [ka] Includes.

[0285] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1.

[0286] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2. In one embodiment, the "masked" IFN is [ka] and further comprising IFNα4.

[0287] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα5.

[0288] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds CD138.

[0289] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds CD20.

[0290] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds Her2.

[0291] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds CSPG4.

[0292] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds PSCA.

[0293] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds CEA.

[0294] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds to RCC.

[0295] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds to 5T4.

[0296] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds mesothelin.

[0297] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds CD138.

[0298] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds CD20.

[0299] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds Her2.

[0300] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds CSPG4.

[0301] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds PSCA.

[0302] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds CEA.

[0303] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds to RCC.

[0304] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds to 5T4.

[0305] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds mesothelin.

[0306] In one embodiment, the "masked" IFN is [ka] Includes.

[0307] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1.

[0308] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2. In one embodiment, the "masked" IFN is [ka] and further comprising IFNα4.

[0309] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα5.

[0310] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds CD138.

[0311] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds CD20.

[0312] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds Her2.

[0313] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds CSPG4.

[0314] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds PSCA.

[0315] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds CEA.

[0316] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds to RCC.

[0317] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds to 5T4.

[0318] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα1 fused to an antibody that binds mesothelin.

[0319] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds CD138.

[0320] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds CD20.

[0321] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds Her2.

[0322] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds CSPG4.

[0323] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds PSCA.

[0324] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds CEA.

[0325] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds to RCC.

[0326] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds to 5T4.

[0327] In one embodiment, the "masked" IFN is [ka] and further comprising IFNα2 fused to an antibody that binds mesothelin. In one embodiment, the "masked" IFN comprises TDVDYYREWSWTQVGG (SEQ ID NO: 30). In one embodiment, the "masked" IFN comprises TDVDYYREWSWTQVSGG (SEQ ID NO:31). In one embodiment, the "masked" IFN comprises TDVDYYREWSWTQ (SEQ ID NO:32). In one embodiment, the "masked" IFN comprises TLFSSSHNFWLAIDMS (SEQ ID NO:33). In one embodiment, the "masked" IFN comprises TDVDYYREWSWTQV (SEQ ID NO:34). In one embodiment, the "masked" IFN comprises TLFSSSHNFWLAIDMSGG (SEQ ID NO:35).

[0328] In one embodiment, the "masked" IFN comprises TDVDYYREWSWTQVGGSGGSGGGKVKAALLTSWKIGVYS (SEQ ID NO: 47). In one embodiment, the "masked" IFN comprises ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTRSFCDLTDEWRSTHEAYVTVLEGFSGNTTLFSCSHNFWLAIDMS (SEQ ID NO: 48).

[0329] In one embodiment, the "masked" IFN comprises 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.

[0330] The resulting embodiment, e.g., masked targeted IFN, offers unique advantages over previous approaches for several reasons. First, the IFN is masked, and thus its activity is significantly reduced and / or eliminated until it reaches the tumor, at which point it is unmasked and reactivated, thereby maximizing its efficacy in the tumor. Second, the masked IFN linked to the C-terminus can be targeted to a specific TAA by attaching the C-terminus of an antibody. Specific targeting allows for a higher probability that the IFN will be directed to the cancer of interest and avoid normal tissues. Third, the masking sequence is derived from the extracellular sequence of a human protein (IFNAR2), thus reducing the potential for immunogenic responses. Fourth, the masks presented herein offer the potential to mask a broader range of type I interferons, and the higher affinity can result in more effective masking.

[0331] General embodiments of the resulting masked targeted IFN are contemplated in this disclosure, including, but not limited to, the following: First, the compositions presented herein: (i) antibody-linker-cytokine (e.g., IFN)-linker-protease cleavage (PC) site-linker-mask; and Second, the compositions provided herein include: (ii) antibody-linker-PC cleavage site-linker-cytokine (e.g., IFN)

[0332] It will be appreciated that those skilled in the art will recognize and understand that the compositions described in (ii) have the additional property of the antibodies acting as a partial mask by sterically hindering the cytokines present in the composition.

[0333] In some embodiments, the protease cleavage site is a tumor-associated protease cleavage site. The "tumor-associated protease cleavage site" presented herein is an amino acid sequence recognized by a protease whose expression is specific to tumor cells or the 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 designated by a specific amino acid sequence. In one embodiment of the disclosure, the protease cleavage site is LSGRSDNH (SEQ ID NO: 15). In one embodiment of the disclosure, the protease cleavage site is KQSRVVNH (SEQ ID NO: 16).

[0334] V.) Treatment of Cancers Expressing Tumor-Associated Antigens (TAA) Also provided herein are fusion proteins, such as masked targeted IFN, or compositions and uses that are useful in various therapeutic, diagnostic and prophylactic methods. For example, the fusion proteins and compositions are useful in treating various diseases and disorders in a subject, such as cancer or tumor. Such methods and uses include, for example, therapeutic methods and uses involving administering the fusion proteins or compositions to a subject having a disease or disorder, such as tumor or cancer. In some embodiments, the fusion proteins or compositions are administered in an effective amount to effect treatment of the disease or disorder. Uses include the use of the fusion proteins or compositions in such methods and treatments, and in the preparation of medicaments for carrying out such therapeutic methods. In some embodiments, the fusion proteins or compositions are for use in treating various diseases and disorders in a subject, for example, according to a therapeutic method. In some embodiments, the method is carried out by administering the fusion proteins or compositions to a subject having or suspected of having a disease or disorder, such as tumor or cancer. In some embodiments, the method thus treats a disease or disorder in a subject.

[0335] In some embodiments, the presented fusion protein, for example, masked targeted IFN, is used in a method or use for treating a disease or disorder, such as a tumor or cancer, including those that express tumor-associated antigens (TAA).By identifying the TAA of the present disclosure as a protein that is normally expressed in a limited set of tissues or cells, but is also expressed in cancer, for example, solid tumor cancer, several therapeutic approaches are available for treating such cancers using the masked fusion protein disclosed herein.

[0336] Of note, targeted antitumor therapy is useful even when the targeted protein is expressed in normal tissue or cell, even if it is a vital normal organ tissue.Vital organs are organs that are necessary to sustain life, such as the heart or colon.Non-vital organs are organs that can be removed and the individual can still survive.Examples of non-vital organs are ovaries, breasts, and prostate.

[0337] The expression of a target protein in normal tissue, even if it is a vital normal tissue, does not overrule the utility of targeting an agent against that protein as a therapeutic agent against a particular tumor in which that protein is also overexpressed. For example, expression in a vital organ is not harmful in itself. Furthermore, removal of dispensable organs such as the prostate and ovaries can be performed without affecting mortality. Finally, some vital organs are not affected by expression in normal organs due to immune privilege. Immune privileged organs are organs that are protected from the blood by the blood-organ barrier and therefore cannot be used for immunotherapy. Examples of immune privileged organs are the brain and testes.

[0338] Thus, therapeutic approaches that inhibit the activity of TAA proteins, including the masked targeted IFN fusion proteins of the present invention, are useful for patients suffering from cancers that express TAA (such as solid tumor cancers of the lung, kidney, prostate, ovary, breast, and other types of cancers known in the art). Therapeutic approaches involve IFNA-induced death (e.g., when the "mask" is removed and IFN is reactivated in the tumor of interest), ADCC, CDC, and / or immune modulation. Furthermore, antibodies that bind to TAAs can also synergistically modulate the function of cancer cells. In addition, "unmasked" or activated IFN can modulate the activity of immune cells involved in anti-tumor or anti-cancer immunity by binding IFN to interferon receptors (e.g., IFNAR). Modulation of antibodies that bind to TAAs generally falls into two classes. The first class is related to tumor cell growth, resulting in inhibition or delay of tumor cell growth, or inducing tumor cell death, thus modulating TAA function. The second class includes various methods for inhibiting the binding or association of a TAA protein with its binding partner or other proteins.

[0339] Therefore, cancer patients can be evaluated for the presence and level of TAA expression, and preferably, the evaluation uses immunohistochemical evaluation of tumor tissue, quantitative TAA imaging, or other techniques that reliably show the presence and the degree of TAA expression.For this purpose, the immunohistochemical analysis of tumor biopsy or resection specimen is preferred, if applicable.The method for immunohistochemical analysis of tumor tissue is well known in the art.

[0340] VI.) Masked Targeted IFN Fusion Protein Cocktails The therapeutic methods of the present invention contemplate the administration of a single masked targeted IFN fusion protein as well as a combination or cocktail of different monoclonal antibodies (i.e., naked monoclonal antibodies that bind to the same TAA as the masked IFN fusion protein or monoclonal antibodies that bind to a different protein or all of the different masked targeted IFN fusion proteins that bind to a different TAA). Such monoclonal antibody cocktails may have certain advantages insofar as they contain monoclonal antibodies that target different epitopes, that exploit different effector mechanisms, or that combine monoclonal antibodies that are directly cytotoxic with monoclonal antibodies that rely on immune effector functionality. Such monoclonal antibody combinations may exhibit synergistic therapeutic effects. Furthermore, the masked targeted IFN fusion protein may be administered simultaneously with other therapeutic modalities, including, but not limited to, various chemotherapeutic and biological agents, androgen blockade, immune modulators (e.g., IL-2, GM-CSF), surgery, or radiation. In a preferred embodiment, the masked targeted IFN is administered in the form of a fusion protein.

[0341] The masked targeted IFN fusion protein formulation is administered by any route capable of delivering the antibody to the tumor cells. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumoral, intradermal, and the like. Treatment generally involves repeated administration of the masked targeted IFN fusion protein preparation by an acceptable route of administration, such as intravenous injection (IV), generally at a dose ranging from, but not limited to, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 15 mg, 20 mg, or 25 mg per kg of body weight. Generally, doses ranging from 10 to 1000 mg of masked targeted IFN fusion protein per week are effective and well tolerated.

[0342] Based on clinical experience with Herceptin® (trastuzumab) in the treatment of metastatic breast cancer, an initial loading dose of approximately 4 mg per kg of patient body weight via IV, followed by weekly doses of approximately 2 mg / kg via IV of monoclonal antibody preparations is an acceptable dosing regimen. The initial loading dose is preferably administered as a 90-minute or longer infusion. If the initial dose is well tolerated, periodic maintenance doses are administered as 30-minute or longer infusions. As will be understood by those skilled in the art, various factors may affect the ideal dosing regimen in a particular case. Such factors include, for example, the binding affinity and half-life of the TAA monoclonal antibody used, the degree of TAA expression in the patient, the degree of excreted TAA antigen in the circulation, the desired steady-state antibody concentration level, the frequency of treatment, and the effect of chemotherapeutic drugs or other agents used in combination with the treatment method of the present invention (i.e., masked targeted IFN), as well as the health status of the particular patient.

[0343] If necessary, patients should be evaluated for levels of TAAs in a given sample (e.g., levels of circulating TAAs and / or TAA-expressing cells), such as to help determine the most effective dosing regimen. Such evaluations are also used for monitoring throughout treatment and are useful in combination with evaluation of other parameters (e.g., urine cytology and / or ImmunoCyt levels in bladder cancer treatment, or similarly, serum PSA levels in prostate cancer treatment) to determine therapeutic success.

[0344] One object of the present invention is to provide masked targeted IFN fusion proteins that inhibit or slow the growth of tumor cells expressing a specific TAA to which the fusion protein binds. A further object of the present invention is to provide methods for using such masked targeted IFN-containing fusion proteins that bind to a TAA, particularly in combination with other drugs or immunologically active treatments, to inhibit angiogenesis and other biological functions, thereby reducing tumor growth in a mammal, preferably a human.

[0345] VII.) Combination Therapy In some embodiments, methods and uses involving combination therapy are also provided, for example, involving the use of any of the presented fusion proteins or compositions and additional therapeutic agents, such as chemotherapeutic agents or radiation. In some embodiments, the presented fusion proteins or compositions can be used in combination with additional therapeutic agents, such as anti-cancer or anti-tumor agents, to treat a disease or disorder.

[0346] In some embodiments, synergy occurs when tumors, including human tumors, are treated with a masked targeted IFN fusion protein that binds to a specific TAA in combination with additional therapeutic agents, such as chemotherapy, radiation, immunomodulatory therapy, or any combination thereof. In other words, the inhibition of tumor growth by the masked targeted IFN fusion protein that binds to a specific TAA is enhanced to a greater extent than would be expected when combined with chemotherapy or radiation, or a combination thereof. Synergy can be shown, for example, by a tumor growth inhibition with combined treatment that is greater than would be expected with treatment with a masked targeted IFN fusion protein that binds to a specific TAA alone, or the additive effect of treatment with a masked targeted IFN fusion protein that binds to a specific TAA and chemotherapy or radiation. Synergy is preferably demonstrated by remission of cancer when remission is not expected with treatment with a masked targeted IFN fusion protein that binds to a specific TAA, or treatment with an additive combination of a masked targeted IFN fusion protein that binds to a specific TAA and chemotherapy or radiation.

[0347] The method for inhibiting tumor cell growth using a combination of a masked targeted IFN fusion protein that binds to a specific TAA and chemotherapy, radiation, or immunomodulatory therapy, or a combination of any one, two, or three, includes administering a masked targeted IFN fusion protein that binds to a specific TAA before, at, or after chemotherapy or radiation therapy, and any combination thereof (i.e., before and at, before and after, at, or before, at, and after chemotherapy and / or radiation therapy). For example, a masked targeted IFN fusion protein that binds to a specific TAA is generally administered between 1 and 60 days, preferably between 3 and 40 days, and more preferably between 5 and 12 days, before radiation therapy and / or chemotherapy is initiated. However, depending on the treatment protocol and the needs of the particular patient, the method is performed in a manner that provides the most effective treatment and ultimately extends the patient's lifespan.

[0348] Administration of the chemotherapeutic agent can be accomplished in a variety of ways, including systemically via parenteral and enteral routes. In one embodiment, the masked targeted IFN fusion protein that binds to a specific TAA and the 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 (taxonomycin), cetaxel (cetaxel ... TAIL), aldesleukin, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon alpha, 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.

[0349] The source of radiation used in combination with the masked targeted IFN fusion protein that binds to a specific TAA can be external or internal to the patient undergoing treatment. When the source is external to the patient, the treatment is known as external beam radiotherapy (EBRT). When the source of radiation is internal to the patient, the treatment is called brachytherapy (BT).

[0350] The above therapeutic regimens can be further combined with additional cancer treatment agents and / or regimens, such as bortezomib, pomalidomide, and / or additional chemotherapy, cancer vaccines, signal transduction inhibitors, agents useful in the treatment of abnormal cell growth or cancer, antibodies (e.g., anti-CTLA-4 antibodies described in WO / 2005 / 092380 (Pfizer)) or other ligands that inhibit tumor growth by binding to IGF-1R, and cytokines.

[0351] Examples of immune modulating therapies 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.

[0352] When subjecting mammal to additional chemotherapy, the above-mentioned chemotherapeutic agents can be used.In addition, growth factor inhibitors, biological response modifiers, antihormonal therapy, selective estrogen receptor modulators (SERMs), angiogenesis inhibitors, and antiandrogens can be used.For example, antihormonal agents, such as antiestrogens such as Nolvadex (tamoxifen) or antiandrogens such as Casodex (4'-cyano-3-(4-fluorophenylsulfonyl)-2-hydroxy-2-methyl-3-'-(trifluoromethyl)propionanilide) can be used.

[0353] The above-mentioned therapeutic approach can be combined with any one of a wide variety of surgical, chemotherapy or radiotherapy regimens.The therapeutic approach of the present invention allows the use of reduced dosages of chemotherapy (or other therapy) and / or less frequent administration, which may be an advantage for all patients, especially those who do not tolerate the toxicity of chemotherapy agents well.

[0354] VIII.) Kits / Manufactured Products Kits, articles of manufacture, systems, and devices are within the scope of the present invention for use in the testing, prognostic, prophylactic, diagnostic, and therapeutic applications described herein. Such kits may include a carrier, package, or container that is compartmentalized to receive one or more containers, such as vials, tubes, etc., each of which includes one of the separate elements used in the method, along with a label or insert that includes instructions for use, such as the uses described herein. For example, a container may include a masked targeted IFN fusion protein that binds to a specific TAA or several TAAs of the present disclosure. A kit may include a container that includes a masked targeted IFN. A kit may include a masked targeted IFN fusion protein that binds to a specific TAA, and / or all or part of a diagnostic assay for detecting cancer and / or other immune disorders.

[0355] Kits of the present invention generally include the above-described container and one or more other containers containing materials desirable from a commercial and user standpoint, including associated buffers, diluents, filters, needles, syringes; carriers, packages, containers, vials and / or tube labels reciting contents and / or instructions for use, and package inserts with instructions for use.

[0356] A label may be present on or with the container to indicate that the composition is used for a particular therapeutic or non-therapeutic application, such as a prognostic, preventative, diagnostic, or testing application, and may indicate instructions for use either in vivo or in vitro, such as those described herein. Instructions and other information may also be included in a package insert(s) or label(s) included with or on the kit. The label may be on or associated with the container. The label may be on the container if the letters, numbers, or other symbols forming the label are embossed or etched into the container itself, or the label may be associated with the container if the label is present in a receptacle or carrier that also holds the container, e.g., as a package insert. The label may indicate that the composition is used to diagnose, treat, prevent, or prognose a condition, such as cancer or other immunological disorder.

[0357] The terms "kit" and "article of manufacture" may be used as synonyms.

[0358] In another embodiment of the present invention, an article of manufacture contains a composition such as a masked targeted IFN fusion protein that binds to a specific TAA of the present disclosure. The article of manufacture generally 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 made of various materials such as glass, metal, or plastic. The container can hold a masked targeted IFN fusion protein that binds to one or several specific TAAs and / or one or more therapeutic doses of masked targeted IFN.

[0359] Alternatively, the container may hold a composition effective for treating, diagnosing, prognosing or preventing a condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition may be a masked targeted IFN fusion protein that binds to a particular TAA of the disclosure.

[0360] The article of manufacture may further include a second container containing a pharma- ceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and / or dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, stirrers, needles, syringes, and / or package inserts with indications and / or instructions for use.

[0361] Exemplary embodiments 1) A composition comprising TLLYTIMSKPEDLK (SEQ ID NO: 25), wherein the composition further comprises a fusion protein that masks the activity of type 1 interferon and is fused to an antibody that binds to a tumor-associated antigen ("TAA").

[0362] 2) A composition comprising STHEATVTVLEGFSG (SEQ ID NO: 26), the composition further comprising a fusion protein that masks the activity of type 1 interferon and is fused to an antibody that binds to a tumor-associated antigen.

[0363] 3) A composition comprising TLFSSSHNFWLAIDMS (SEQ ID NO: 27), the composition further comprising a fusion protein that masks the activity of type 1 interferon and is fused to an antibody that binds to a tumor-associated antigen.

[0364] 4) A composition comprising STHEAYVTVLEGFSNTTLFSSSHNFWLAIDMS (SEQ ID NO: 28), the composition further comprising a fusion protein that masks the activity of type 1 interferon and is fused to an antibody that binds to a tumor-associated antigen.

[0365] 5) A composition comprising TDVDYYREWSWTQVGG (SEQ ID NO: 30), the composition further comprising a fusion protein that masks the activity of type 1 interferon and is fused to an antibody that binds to a tumor-associated antigen.

[0366] 6) A composition comprising TDVDYYREWSWTQVSGG (SEQ ID NO: 31), the composition further comprising a fusion protein that masks the activity of type 1 interferon and is fused to an antibody that binds to a tumor-associated antigen.

[0367] 7) A composition comprising TDVDYYREWSWTQ (sequence number 32), the composition further comprising a fusion protein that masks the activity of type 1 interferon and is fused to an antibody that binds to a tumor-associated antigen.

[0368] 8) A composition comprising TLFSSSHNFWLAIDMS (SEQ ID NO: 33), the composition further comprising a fusion protein that masks the activity of type 1 interferon and is fused to an antibody that binds to a tumor-associated antigen.

[0369] 9) A composition comprising TDVDYYREWSWTQV (sequence number 34), the composition further comprising a fusion protein that masks the activity of type 1 interferon and is fused to an antibody that binds to a tumor-associated antigen.

[0370] 10) A composition comprising TLFSSSHNFWLAIDMSGG (sequence number 35), the composition further comprising a fusion protein that masks the activity of type 1 interferon and is fused to an antibody that binds to a tumor-associated antigen.

[0371] 11) A composition comprising TDVDYYREWSWTQVGGSGGSGGGKVKAALLTSWKIGVYS (sequence number 47), wherein the composition further comprises a fusion protein that masks the activity of type 1 interferon and is fused to an antibody that binds to a tumor-associated antigen.

[0372] 12) A composition comprising ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTRSFCDLTDEWRSTHEAYVTVLEGFSGNTTLFSSCSHNFWLAIDMS (sequence number 48), wherein the composition further comprises a fusion protein that masks the activity of type 1 interferon and is fused to an antibody that binds to a tumor-associated antigen.

[0373] 13) The composition according to any one of items 1 to 12, further comprising a flexible peptide linker.

[0374] 14) The composition according to item 13, further comprising a tumor-associated protease cleavage site.

[0375] 15) The composition according to any one of items 1 to 12, wherein the type 1 interferon comprises IFNα1.

[0376] 16) The composition according to any one of items 1 to 12, wherein the type 1 interferon includes IFNα2.

[0377] 17) The composition according to any one of items 1 to 12, wherein the type 1 interferon comprises IFNα4.

[0378] 18) The composition according to any one of items 1 to 12, wherein the type 1 interferon comprises IFNα5.

[0379] 19) The composition according to any one of items 1 to 12, wherein the type 1 interferon comprises IFNα6.

[0380] 20) The composition according to any one of items 1 to 12, wherein the type 1 interferon includes IFNα14.

[0381] 21) The composition according to any one of items 1 to 12), wherein the type 1 interferon comprises IFNβ1.

[0382] 22) The composition according to any one of items 1 to 12, wherein the type 1 interferon or functionally active variant is selected from the type 1 interferons shown in Table II.

[0383] 23) The composition according to any one of items 1 to 12, wherein the TAA comprises CD138.

[0384] 24) The composition according to any one of items 1 to 12, wherein the TAA comprises CD20.

[0385] 25) The composition according to any one of items 1 to 12, wherein the TAA comprises PSCA.

[0386] 26) The composition according to any one of items 1 to 12, wherein the TAA comprises a FAP.

[0387] 27) The composition according to any one of items 1 to 12, wherein the TAA is selected from the tumor-associated antigens shown in Table I.

[0388] 28) a. an antibody that specifically binds to a tumor-associated antigen; b. a type 1 interferon, wherein the N-terminus of the type 1 interferon is fused to the C-terminus of an antibody heavy and / or light chain; and c. An interferon mask comprising (SEQ ID NO:25), whereby the interferon mask is linked to the C-terminus of a type 1 interferon. "Masked targeted IFN."

[0389] 29) a. an antibody that specifically binds to a tumor-associated antigen; b. a type 1 interferon, wherein the N-terminus of the type 1 interferon is fused to the C-terminus of an antibody heavy and / or light chain; and c. An interferon mask comprising (SEQ ID NO:26), whereby the interferon mask is linked to the C-terminus of a type 1 interferon. "Masked targeted IFN."

[0390] 30) a. an antibody that specifically binds to a tumor-associated antigen; b. a type 1 interferon, wherein the N-terminus of the type 1 interferon is fused to the C-terminus of an antibody heavy and / or light chain; and c. An interferon mask comprising (SEQ ID NO:27), whereby the interferon mask is linked to the C-terminus of a type 1 interferon. "Masked targeted IFN."

[0391] 31) a. an antibody that specifically binds to a tumor-associated antigen; b. a type 1 interferon, wherein the N-terminus of the type 1 interferon is fused to the C-terminus of an antibody heavy and / or light chain; and c. An interferon mask comprising (SEQ ID NO:28), whereby the interferon mask is linked to the C-terminus of a type 1 interferon. "Masked targeted IFN."

[0392] 32) a. an antibody that specifically binds to a tumor-associated antigen; b. a type 1 interferon, wherein the N-terminus of the type 1 interferon is fused to the C-terminus of an antibody heavy and / or light chain; and c. An interferon mask comprising (SEQ ID NO:30), whereby the interferon mask is linked to the C-terminus of a type 1 interferon. "Masked targeted IFN."

[0393] 33) a. an antibody that specifically binds to a tumor-associated antigen; b. a type 1 interferon, wherein the N-terminus of the type 1 interferon is fused to the C-terminus of an antibody heavy and / or light chain; and c. An interferon mask comprising (SEQ ID NO:31), whereby the interferon mask is linked to the C-terminus of a type 1 interferon. "Masked targeted IFN."

[0394] 34) a. an antibody that specifically binds to a tumor-associated antigen; b. a type 1 interferon, wherein the N-terminus of the type 1 interferon is fused to the C-terminus of an antibody heavy and / or light chain; and c. An interferon mask comprising (SEQ ID NO:32), whereby the interferon mask is linked to the C-terminus of a type 1 interferon. "Masked targeted IFN."

[0395] 35) a. an antibody that specifically binds to a tumor-associated antigen; b. a type 1 interferon, wherein the N-terminus of the type 1 interferon is fused to the C-terminus of an antibody heavy and / or light chain; and c. An interferon mask comprising (SEQ ID NO:33), whereby the interferon mask is linked to the C-terminus of a type 1 interferon. "Masked targeted IFN."

[0396] 36) a. an antibody that specifically binds to a tumor-associated antigen; b. a type 1 interferon, wherein the N-terminus of the type 1 interferon is fused to the C-terminus of an antibody heavy and / or light chain; and c. An interferon mask comprising (SEQ ID NO:34), whereby the interferon mask is linked to the C-terminus of a type 1 interferon. "Masked targeted IFN."

[0397] 37) a. an antibody that specifically binds to a tumor-associated antigen; b. a type 1 interferon, wherein the N-terminus of the type 1 interferon is fused to the C-terminus of an antibody heavy and / or light chain; and c. An interferon mask comprising (SEQ ID NO:35), whereby the interferon mask is linked to the C-terminus of a type 1 interferon. "Masked targeted IFN."

[0398] 38) a. an antibody that specifically binds to a tumor-associated antigen; b. a type 1 interferon, wherein the N-terminus of the type 1 interferon is fused to the C-terminus of an antibody heavy and / or light chain; and c. An interferon mask comprising (SEQ ID NO: 47), whereby the interferon mask is linked to the C-terminus of a type 1 interferon. "Masked targeted IFN."

[0399] 39) a. an antibody that specifically binds to a tumor-associated antigen; b. a type 1 interferon, wherein the N-terminus of the type 1 interferon is fused to the C-terminus of an antibody heavy and / or light chain; and c. An interferon mask comprising (SEQ ID NO: 48), whereby the interferon mask is linked to the C-terminus of a type 1 interferon. "Masked targeted IFN."

[0400] 40) The "masked targeted IFN" according to any one of items 28 to 39, wherein the N-terminus of type 1 interferon is fused to the C-terminus of an antibody heavy chain and / or light chain further comprising a flexible peptide linker.

[0401] 41) The "masked targeted IFN" according to any one of items 28 to 39, wherein the interferon mask is attached to the C-terminus of a type 1 interferon that further comprises a flexible peptide linker.

[0402] 42) The "masked targeted IFN" according to paragraph 40, further comprising a tumor-associated protease cleavage site inserted between the antibody and the flexible peptide linker.

[0403] 43) The "masked targeted IFN" according to item 41, further comprising a tumor-associated protease cleavage site inserted between the IFN and the IFN mask.

[0404] 44) The masked targeted interferon according to any one of items 28 to 39, wherein the antibody binds to a tumor-associated antigen listed in Table I.

[0405] 45) The masked targeted interferon according to any one of items 28 to 39, wherein the type 1 interferon or functionally active variant is listed in Table II.

[0406] 46) The masked targeted interferon according to any one of items 28 to 39, wherein the functionally active mutant is a YNS mutant.

[0407] 47) The masked targeted interferon of any one of items 28 to 39, wherein the antibody binds to CD138.

[0408] 48) The masked targeted interferon of any of items 28 to 39, wherein the antibody binds to CD20.

[0409] 49) A method for producing a masked targeted interferon according to any one of items 28 to 39.

[0410] 50) A pharmaceutical composition comprising a therapeutically effective amount of the masked targeted IFN according to any one of items 28 to 39, wherein (i) optionally, the pharmaceutical composition is for use in therapy, including the treatment of cancer, 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-cancer agents.

[0411] 51) A kit comprising the masked targeted IFN according to any one of items 28 to 39.

[0412] 52) A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a masked targeted IFN according to item 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39, wherein optionally the subject is a human subject.

[0413] 53) A composition comprising TDVDYYREWSWTQV (SEQ ID NO: 34), wherein the composition masks the activity of type 1 interferon, the composition further comprises a fusion protein comprising (SEQ ID NO: 5) fused to an antibody that binds CD138, the antibody comprising a heavy chain set forth in (SEQ ID NO: 6) and further comprising a light chain set forth in (SEQ ID NO: 8).

[0414] 54) [ka] (SEQ ID NO: 38), wherein the flexible peptide linker is [ka] 54. The composition of any of paragraphs 53, further comprising a cleavable linker set forth as (SEQ ID NO: 16).

[0415] 55) The composition of any of paragraphs 53, wherein the antibody comprises a variable heavy chain as set forth in (SEQ ID NO: 7).

[0416] 56) The composition of any of paragraphs 53, wherein the antibody comprises a variable light chain set forth in (SEQ ID NO: 9).

[0417] 57) The composition according to any one of items 53 to 56, wherein the type 1 interferon comprises IFNα1.

[0418] 58) The composition according to any one of items 53 to 56, wherein the type 1 interferon includes IFNα2.

[0419] 59) The composition according to any one of items 53 to 56, wherein the type 1 interferon includes IFNα4.

[0420] 60) The composition according to any one of items 53 to 56, wherein the type 1 interferon includes IFNα5.

[0421] 61) The composition according to any one of items 53 to 56, wherein the type 1 interferon comprises IFNα6.

[0422] 62) The composition according to any one of items 53 to 56, wherein the type 1 interferon includes IFNα14.

[0423] 63) The composition according to any one of items 53 to 56, wherein the type 1 interferon includes IFNβ1.

[0424] 64) The composition according to any one of items 53 to 56, wherein the type 1 interferon or functionally active variant is selected from the type 1 interferons shown in Table II.

[0425] 65) The composition according to any one of items 53 to 56, wherein the type 1 interferon contains a YNS mutation.

[0426] 66) a. An antibody that specifically binds to the CD138 antigen, said antibody comprising a variable heavy chain set forth in (SEQ ID NO:7) and a variable light chain set forth in (SEQ ID NO:9); b. a type 1 interferon, wherein the N-terminus of the type 1 interferon is fused to the C-terminus of an antibody heavy and / or light chain; and c. An interferon mask comprising (SEQ ID NO:30), whereby the interferon mask is linked to the C-terminus of a type 1 interferon. "Masked targeted IFN."

[0427] 67) The "masked targeted IFN" according to paragraph 66, wherein the N-terminus of type 1 interferon is fused to the C-terminus of an antibody heavy and / or light chain further comprising a flexible peptide linker as set forth in (SEQ ID NO: 38).

[0428] 68) The "masked targeted IFN" according to paragraph 67, wherein the interferon mask is attached to the C-terminus of a type 1 interferon which further comprises a flexible peptide linker.

[0429] 69) The "masked targeted IFN" according to paragraph 67, further comprising a tumor-associated protease cleavage site inserted between the antibody and the flexible peptide linker.

[0430] 70) The "masked targeted IFN" according to paragraph 67, further comprising a tumor-associated protease cleavage site inserted between the IFN and the IFN mask.

[0431] 71) The masked targeted interferon of item 67, wherein the type 1 interferon or functionally active variant is listed in Table II.

[0432] 72) The masked targeted IFN of paragraph 65, wherein the functionally active mutant is a YNS mutant.

[0433] 73) The masked targeted interferon of paragraph 67, wherein the antibody binds to CD138.

[0434] 74) A method for producing a masked targeted interferon according to any one of items 67.

[0435] 75) A pharmaceutical composition comprising a therapeutically effective amount of the masked targeted IFN according to any of paragraphs 67, wherein (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-cancer agents.

[0436] 76) A kit comprising a masked targeted IFN according to any of items 67.

[0437] 77) A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a masked targeted IFN described in paragraph 65, wherein optionally the subject is a human subject.

[0438] 78) A composition comprising the sequence depicted in Figure 25 (sequence number 41).

[0439] 79) A kit comprising the composition according to item 78.

[0440] 80) A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the composition described in paragraph 78, optionally wherein the subject is a human subject.

[0441] 81) a. An antibody that specifically binds to the CD138 antigen, comprising a heavy chain variable region (SEQ ID NO:7) and a heavy chain constant region (SEQ ID NO:54), and a variable light chain as set forth in (SEQ ID NO:9); b. a type 1 interferon, wherein the N-terminus of the type 1 interferon is fused to the C-terminus of an antibody heavy and / or light chain; and c. An interferon mask comprising (SEQ ID NO: 48), whereby the interferon mask is linked to the C-terminus of a type 1 interferon. "Masked targeted IFN."

[0442] 82) The "masked targeted IFN" according to paragraph 81, wherein the N-terminus of a type 1 interferon is fused to the C-terminus of an antibody heavy and / or light chain further comprising a flexible peptide linker as set forth in (SEQ ID NO: 37).

[0443] 83) The "masked targeted IFN" according to paragraph 81, wherein the interferon mask is attached to the C-terminus of a type 1 interferon which further comprises a flexible peptide linker.

[0444] 84) The "masked targeted IFN" according to paragraph 81, further comprising a tumor-associated protease cleavage site inserted between the antibody and the flexible peptide linker.

[0445] 85) The "masked targeted IFN" according to paragraph 81, further comprising a tumor-associated protease cleavage site inserted between the IFN and the IFN mask.

[0446] 86) The masked targeted interferon of item 81, wherein the type 1 interferon or functionally active variant is listed in Table II.

[0447] 87) The masked targeted IFN of paragraph 81, wherein the functionally active mutant is a YNS mutant.

[0448] 88) The masked targeted interferon of paragraph 81, wherein the antibody binds to CD138.

[0449] 89) A method for producing a masked targeted interferon according to any one of items 81.

[0450] 90) A pharmaceutical composition comprising a therapeutically effective amount of the masked targeted IFN according to any of paragraphs 81, wherein (i) optionally the pharmaceutical composition is for use in therapy, including the treatment of cancer, 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-cancer agents.

[0451] 91) A kit comprising a masked targeted IFN according to any of items 81.

[0452] 92) A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a masked targeted IFN described in paragraph 81, optionally wherein the subject is a human subject.

[0453] 93) A composition comprising the sequence set forth in Figure 59 (sequence number 49).

[0454] 94) A kit comprising the composition according to item 93.

[0455] 95) A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the composition described in item 93, optionally wherein the subject is a human subject.

[0456] 96) a. An antibody that specifically binds to the CD138 antigen, comprising a heavy chain variable region (SEQ ID NO:7) and a heavy chain constant region (SEQ ID NO:55), and a variable light chain as set forth in (SEQ ID NO:9); b. a type 1 interferon, wherein the N-terminus of the type 1 interferon is fused to the C-terminus of an antibody heavy and / or light chain; and c. An interferon mask comprising (SEQ ID NO: 48), whereby the interferon mask is linked to the C-terminus of a type 1 interferon. "Masked targeted IFN."

[0457] 97) The "masked targeted IFN" of paragraph 96, wherein the N-terminus of a type 1 interferon is fused to the C-terminus of an antibody heavy and / or light chain further comprising a flexible peptide linker as described in (SEQ ID NO: 37).

[0458] 98) The "masked targeted IFN" according to paragraph 97, wherein the interferon mask is attached to the C-terminus of a type 1 interferon which further comprises a flexible peptide linker.

[0459] 99) The "masked targeted IFN" described in paragraph 96, further comprising a tumor-associated protease cleavage site inserted between the antibody and the flexible peptide linker.

[0460] 100) The "masked targeted IFN" according to paragraph 96, further comprising a tumor-associated protease cleavage site inserted between the IFN and the IFN mask.

[0461] 101) The masked targeted interferon of item 96, wherein the type 1 interferon or functionally active variant is listed in Table II.

[0462] 102) The masked targeted IFN of paragraph 96, wherein the functionally active mutant is a YNS mutant.

[0463] 103) The masked targeted interferon of paragraph 96, wherein the antibody binds to CD138.

[0464] 104) A method for producing a masked targeted interferon according to any of items 96.

[0465] 105) A pharmaceutical composition comprising a therapeutically effective amount of a masked targeted IFN according to any of paragraphs 96, wherein (i) optionally the pharmaceutical composition is for use in therapy, including the treatment of cancer, 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-cancer agents.

[0466] 106) A kit comprising a masked targeted IFN according to any of items 96.

[0467] 107) A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the masked targeted IFN described in paragraph 96, wherein optionally the subject is a human subject.

[0468] 108) A composition comprising the sequence set forth in Figure 61 (sequence number 51).

[0469] 109) A kit comprising the composition according to item 108.

[0470] 110) A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the composition described in paragraph 108, optionally wherein the subject is a human subject. EXAMPLES

[0471] Various aspects of the present invention are further described and illustrated by the following several examples, none of which are intended to limit the scope of the invention.

[0472] Example 1: Characterization of masked targeted IFNα2 fused with anti-CD138 (anti-CD138-IFNα2). In this example, it is shown that the IFN mask can be cleaved from the H chain using Matripase ST 14 ("MST 14"). 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 then denatured by heating to 95°C, reduced with approximately 2% beta-mercaptoethanol (Thermofisher), and run 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 run on a 5% PO4 SDS-PAGE gel.

[0473] The analytical results obtained show that the IFN mask on the anti-CD138 fusion antibody is efficiently cleaved by MST 14. Unmasked anti-CD138-IFNα was used as a control (see FIG. 5).

[0474] Example 2: Binding of masked fusion antibodies (using mask 1 and mask 2) to the IFNα2 receptor. This example shows that the masked fusion antibody of the present disclosure (using mask 2) can bind to the IFNα2 receptor. Briefly, Immulon 2 HB plates (Thermofisher) were coated with 10 μg / mL IFNαR2 (R&D Systems) overnight at 4° C. and blocked with 2% BSA (Fisher) for a minimum of 2 hours at room temperature. Wells were then washed three times with PBS+0.05% Tween® (Sigma). Indicated antibody concentrations were loaded overnight at 4° C. Wells were then washed three times with PBS+0.05% Tween®. Bound antibodies were detected with anti-human Kappa-AP (Southern Biotech) diluted 1:3000 in PBS+1% BSA. The change in absorbance after addition of AP substrate (Sigma) was assayed at 410 nm using a Biotek EPOCH ELISA reader.

[0475] The results show that both mask 1 and mask 2 can inhibit the binding of the fusion antibody to IFNαR2 (see FIG. 6).

[0476] Example 3: Binding of masked fusion antibodies (using mask 1, mask 2, and mask 3) to the IFNα2 receptor. In a separate experiment, masked fusion antibodies (utilizing mask 1, mask 2, and mask 3) were analyzed to assess binding to the IFNα2 receptor. Briefly, Immulon 2 HB plates (Thermofisher) were coated with 10 μg / mL IFNαR2 (R&D Systems) overnight at 4° C. and blocked with 2% BSA (Fisher) for a minimum of 2 hours at room temperature. Wells were then washed three times with PBS+0.05% Tween® (Sigma). Indicated antibody concentrations were loaded overnight at 4° C. Wells were then washed three times with PBS+0.05% Tween®. Bound antibodies were detected with anti-human Kappa-AP (Southern Biotech) diluted 1:3000 in PBS+1% BSA. The change in absorbance after addition of AP substrate (Sigma) was assayed at 410 nm using a Biotek EPOCH ELISA reader.

[0477] The results show that both mask 1 and mask 2 can inhibit the binding of fusion antibody to IFNαR2. However, mask 3 does not appear to significantly inhibit the binding of fusion antibody to IFNAR (see FIG. 7).

[0478] Example 4: Binding of masked fusion antibodies (using mask 1, mask 2, mask 2.2, and mask 3) to the IFNα2 receptor. In a separate experiment, masked fusion antibodies (utilizing mask 1, mask 2, mask 2.2, and mask 3) were analyzed to assess binding to the IFNα2 receptor. Briefly, Immulon 2 HB plates (Thermofisher) were coated with 10 μg / mL IFNαR2 (R&D Systems) overnight at 4° C. and blocked with 2% BSA (Fisher) for a minimum of 2 hours at room temperature. Wells were then washed three times with PBS+0.05% Tween® (Sigma). Indicated antibody concentrations were loaded overnight at 4° C. Wells were then washed three times with PBS+0.05% Tween®. Bound antibodies were detected with anti-human Kappa-AP (Southern Biotech) diluted 1:3000 in PBS+1% BSA. The change in absorbance after addition of AP substrate (Sigma) was assayed at 410 nm using a Biotek EPOCH ELISA reader.

[0479] The results show that mask 1, mask 2 and mask 2.2 can inhibit the binding of fusion antibody to IFNαR2. However, mask 3 does not seem to inhibit the binding of fusion antibody to IFNAR. The results seem to show that mask 3 enhances the binding with IFNAR2 (see FIG. 8).

[0480] (Example 5: Method for binding a fusion antibody to a masking peptide) In this example, it is shown that multiple fusion antibodies specifically bind to additional peptide masks of the present disclosure. As a reference, one peptide mask was tested: Peptide 6 (Mask 3): TLFSSSHNFWLAIDMS (SEQ ID NO:27);

[0481] Briefly, streptavidin-coated plates (Pierce) were loaded with 50 μM of each indicated peptide (Thermofisher) for a minimum of 2 hours at room temperature. Wells were then washed three times with PBS+0.05% Tween®. Antibodies at the indicated concentrations were then allowed to bind overnight at 4° C. Wells were washed three times with PBS+0.05% Tween®. Bound antibodies were detected using anti-human Kappa-AP (Southern Biotech) diluted 1:3000 in PBS+1% BSA. Changes in absorbance following addition of AP substrate (Sigma) were assayed at 410 nm using a Biotek EPOCH ELISA reader.

[0482] The results show that peptide 6 binds to all IFN fusion antibodies tested (see FIG. 9). Furthermore, compared to peptide 1 (see Example 3), peptide 6 binds to all IFN fusion antibodies with a similar Kd (see FIG. 9).

[0483] Example 6: Binding of masked fusion antibodies (using antibodies with different targets) to the IFNα2 receptor. In this example, various fusion antibodies (anti-CD138 IgG1-IFNα, anti-5T4 IgG1-IFNα masked, and anti-mesothelin IgG1-IFNα masked) were tested to compare the IFNAR2 binding affinity of masked anti-5T4 and anti-mesothelin fusion antibodies with unmasked anti-CD138 fusion antibodies. Briefly, Immulon 2 HB plates (Thermofisher) were coated with 10 μg / mL IFNαR2 (R&D Systems) overnight at 4° C. and blocked with 2% BSA (Fisher) for a minimum of 2 hours at room temperature. Wells were then washed three times with PBS+0.05% Tween® (Sigma). The indicated antibody concentrations were loaded overnight at 4° C. Wells were then washed three times with PBS+0.05% Tween®. Bound antibodies were detected with anti-human Kappa-AP (Southern Biotech) diluted 1:3000 in PBS+1% BSA. The change in absorbance after addition of AP substrate (Sigma) was assayed at 410 nm using a Biotek EPOCH ELISA reader.

[0484] The results show that the masked anti-5T4 and anti-mesothelin antibodies bind to IFNAR with approximately 40-80 fold lower affinity compared to the unmasked (anti-CD138 IgG1-IFNα) fusion antibody (see FIG. 10).

[0485] Example 7: 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 restore IFNα activity. Briefly, HEK Blue IFNα / β cells (Invivogen) were plated in 96-well tissue culture plates (Fisher) at 1×10 cells per well. 4Cells were seeded at a density of 1000 μL / well (50 μL). 50 μL / well of recombinant IFNα (Novus Biologicals) or the indicated antibodies (5T4 or mesothelin) were incubated with the cells at the indicated concentrations overnight at 37° C. Antibodies cleaved with MST14 (R&D Systems) were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 h at 37° C. 10 μL of supernatant was then added to the plate containing 90 μL / well Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader.

[0486] The results show that the IFNα activity of masked anti-CD138-IFNα (Mask 1) and masked anti-CD138-IFNα (Mask 2) was reduced compared to when the mask was cleaved (see Figures 11(A) and 11(B)).

[0487] Example 8: Methods for reducing and restoring masked IFNα activity. In this example, further testing was performed to demonstrate that the masks of the present disclosure can reduce and restore IFNα activity. Briefly, HEK Blue IFNα / β cells (Invivogen) were plated in 96-well tissue culture plates (Fisher) at 1×10 cells per well. 4 Cells were seeded at a density of 1000 μL / well (50 μL). 50 μL / well of recombinant IFNα (Novus Biologicals) or the indicated antibodies (5T4 or mesothelin) were incubated with the cells at the indicated concentrations overnight at 37° C. Antibodies cleaved with MST14 (R&D Systems) were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 h at 37° C. 10 μL of supernatant was then added to the plate containing 90 μL / well Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader.

[0488] The results show that the IFNα masks (anti-5T4 and anti-mesothelin) reduce IFNα activity by 1-2 logs compared to when the masks are cleaved (see Figures 12(A) and 12(B)).

[0489] Example 9: Methods for reducing and restoring masked IFNα activity. In this example, further testing was performed to demonstrate that the masks disclosed herein (IFNα Mask 1, IFNα Mask 2.2, and IFNα Mask 3) can reduce and restore IFNα activity. Briefly, HEK Blue IFNα / β cells (Invivogen) were plated in 96-well tissue culture plates (Fisher) at 1×10 cells per well. 4 Cells were seeded at a density of 1000 μL / well (50 μL). 50 μL / well of recombinant IFNα (Novus Biologicals) or the indicated antibodies (5T4 or mesothelin) were incubated with the cells at the indicated concentrations overnight at 37° C. Antibodies cleaved with MST14 (R&D Systems) were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 h at 37° C. 10 μL of supernatant was then added to the plate containing 90 μL / well Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader.

[0490] The results show that IFNα mask 1 and IFNα mask 2 reduce IFNα activity, but IFNα mask 3 did not effectively reduce activity (see Figures 13(A) and 13(B)).

[0491] Example 10: Methods for reducing and restoring masked IFNα activity. In this example, further testing was performed to demonstrate that the masks disclosed herein (IFNα Mask 1, IFNα Mask 2, IFNα Mask 2.2, and IFNα Mask 3) can reduce and restore IFNα activity. Briefly, HEK Blue IFNα / β cells (Invivogen) were plated in 96-well tissue culture plates (Fisher) at 1×10 cells per well. 4 Cells were seeded at a density of 1000 μL / well (50 μL). 50 μL / well of recombinant IFNα (Novus Biologicals) or the indicated antibodies (5T4 or mesothelin) were incubated with the cells at the indicated concentrations overnight at 37° C. Antibodies cleaved with MST14 (R&D Systems) were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 h at 37° C. 10 μL of supernatant was then added to the plate containing 90 μL / well Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader.

[0492] The results show that IFNα mask 1, IFNα mask 2, and IFNα mask 2.2 reduce IFNα activity. However, IFNα mask 3 did not effectively reduce activity (see Figures 14(A) and 14(B)).

[0493] Example 11: Methods for reducing and restoring masked IFNα activity. In this example, further testing was performed to show that the masks of the present disclosure (IFNα Mask 1, IFNα Mask 1 N297Q, IFNα Mask 2.2, IFNα Mask 2.2 (N297Q), IFNα Mask 3, and IFNα Mask 3.2 N297Q) can reduce and restore IFNα activity. Briefly, HEK Blue IFNα / β cells (Invivogen) were plated in 96-well tissue culture plates (Fisher) at 1×10 cells per well. 4Cells were seeded at a density of 1000 μL / well (50 μL). 50 μL / well of recombinant IFNα (Novus Biologicals) or the indicated antibodies (5T4 or mesothelin) were incubated with the cells at the indicated concentrations overnight at 37° C. Antibodies cleaved with MST14 (R&D Systems) were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 h at 37° C. 10 μL of supernatant was then added to the plate containing 90 μL / well Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader.

[0494] The results show that the introduction of the N297Q mutation did not affect the IFNα activity of the fusion protein compared to its wild-type counterpart. Moreover, unmasking restores the IFNα activity of the N297Q fusion protein to the level observed in its wild-type counterpart (see Figures 15(A), 15(B), and 15(C)).

[0495] Example 12: Methods for reducing and restoring masked IFNα activity. In this example, further studies were performed to show that the masks of the present disclosure (IFNα Mask 1 N297Q and IFNα Mask 3.2 N297Q) can reduce and restore IFNα activity. Briefly, HEK Blue IFNα / β cells (Invivogen) were plated in 96-well tissue culture plates (Fisher) at 1×10 cells per well. 4Cells were seeded at a density of 1000 μL / well (50 μL). 50 μL / well of recombinant IFNα (Novus Biologicals) or the indicated antibodies (5T4 or mesothelin) were incubated with the cells at the indicated concentrations overnight at 37° C. Antibodies cleaved with MST14 (R&D Systems) were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 h at 37° C. 10 μL of supernatant was then added to the plate containing 90 μL / well Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader.

[0496] The results show that IFNα mask 3.2 reduces the IFNα activity of the fusion protein whether or not it is unmasked (see FIG. 16).

[0497] Example 13: Methods for reducing induction of IP-10 in PBMCs. This example shows that several masked fusion antibodies of the present disclosure (anti-5T4 IFNα mask 1 and anti-mesothelin IFNα mask 1) can reduce the induction of IP-10. Briefly, freshly thawed human PBMCs (Human Cells Biosciences) were washed once with cold RPMI + 10% FBS (Invitrogen) and plated into 12-well plates (Themofisher) with approximately 1 x 10 cells per well. 6Cells were seeded at a density of 1000 μg / well (1 mL / well). Any Fc receptor and / or CD138 antigen expression was blocked / reduced by adding 300 nM of unfused anti-CD138 IgG1 to the cells for 1 h before proceeding with the described experiments. Recombinant human IFNα (Novus Biologicals) or the indicated antibodies (5T4 or mesothelin) were then added to the cells and incubated for an additional 7 h at 37°C. MST14 (R&D Systems) cleaved antibodies were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 h at 37°C. After 7 h incubation, cells were spun down at 500×g for 3 min and 20 μL of supernatant from each sample was assayed for IP-10 (Abcam) by ELISA according to the manufacturer's protocol.

[0498] The results show that masking both the anti-5T4 and anti-mesothelin fusion antibodies reduces the induction of IP-10 (see FIG. 17).

[0499] Example 14: Methods for reducing induction of IP-10 in PBMCs. In this example, several masked fusion antibodies, both glycosylated and non-glycosylated (anti-CD138 IFNα mask 1, anti-CD138 IFNα mask 1 N297Q) were tested for reducing induction of IP-10.

[0500] Briefly, freshly thawed human PBMCs (Human Cells Biosciences) were washed once with cold RPMI + 10% FBS (Invitrogen) and plated into 12-well plates (Themofisher) at approximately 1 × 10 cells per well. 6Cells were seeded at a density of 1000 μg / well (1 mL / well). Any Fc receptor and / or CD138 antigen expression was blocked / reduced by adding 300 nM of unfused anti-CD138 IgG1 to the cells for 1 h before proceeding with the described experiments. MST14 (R&D Systems) cleaved antibodies were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 h at 37° C. After 7 h of incubation, cells were spun down at 500×g for 3 min and 20 μL of supernatant from each sample was assayed for IP-10 (Abcam) by ELISA according to the manufacturer's protocol.

[0501] The results show that the masked, non-glycosylated fusion protein does not induce IP-10 expression in PBMCs at the concentrations tested (i.e., 1.5 nM and 0.15 nM), while the masked and unmasked fusion proteins induce significant amounts of IP-10 at 1.5 nM (see FIG. 18).

[0502] Example 15: Methods for reducing induction of IP-10 in PBMCs. In this example, several masked fusion antibodies, both glycosylated and non-glycosylated (anti-CD138 IFNα mask 1, anti-CD138 IFNα mask 1 N297Q) were tested for reducing induction of IP-10.

[0503] Briefly, freshly thawed human PBMCs (Human Cells Biosciences) were washed once with cold RPMI + 10% FBS (Invitrogen) and plated into 12-well plates (Themofisher) at approximately 1 × 10 cells per well. 6Cells were seeded at a density of 1000 μg / well (1 mL / well). Any Fc receptor and / or CD138 antigen expression was blocked / reduced by adding 300 nM of unfused anti-CD138 IgG1 to the cells for 1 h before proceeding with the described experiments. MST14 (R&D Systems) cleaved antibodies were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 h at 37° C. After 7 h of incubation, cells were spun down at 500×g for 3 min and 20 μL of supernatant from each sample was assayed for IP-10 (Abcam) by ELISA according to the manufacturer's protocol.

[0504] The results show that the masked, non-glycosylated fusion protein does not induce IP-10 expression in PBMCs at the concentrations tested (i.e., 1.5 nM and 15 nM), whereas the masked, glycosylated anti-CD138 IFNα fusion protein induces IP-10 at 1.5 nM and 15 nM (see FIG. 19).

[0505] Example 16: Methods for reducing induction of IP-10 in PBMCs. In this example, several masked fusion antibodies (anti-CD138 IFNα Mask 1, anti-CD138 IFNα Mask 2, anti-CD138 IFNα Mask 2.2, and anti-CD138 IFNα Mask 3 (with and without MST)) were tested for reducing induction of IP-10.

[0506] Briefly, freshly thawed human PBMCs (HumanCells Biosciences) were washed once with cold RPMI + 10% FBS (Invitrogen) and plated into 12-well plates (Themofisher) at approximately 1 × 10 cells per well. 6Cells were seeded at a density of 1 mL / well. 167 nM Human Fc Block (BD Biosciences) + 300 nM unfused anti-CD138 IgG1 was added to the cells for 1 h before proceeding with the experiment to block / reduce any Fc receptor and / or CD138 antigen expression. Recombinant human IFNα (Novus Biologicals) or the indicated antibodies were added to the cells and incubated for an additional 7 h at 37°C. MST14 (R&D Systems) cleaved antibodies were prepared by incubating 50 μg of antibody with 0.5 μg MST14 for 1 h at 37°C. After 7 h incubation, cells were spun down at 500 × g for 3 min and 20 μL of supernatant from each sample was assayed for IP-10 (Abcam) by ELISA according to the manufacturer's protocol.

[0507] The results show that at low concentrations, all masked fusion proteins are able to reduce the induction of IP-10. Except for IFNα mask 3, MST14 treatment restores the ability of the masked fusion proteins to induce IP-10 (Figure 20).

[0508] Example 17: Methods for reducing induction of IP-10 in PBMCs. In this example, several masked fusion antibodies (anti-CD138 IFNα mask 1, anti-CD138 IFNα mask 1 N297Q and anti-CD138 IFNα mask 2.2) were tested for reducing induction of IP-10.

[0509] Briefly, freshly thawed human PBMCs (HumanCells Biosciences) were washed once with cold RPMI + 10% FBS (Invitrogen) and plated into 12-well plates (Themofisher) at approximately 1 × 10 cells per well. 6Cells were seeded at a density of 1 mL / well. 667 nM human IgG was added to the cells for 1 h before proceeding with the experiment to block / reduce any Fc receptors. Recombinant human IFNα (Novus Biologicals) or the indicated antibodies were added to the cells and incubated for a further 7 h at 37°C. MST14 (R&D Systems) cleaved antibodies were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 h at 37°C. After 7 h incubation, cells were spun down at 500 × g for 3 min and 20 μL of supernatant from each sample was assayed for IP-10 (Abcam) by ELISA according to the manufacturer's protocol.

[0510] The results show that approximately 10-fold more non-glycosylated mask 1 fusion protein is required to inhibit the same amount of IP-10 as the wild-type mask 1 fusion protein. Furthermore, approximately 100-fold more non-glycosylated mask 1 fusion protein is required to inhibit the same amount of IP-10 as the wild-type mask 2.2 fusion protein (Figure 21).

[0511] Example 18: Methods for reducing induction of IP-10 in PBMCs. In this example, several masked fusion antibodies (anti-CD138 IFNα mask 1, anti-CD138 IFNα mask 1 N297Q and anti-CD138 IFNα mask 2.2) were tested for reducing induction of IP-10.

[0512] Briefly, freshly thawed human PBMCs (HumanCells Biosciences) were washed once with cold RPMI + 10% FBS (Invitrogen) and plated into 12-well plates (Themofisher) at approximately 1 × 10 cells per well. 6Cells were seeded at a density of 1 mL / well. 1 μM human IgG was added to the cells for 1 h before proceeding with the experiment to block / reduce any Fc receptors. Recombinant human IFNα (Novus Biologicals) or the indicated antibodies were added to the cells and incubated for a further 7 h at 37°C. MST14 (R&D Systems) cleaved antibodies were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 h at 37°C. After 7 h incubation, cells were spun down at 500 × g for 3 min and 20 μL of supernatant from each sample was assayed for IP-10 (Abcam) by ELISA according to the manufacturer's protocol.

[0513] The results show that removal of the glycosylation site significantly reduces the induction of IP-10 by the fusion protein (FIG. 22).

[0514] Example 19: Methods of reducing induction of MCP-1 in PBMCs. In this example, several masked fusion antibodies (glycosylated and non-glycosylated) (anti-CD138 IFNα mask 1, anti-CD138 IFNα mask 1 N297Q, and anti-CD138 IFNα mask 2.2) were tested for reducing the induction of MCP-1. MCP-1 is a chemokine involved in regulating the migration and infiltration of monocytes, memory T lymphocytes, and NK cells. Expression of MCP-1 can be induced by cytokines, including IFNα.

[0515] Briefly, freshly thawed human PBMCs (HumanCells Biosciences) were washed once with cold RPMI + 10% FBS (Invitrogen) and plated into 12-well plates (Themofisher) at approximately 1 × 10 cells per well. 6Cells were seeded at a density of 1 mL / well. 667 nM human IgG was added to the cells for 1 h before proceeding with the experiment to block / reduce any Fc receptors. Recombinant human IFNα (Novus Biologicals) or the indicated antibodies were added to the cells and incubated for a further 7 h at 37°C. MST14 (R&D Systems) cleaved antibodies were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 h at 37°C. After 7 h incubation, cells were spun down at 500 × g for 3 min and 20 μL of supernatant from each sample was assayed for IP-10 (Abcam) by ELISA according to the manufacturer's protocol.

[0516] The results show that IFNα and IFNα fusion proteins (at a concentration of 15 nM) can induce MCP-1 expression, but the induction of MCP-1 by the masked fusion proteins is lower than that of recombinant IFNα or unmasked IFNα fusion proteins. Furthermore, the results show that the masked non-glycosylated fusion proteins negligibly induce MCP-1 compared to untreated samples (Figure 23).

[0517] Example 20: Methods for making masked fusion proteins. This example shows the construct design and characterization of QXL138AM2.2. Briefly, anti-CD138 IgG1 is generated using standard methods in the art. The heavy chain isotype is human gamma 1 and the light chain isotype is human kappa. The sequence of the QXL138AM2.2 heavy chain is described and disclosed as (SEQ ID NO:6). For the purposes of this disclosure, the heavy chain contains an amino acid substitution at position 297 (N297Q) to remove the glycosylation site by mutation. This prevents the fusion protein from binding to endogenous Fc receptors.

[0518] The mask was generated as described herein. See the method of masking IFN of this disclosure. The resulting construct, designated QXL138AM2.2 (anti-CD138-linker-IFNα2-cleavable linker-mask), contains an IgG heavy chain variable region (shown in red) depicted as (SEQ ID NO:7), a fusion protein linker SGGAGGS (SEQ ID NO:5), IFNα (shown in light blue) (SEQ ID NO:10), a second fusion protein linker [ka] (Cleavable linker shown in bold and underlined black. [ka] (SEQ ID NO: 16), and the mask of the present disclosure [ka] (See Figure 25 and SEQ ID NO:41). The nucleic acid sequence of the QXL138AM2.2. heavy chain is set forth in Figure 26 and is set forth as (SEQ ID NO:39).

[0519] The sequence of the QXL138AM2.2 light chain is set forth in Figure 24 and is disclosed as (SEQ ID NO: 8). The resulting construct contains the variable region (shown in red) and is disclosed as (SEQ ID NO: 9). The nucleic acid sequence of the QXL138AM2.2. light chain is set forth in Figure 24 and is disclosed as (SEQ ID NO: 40).

[0520] The constructs described in Figures 24 and 25 are transiently expressed in CHO cells using methods known in the art. Further analysis of the heavy and light chains is assessed by mass spectrometry using standard methods.

[0521] Example 21: Methods for reducing and restoring masked IFNα activity. In this example, further testing was performed to demonstrate that the masks of the present disclosure (IFNα Mask 2.2 N297Q and IFNα Mask 2.2 N297Q w / MST) can reduce and restore IFNα activity. Briefly, HEK Blue IFNα / β cells (Invivogen) were plated in 96-well tissue culture plates (Fisher) at 1×10 cells per well. 4 Cells were seeded at a density of 100 μL / well. 100 μL / well of recombinant IFNα (Novus Biologicals) or the indicated antibodies (FAP) were incubated with the cells at sufficient concentrations to produce the final concentrations indicated at 37° C. overnight. Antibodies cleaved with MST14 (R&D Systems) were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 hour at 37° C. 20 μL of supernatant was then added to the plate containing 180 μL / well of Quanti-Blue substrate (Invivogen). The change in absorbance was read at 630 nm using a Biotek EPOCH ELISA reader.

[0522] The results show that the EC50 of the masked anti-FAP fusion protein is at least 100-fold higher than the masked anti-CD138 fusion protein, compared to previous data. This is due to the expression of CD138 antigen in HEK-Blue IFNα / β cells, and the targeting of the anti-CD138 fusion protein affects the local IFNα concentration compared to the non-targeted anti-FAP fusion protein. Removing the mask from the anti-FAP fusion antibody reduced the EC50 by approximately 50-fold (see Figure 27).

[0523] Example 22: Methods for reducing induction of IP-10 in PBMCs. In this example, masked fusion antibodies (anti-CD138 IFNα mask 2.2 N297Q, anti-CD138 IFNα N297Q) were tested for reducing induction of IP-10.

[0524] Briefly, freshly thawed human PBMCs (HumanCells Biosciences) were washed once with cold RPMI + 10% FBS (Invitrogen) and plated into 12-well plates (Themofisher) at approximately 1 × 10 cells per well. 6 Cells were seeded at a density of 1000 μg / well (1 mL / well). 1 μM hIgG (Fisher) was added to the cells for 1 h before proceeding with the experiment to block / reduce any Fc receptor and / or CD138 antigen expression. Recombinant human IFNα (Novus Biologicals) or the indicated antibodies were then added to the cells and incubated for an additional 7 h at 37°C. MST14 (R&D Systems) cleaved antibodies were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 h at 37°C. After 7 h of incubation, cells were then spun down at 500×g for 3 min and 20 μL of supernatant from each sample was assayed for IP-10 (Abcam) by ELISA according to the manufacturer's protocol.

[0525] The results show that approximately 1000-fold more masked, unglycosylated 2.2 FP than recombinant hIFNα is required to induce the same amount of IP-10. Furthermore, approximately 10-fold more masked, unglycosylated 2.2 FP is required than unglycosylated unmasked FP to induce the same amount of IP-10 (Figure 28).

[0526] (Example 23: Masked non-glycosylated 2.2 fusion protein binds to IFNα2 receptor with reduced affinity compared to unmasked non-glycosylated 2.2 fusion protein.) In this example, various fusion antibodies (anti-huCD138 IFNα, anti-huCD138 IFNα mask 1.1, anti-huCD138 IFNα N297Q, and anti-huCD138 IFNα mask 2.2 N297Q) were tested to compare their binding affinity to IFNAR2 with that of unmasked fusion antibodies. Briefly, Immulon 2 HB plates (Thermofisher) were coated with 10 μg / mL of IFNαR2 (R&D Plates were coated with 100x100mm thick PBS (Sigma) overnight at 4°C and blocked with 2% BSA (Fisher) for a minimum of 2 hours at room temperature. Wells were washed 3 times with PBS+0.05% Tween® (Sigma). The indicated antibody concentrations were loaded overnight at 4°C. Wells were then washed 3 times with PBS+0.05% Tween®. Bound antibodies were detected with anti-human Kappa-AP (Southern Biotech) diluted 1:3000 in PBS+1% BSA. Changes in absorbance following addition of AP substrate (Sigma) were assayed at 410 nm using a Biotek EPOCH ELISA reader.

[0527] The results show that the Kd value of the unglycosylated masked 2.2 fusion protein is about 4-fold higher than that of the unmasked unglycosylated fusion protein. As shown above, the Kd value of the wild-type masked 2.2 fusion protein is about 30-fold higher than that of the unmasked wild-type fusion protein (see FIG. 29).

[0528] Example 24: Methods for reducing induction of IP-10 in PBMCs. In this example, masked fusion antibodies (anti-CD138 IFNα mask 2.2 N297Q, anti-CD138 IFNα N297Q) were tested for dose-dependent reduction in induction of IP-10.

[0529] Briefly, freshly thawed human PBMCs (HumanCells Biosciences) were washed once with cold RPMI + 10% FBS (Invitrogen) and plated into 12-well plates (Themofisher) at approximately 1 × 10 cells per well.6 Cells were seeded at a density of 1000 μg / well (1 mL / well). 1 μM hIgG (Fisher) was added to the cells for 1 h before proceeding with the experiment to block / reduce any Fc receptor and / or CD138 antigen expression. Recombinant human IFNα (Novus Biologicals) or the indicated antibodies were then added to the cells and incubated for an additional 7 h at 37°C. MST14 (R&D Systems) cleaved antibodies were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 h at 37°C. After 7 h of incubation, cells were then spun down at 500×g for 3 min and 20 μL of supernatant from each sample was assayed for IP-10 (Abcam) by ELISA according to the manufacturer's protocol.

[0530] The results show that approximately 10-fold more masked fusion protein is required to induce the same level of IP-10 in PBMCs compared to the unmasked fusion protein (FIG. 30).

[0531] Example 25: Binding of QXL138AM2.2-N297Q to soluble CD138. In this example, it was shown that QXL138AM2.2-N297Q binds to CD138 in a dose-dependent manner.

[0532] Briefly, Immulon 2 HB plates (Thermofisher) were coated with soluble CD138 (R&D Systems) at 10 μg / mL overnight at 4° C. and blocked with 2% BSA (Fisher) for a minimum of 2 hours at room temperature. Wells were washed three times with PBS+0.05% Tween® (Sigma). Indicated antibody concentrations were loaded overnight at 4° C. Wells were washed three times with PBS+0.05% Tween®. Bound antibodies were detected with anti-human Kappa-AP (Southern Biotech) diluted 1:3000 in PBS+1% BSA. Changes in absorbance following addition of AP substrate (Sigma) were assayed at 410 nm using a Biotek EPOCH ELISA reader.

[0533] The results show that QXL138AM2.2-N297Q specifically binds to soluble CD138 in a dose-dependent manner (Figure 31).

[0534] Example 26: Comparison of binding of multiple production lots (Lot 2 and Lot 3) of QXL138AM2.2-N297Q to soluble CD138. In this example, multiple lots of QXL138AM2.2-N297Q were shown to bind to CD138 in a consistent manner.

[0535] Briefly, Immulon 2 HB plates (Thermofisher) were coated with 10 μg / mL soluble CD138 (R&D Systems) overnight at 4° C. and blocked with 2% BSA (Fisher) for a minimum of 2 hours at room temperature. Wells were washed three times with PBS+0.05% Tween® (Sigma). Indicated antibody concentrations were loaded overnight at 4° C. Wells were washed three times with PBS+0.05% Tween®. Bound antibodies were detected with anti-human Kappa-AP (Southern Biotech) diluted 1:3000 in PBS+1% BSA. Changes in absorbance following addition of AP substrate (Sigma) were assayed at 410 nm using a Biotek EPOCH ELISA reader.

[0536] The results show that the Kd values ​​of multiple manufacturing lots of QXL138AM2.2-N297Q are similar (Figure 32).

[0537] Example 27: Methods for reducing and restoring masked IFNα activity. In this example, studies were performed to demonstrate that QXL138AM can reduce and restore IFNα activity. Briefly, HEK Blue IFNα / β cells (Invivogen) were plated in 96-well tissue culture plates (Fisher) at 1×10 cells per well. 4 Cells were seeded at a density of 100 μL / well. 100 μL / well of recombinant IFNα (Novus Biologicals) or the indicated antibodies were incubated with the cells at sufficient concentrations to produce the indicated final concentrations overnight at 37° C. Antibodies cleaved with MST14 (R&D Systems) were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 hour at 37° C. 20 μL of supernatant was then added to the plate containing 180 μL / well of Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader.

[0538] The results show that the EC50 of QXL138AM is approximately 4-fold higher than recombinant IFNα. Removal of the mask restores IFNα activity (see Figure 33).

[0539] Example 28: Methods for reducing and restoring masked IFNα activity of QXL138AM2.2-N297Q. In this example, studies were performed to demonstrate that QXL138AM2.2-N297Q can reduce and restore IFNα activity binding to IFNAR2. Briefly, HEK Blue IFNα / β cells (Invivogen) were plated in 96-well tissue culture plates (Fisher) at 1×10 cells per well. 4 Cells were seeded at a density of 100 μL / well. 100 μL / well of recombinant IFNα (Novus Biologicals) or the indicated antibodies were incubated with the cells at sufficient concentrations to produce the indicated final concentrations overnight at 37° C. Antibodies cleaved with MST14 (R&D Systems) were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 hour at 37° C. 20 μL of supernatant was then added to the plate containing 180 μL / well of Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader.

[0540] The results show that the EC50 of QXL138AM2.2-N297Q is approximately 4-fold higher than that of the unmasked fusion protein. Removal of the mask from QXL138AM2.2-N297Q restores binding to IFNAR2 to the level of the unmasked fusion protein (see FIG. 34).

[0541] Example 29: Methods for reducing and restoring induction of IP-10 in PBMCs. In this example, QXL138A, QXL138AM, and QXL138AM+MST were tested for reducing and restoring the induction of IP-10.

[0542] Briefly, freshly thawed human PBMCs (HumanCells Biosciences) were washed once with cold RPMI + 10% FBS (Invitrogen) and plated into 12-well plates (Themofisher) at approximately 1 × 10 cells per well. 6 Cells were seeded at a density of 1000 μg / well (1 mL / well). 1 μM human IgG (Fisher) was added to the cells for 1 h before proceeding with the experiment to block / reduce any Fc receptor and / or CD138 antigen expression. Recombinant human IFNα (Novus Biologicals) or the indicated antibodies were added to the cells and incubated for an additional 7 h at 37°C. MST14 (R&D Systems) cleaved antibodies were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 h at 37°C. After 7 h incubation, cells were spun down at 500 × g for 3 min and 20 μL of supernatant from each sample was assayed for IP-10 (Abcam) by ELISA according to the manufacturer's protocol.

[0543] The results show that the induction of IP-10 by the masked fusion protein QXL138AM is significantly reduced compared to the unmasked fusion protein. Moreover, removal of the mask using MST restores the induction of IP-10 (Figure 35).

[0544] Example 30: Methods for implementing tumor inhibition by QXL138AM in OVCAR3 cells in vivo. In this example, QXL138A and QXL138AM were tested for their ability to inhibit tumor growth in vivo in OVCAR3 cells.

[0545] Briefly, 5 × 10 OVCAR3 cells 6 Each piece was injected subcutaneously in a volume of 200 μl into female NSG mice approximately 6-8 weeks of age. Tumors were approximately 0.15-0.2 cm 2Treatment was initiated when tumor size reached 100%. Mice were then treated intravenously (iv) with either PBS, 5 mg / kg QXL138AM, or 5 mg / kg QXL138A on days 49, 52, 56, 59, 69, 72, 76, 79, 83, 86, 90, 93, and 97 (i.e., twice weekly for 7 weeks). Tumor sizes were recorded and compared using standard methods.

[0546] The results indicate that in vivo tumor inhibition of both QXL138A and QXL138AM is similar, and that QXL138AM is unmasked and active at the tumor site (FIG. 36).

[0547] Example 31: Methods for implementing tumor inhibition by QXL138AM in vivo in H929 cells. In this example, QXL138AM was tested for its ability to inhibit tumor growth in vivo in H929 cells.

[0548] Briefly, 1 × 10 H929 cells were 6 The individual pieces were injected subcutaneously with Matrigel in a volume of 200 μl into female NSG mice between 6 and 8 weeks of age. Tumors were 0.15–0.2 cm 2 Treatment was initiated when tumor size reached 100%. Mice were treated intravenously (iv) with either PBS, 0.1 mg / kg QXL138AM, 0.03 mg / kg QXL138AM, or 0.01 mg / kg QXL138AM on days 14, 18, 21, 25, 28, 32, 35, 39, 46, and 50 (i.e., twice weekly for 5 weeks). Tumor sizes were recorded and compared using standard methods.

[0549] The results indicate that the minimum predicted biological effect level is a dose of 0.01 mg / kg during treatment, with a complete response occurring at a dose of 0.1 mg / kg (Figure 37).

[0550] Example 32: Methods for demonstrating tumor inhibition by QXL138AM in vivo in Capan-2 cells. In this example, QXL138AM was tested for its ability to inhibit tumor growth in vivo in Capan-2 cells.

[0551] Briefly, 5 × 10 Capan-2 cells 6 The individual pieces were injected subcutaneously with Matrigel in a volume of 200 μl into female NSG mice between 6 and 8 weeks of age. Tumors were 0.15–0.2 cm 2 Treatment was initiated when tumor size reached 100%. Mice were treated intravenously (iv) with either PBS or 5.0 mg / kg QXL138AM on days 9, 13, 16, 20, 23, 27, 30, 34, 37, 41, 44, 48, 51, 55, 58, 62, 65, 69, 72, 76, 79, 83, 86, 90, and 93 (i.e., twice weekly for 13 weeks). Tumor sizes were recorded and compared using standard methods.

[0552] The results show that tumor growth is inhibited by the masked therapeutic agent of QXL138AM during the treatment period, and that tumors continue to grow when treatment is stopped (Figure 38).

[0553] Example 33: Characterization of masked targeted YNS mutants fused to anti-CD138. In this example, it is shown that multiple masked YNS variants can be cleaved from the heavy chain using matriptase ST 14 ("MST 14"). 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. Additionally, 1 μg of each purified antibody was denatured by heating to 95°C, reduced with approximately 2% beta-mercaptoethanol (Thermofisher), and run on a 4-12% Bis-Tris SDS-PAGE gel (Invitrogen). The gel was stained with EZ Stain (Fisher) according to the manufacturer's protocol.

[0554] The analytical results obtained indicate that the mask of the masked YNS mutant can be cleaved by MST, and MST treatment also leads to partial cleavage of the IFN / mask moiety, as observed by an increase in the band of the size of the unfused antibody (see Figure 42).

[0555] Example 34: Methods for conjugating a fusion protein to a masking peptide. In this example, multiple fusion antibodies are shown to specifically bind to additional peptide masks of the present disclosure. For reference, the peptide sequences of PEP1, PEP21, PEP22, PEP23, and PEP24 are set forth in FIG.

[0556] Briefly, Immulon 2 HB plates (Thermofisher) were plated with 10 μg / mL of anti-CD138 IFNα YNSFusion proteins were coated overnight at 4° C. and blocked with 2% BSA (Fisher) for a minimum of 2 hours at room temperature. Wells were washed three times with PBS+0.05% Tween® (Sigma). The indicated peptide (Thermofisher) concentrations were loaded overnight at 4° C. Wells were washed three times with PBS+0.05% Tween®. Bound peptides were detected with streptavidin-HRP (Pierce) diluted 1:5000 in PBS+1% BSA. After washing wells three times with PBS+0.05% Tween®, HRP substrate TMB (Fisher) was added to the wells. The reaction was terminated by adding 0.1 M sulfuric acid (Fisher) and the change in absorbance was assayed at 450 nm using a Biotek EPOCH ELISA reader.

[0557] The results show that peptide 22 and peptide 23 bind most strongly to the immobilized fusion protein of all IFN fusion antibodies tested (see Figure 43).

[0558] Example 35: Methods for conjugating a fusion protein to a masking peptide. In this example, multiple fusion antibodies are shown to specifically bind to additional peptide masks of the present disclosure. For reference, the peptide sequences of PEP22 and PEP23 are set forth in FIG.

[0559] Briefly, Immulon 2 HB plates (Thermofisher) were plated with 10 μg / mL of anti-CD138 IFNα YNSFusion proteins were coated overnight at 4° C. and blocked with 2% BSA (Fisher) for a minimum of 2 hours at room temperature. Wells were washed three times with PBS+0.05% Tween® (Sigma). Indicated fusion protein concentrations were mixed with 10 μM peptide (Thermofisher) and loaded overnight at 4° C. Wells were washed three times with PBS+0.05% Tween®. Bound peptide was detected with streptavidin-HRP (Pierce) diluted 1:5000 in PBS+1% BSA. After washing wells three times with PBS+0.05% Tween®, HRP substrate TMB (Fisher) was added to the wells. Reactions were terminated by the addition of 0.1 M sulfuric acid (Fisher) and changes in absorbance were assayed at 450 nm using a Biotek EPOCH ELISA reader.

[0560] The results show that free fusion protein is able to compete off the binding of PEP22 and PEP23 to the immobilized fusion protein in a dose-dependent manner (see FIG. 44).

[0561] Example 36: Methods for binding fusion proteins to immobilized peptides. In this example, multiple fusion proteins are shown to specifically bind to additional peptide masks of the present disclosure. For reference, the peptide sequences of PEP21, PEP22, and PEP23 are set forth in FIG.

[0562] Briefly, streptavidin-coated plates (Pierce) were loaded with 50 μM of each indicated peptide (Thermofisher) for a minimum of 2 hours at room temperature. Wells were washed three times with PBS+0.05% Tween®. Antibodies at the indicated concentrations were then allowed to bind overnight at 4° C. Wells were washed three times with PBS+0.05% Tween®. Bound antibodies were detected using anti-human Kappa-AP (Southern Biotech) diluted 1:3000 in PBS+1% BSA. Changes in absorbance following addition of AP substrate (Sigma) were assayed at 410 nm using a Biotek EPOCH ELISA reader.

[0563] The results show that the fusion protein is able to bind to immobilized PEP23, but not to PEP21 or PEP22 (see FIG. 45).

[0564] Example 37: Methods for reducing and restoring masked IFNα activity. In this example, further tests were performed to show that the fusion proteins of the present disclosure (QXL138YNS, QXL138YNSM1.2-N297Q, and QXL138YNSM1.2-N297Q w / MST) can reduce and restore IFNα activity.

[0565] Briefly, HEK Blue IFNα / β cells (Invivogen) were plated in 96-well tissue culture plates (Fisher) at 1 × 10 cells per well. 4Cells were seeded at a density of 100 μL / well. 100 μL / well of recombinant IFNα (Novus Biologicals) or the indicated antibodies were incubated with the cells at sufficient concentrations to produce the indicated final concentrations overnight at 37° C. Antibodies cleaved with MST14 (R&D Systems) were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 hour at 37° C. 20 μL of supernatant was then added to the plate containing 180 μL / well of Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader.

[0566] The results show that the YNS mutant is active, but a mask of the YNS mutant (based on PEP23) appears to be active (see FIG. 46).

[0567] Example 38: Methods for reducing and restoring masked IFNα activity. In this example, further tests were performed to show that the fusion proteins of the present disclosure (QXL138YNS and QXL138YNSM2.2-N297Q) can reduce and restore IFNα activity.

[0568] Briefly, HEK Blue IFNα / β cells (Invivogen) were plated in 96-well tissue culture plates (Fisher) at 1 × 10 cells per well. 4Cells were seeded at a density of 100 μL / well. 100 μL / well of recombinant IFNα (Novus Biologicals) or the indicated antibodies were incubated with the cells at sufficient concentrations to produce the indicated final concentrations overnight at 37° C. Antibodies cleaved with MST14 (R&D Systems) were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 hour at 37° C. 20 μL of supernatant was then added to the plate containing 180 μL / well of Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader.

[0569] The results show that QXL138YNSM2.2-N297Q (based on the combination of PEP2 and PEP23) appears to exhibit a masking effect, with an EC50 approximately 3-fold higher than that of the unmasked fusion protein (see Figure 47).

[0570] Example 39: Methods for reducing and restoring masked IFNα activity. In this example, further tests were performed to show that the fusion proteins of the present disclosure (QXL138YNS, QXL138YNSM2.2-N297Q, and QXL138YNSM2.2-N297Q w / MST) can reduce and restore IFNα activity.

[0571] Briefly, HEK Blue IFNα / β cells (Invivogen) were plated in 96-well tissue culture plates (Fisher) at 1 × 10 cells per well. 4Cells were seeded at a density of 100 μL / well. 100 μL / well of recombinant IFNα (Novus Biologicals) or the indicated antibodies were incubated with the cells at sufficient concentrations to produce the indicated final concentrations overnight at 37° C. Antibodies cleaved with MST14 (R&D Systems) were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 hour at 37° C. 20 μL of supernatant was then added to the plate containing 180 μL / well of Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader.

[0572] The results show that MST treatment of QXL138YNSM2.2-N297Q does not appear to restore IFN activity, even though SDS-PAGE shows that the majority of the mask is cleaved (see FIG. 48).

[0573] Example 40: Methods for reducing and restoring masked IFNα activity. In this example, further tests were performed to show that the fusion proteins of the present disclosure (QXL138YNS and QXL138YNSM2.2-N297Q) can reduce and restore IFNα activity.

[0574] Briefly, Immulon 2 HB plates (Thermofisher) were coated with 10 μg / mL IFNαR2 (R&D Systems) overnight at 4° C. and blocked with 2% BSA (Fisher) for a minimum of 2 hours at room temperature. Wells were washed three times with PBS+0.05% Tween® (Sigma). Indicated antibody concentrations were loaded overnight at 4° C. Wells were washed three times with PBS+0.05% Tween®. Bound antibodies were detected with anti-human Kappa-AP (Southern Biotech) diluted 1:3000 in PBS+1% BSA. Changes in absorbance following addition of AP substrate (Sigma) were assayed at 410 nm using a Biotek EPOCH ELISA reader.

[0575] The results show that QXL138YNSM2.2-N297Q binds to IFNAR2 less strongly than QXL138YNS, with an EC50 that appears to be approximately 15-fold higher than the unmasked fusion protein (see FIG. 49).

[0576] Example 41: Methods for reducing and restoring masked IFNα activity. In this example, further tests were performed to show that the fusion proteins of the present disclosure (QXL138YNS, QXL138YNSM2.2-N297Q, QXL138YNSM2.2-N297Q w / MST and QXL138) can reduce and restore IFNα activity.

[0577] Briefly, Immulon 2 HB plates (Thermofisher) were coated with 10 μg / mL IFNαR2 (R&D Systems) overnight at 4° C. and blocked with 2% BSA (Fisher) for a minimum of 2 hours at room temperature. Wells were washed three times with PBS+0.05% Tween® (Sigma). Indicated antibody concentrations were loaded overnight at 4° C. Wells were washed three times with PBS+0.05% Tween®. Bound antibodies were detected with anti-human Kappa-AP (Southern Biotech) diluted 1:3000 in PBS+1% BSA. Changes in absorbance following addition of AP substrate (Sigma) were assayed at 410 nm using a Biotek EPOCH ELISA reader.

[0578] The results indicate that removal of the mask from QXL138YNSM2.2-N297Q w / MST appears to restore essentially all of its binding ability to IFNAR2 (see Figure 50).

[0579] Example 42: Methods for reducing and restoring induction of IP-10 in PBMCs. In this example, QXL138YNS and QXL138YNSM2.2-N297Q were tested for reducing and restoring the induction of IP-10.

[0580] Briefly, freshly thawed human PBMCs (HumanCells Biosciences) were washed once with cold RPMI + 10% FBS (Invitrogen) and plated into 12-well plates (Themofisher) at approximately 1 × 10 cells per well. 6Cells were seeded at a density of 1000 μg / well (1 mL / well). 1 μM human IgG (Fisher) was added to the cells for 1 h before proceeding with the experiment to block / reduce any Fc receptor and / or CD138 antigen expression. Recombinant human IFNα (Novus Biologicals) or the indicated antibodies were added to the cells and incubated for an additional 7 h at 37°C. MST14 (R&D Systems) cleaved antibodies were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 h at 37°C. After 7 h incubation, cells were spun down at 500 × g for 3 min and 20 μL of supernatant from each sample was assayed for IP-10 (Abcam) by ELISA according to the manufacturer's protocol.

[0581] By comparing the level of IP-10 induction produced by 0.01 nM QXL138YNS with 1 nM QXL138YNSM2.2-N297Q, the results show that 100-fold more masked fusion protein was required to achieve the same level of induction as the unmasked fusion protein (Figure 51).

[0582] Example 43: Methods for reducing and restoring masked IFNα activity. In this example, further tests were performed to show that the fusion proteins of the present disclosure (QXL138YNS-N297Q, QXL138YNSM1.2-N297Q, QXL138YNSM1.2-N297Q w / MST, QXL138YNSM2.2-N297Q and QXL138YNSM2.2-N297Q w / MST) can reduce and restore IFNα activity.

[0583] Briefly, HEK Blue IFNα / β cells (Invivogen) were plated in 96-well tissue culture plates (Fisher) at 1 × 10 cells per well. 4Cells were seeded at a density of 100 μL / well. 100 μL / well of recombinant IFNα (Novus Biologicals) or the indicated antibodies were incubated with the cells at sufficient concentrations to produce the indicated final concentrations overnight at 37° C. Antibodies cleaved with MST14 (R&D Systems) were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 hour at 37° C. 20 μL of supernatant was then added to the plate containing 180 μL / well of Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader.

[0584] The results show that QXL138YNSM1.2-N297Q (based on PEP23) does not appear to have significant masking activity compared to the unmasked fusion protein. QXL138YNS2.2-N297Q (based on PEP23 and PEP2) shows only moderate masking activity compared to the unmasked fusion protein (see Figure 52).

[0585] Example 44: Characterization of QXL138AM4.2-N297Q. In this example, it is shown that QXL138AM4.2-N297Q can be cleaved from the heavy chain using matriptase ST 14 ("MST 14"). 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% beta-mercaptoethanol (Thermofisher), and run on a 4-12% Bis-Tris SDS-PAGE gel (Invitrogen). The gel was stained with EZ Stain (Fisher) according to the manufacturer's protocol.

[0586] The analytical results obtained show that mask 4 of QXL138AM4.2-N297Q can be cleaved by MST (see FIG. 53).

[0587] Example 45: Methods for reducing and restoring masked IFNα activity. In this example, further testing was performed to show that fusion proteins of the present disclosure from multiple manufacturing lots (QXL138AM2.2-N297Q(Lot 10), QXL138AM2.2-N297Q(Lot 1), QXL138AM2.2-N297Q(Lot 10)+MST, and QXL138AM2.2-N297Q(Lot 1)+MST) can reduce and restore IFNα activity.

[0588] Briefly, HEK Blue IFNα / β cells (Invivogen) were plated in 96-well tissue culture plates (Fisher) at 1 × 10 cells per well. 4 Cells were seeded at a density of 100 μL / well. 100 μL / well of recombinant IFNα (Novus Biologicals) or the indicated antibodies were incubated with the cells at sufficient concentrations to produce the indicated final concentrations overnight at 37° C. Antibodies cleaved with MST14 (R&D Systems) were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 hour at 37° C. 20 μL of supernatant was then added to the plate containing 180 μL / well of Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader.

[0589] The results show that the EC50 of QXL138AM4.2-N297Q (based on the D1 loop of IFNAR2) appears to be about 50-100 times higher than when the mask is removed, indicating a significant masking effect compared to QXL138AM2.2-N297Q. Furthermore, the EC50 of QXL138AM4.2-N297Q is about 20 times higher than the EC50 of QXL138AM2.2, indicating an improvement over mask2.2 (see Figure 54).

[0590] Example 46: Methods for reducing and restoring masked IFNα activity. In this example, further tests were performed to show that the fusion proteins of the present disclosure (QXL138YNS-N297Q, QXL138AM4.2-N297Q, QXL138YNSM4.2-N297Q and QXL138YNSM4.2-N297Q+MST) can reduce and restore IFNα activity.

[0591] Briefly, HEK Blue IFNα / β cells (Invivogen) were plated in 96-well tissue culture plates (Fisher) at 1 × 10 cells per well. 4 Cells were seeded at a density of 100 μL / well. 100 μL / well of recombinant IFNα (Novus Biologicals) or the indicated antibodies were incubated with the cells at sufficient concentrations to produce the indicated final concentrations overnight at 37° C. Antibodies cleaved with MST14 (R&D Systems) were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 hour at 37° C. 20 μL of supernatant was then added to the plate containing 180 μL / well of Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader.

[0592] The results show that mask 4.2 (based on the D1 loop of IFNAR2) can be an effective mask for the YNS mutant of IFNα. The EC50 of QXL138YNS4.2-N297Q appears to be about 30-fold higher than when the mask is removed, showing a significant masking effect compared to the unmasked QXL138YNS-N297Q. Removal of the mask restores IFNα activity to the level of the unmasked fusion protein. The EC50 of QXL138AM4.2-N297Q is about 4-fold higher than that of QXL138YNSM4.2-N297Q, indicating that mask 4.2 reduces IFNα activity more efficiently when using wild-type IFNα as opposed to the YNS mutant (see Figure 55).

[0593] Example 47: Methods for reducing and restoring masked IFNα activity. In this example, further testing was performed to show that the fusion proteins of the present disclosure (QXL138A-N297Q, QXL138AM2.2N297Q(Lot 10), QXL138AM4.2-N297Q, QXL138AM2.2N297Q(Lot 10)+MST and QXL138AM4.2-N297Q+MST) can reduce and restore IFNα activity.

[0594] Briefly, Immulon 2 HB plates (Thermofisher) were coated with 10 μg / mL IFNαR2 (R&D Systems) overnight at 4° C. and blocked with 2% BSA (Fisher) for a minimum of 2 hours at room temperature. Wells were washed three times with PBS+0.05% Tween® (Sigma). Indicated antibody concentrations were loaded overnight at 4° C. Wells were washed three times with PBS+0.05% Tween®. Bound antibodies were detected with anti-human Kappa-AP (Southern Biotech) diluted 1:3000 in PBS+1% BSA. Changes in absorbance following addition of AP substrate (Sigma) were assayed at 410 nm using a Biotek EPOCH ELISA reader.

[0595] The results show that the EC50 of QXL138AM4.2-N297Q for binding to IFNAR2 is approximately 50-fold higher than the EC50 of the unmasked fusion protein QXL138A-N297Q and approximately 10-fold higher than the EC50 of QXL138AM2.2, showing an improvement over masked 2.2 (see Figure 56).

[0596] Example 40: Methods for reducing and restoring masked IFNα activity. In this example, further tests were performed to show that the fusion proteins of the present disclosure (QXL138YNS-N297Q, QXL138YNS4.2-N297Q, QXL138YNS4.2-N297Q+MST and QXL138AM4.2-N297Q) can reduce and restore IFNα activity.

[0597] Briefly, Immulon 2 HB plates (Thermofisher) were coated with 10 μg / mL IFNαR2 (R&D Systems) overnight at 4° C. and blocked with 2% BSA (Fisher) for a minimum of 2 hours at room temperature. Wells were washed three times with PBS+0.05% Tween® (Sigma). Indicated antibody concentrations were loaded overnight at 4° C. Wells were washed three times with PBS+0.05% Tween®. Bound antibodies were detected with anti-human Kappa-AP (Southern Biotech) diluted 1:3000 in PBS+1% BSA. Changes in absorbance following addition of AP substrate (Sigma) were assayed at 410 nm using a Biotek EPOCH ELISA reader.

[0598] The results show that the IFNAR2 D1 mask increases the EC50 of the wild-type IFNα fusion protein and the YNS mutant by about 40-fold and about 20-fold, respectively, compared to the unmasked fusion protein. Removal of the mask restores the activity of the YNS mutant to the level of the unmasked fusion protein (see Figure 57).

[0599] Example 49: Methods for reducing and restoring induction of IP-10 in PBMCs. In this example, QXL138A-N297Q, QXL138AM2.2-N297Q, QXL138AM2.2-N297Q+MST, QXL138AM4.2-N297Q, QXL138AM4.2-N297Q+MST were tested for reducing and restoring induction of IP-10.

[0600] Briefly, freshly thawed human PBMCs (HumanCells Biosciences) were washed once with cold RPMI + 10% FBS (Invitrogen) and plated into 12-well plates (Themofisher) at approximately 1 × 10 cells per well. 6 Cells were seeded at a density of 1000 μg / well (1 mL / well). 1 μM human IgG (Fisher) was added to the cells for 1 h before proceeding with the experiment to block / reduce any Fc receptor and / or CD138 antigen expression. Recombinant human IFNα (Novus Biologicals) or the indicated antibodies were added to the cells and incubated for an additional 7 h at 37°C. MST14 (R&D Systems) cleaved antibodies were prepared by incubating 50 μg of antibody with 0.5 μg of MST14 for 1 h at 37°C. After 7 h incubation, cells were spun down at 500 × g for 3 min and 20 μL of supernatant from each sample was assayed for IP-10 (Abcam) by ELISA according to the manufacturer's protocol.

[0601] The results showed that the EC50 for induction of IP-10 in PBMCs of QXL138AM4.2-N297Q was approximately 30-fold higher than that of unmasked QXL138A-N297Q and approximately 3-fold higher than that of QXL138AM2.2-N297Q, showing an improvement over masked 2.2 (Figure 58).

[0602] Example 50: Methods for making masked fusion proteins. In this example, construct design and characterization of QXL138AM4.2-N297Q (or alternatively QXL138AM4.2) are shown. Briefly, anti-CD138 IgG1 is generated using standard methods in the art. The heavy chain isotype is human gamma 1 and the light chain isotype is human kappa. The sequence of QXL138AM4.2-N297Q heavy chain is described and disclosed as (SEQ ID NO:6). For the purposes of this disclosure, the heavy chain further comprises an amino acid substitution at position 297 (N297Q) to remove the glycosylation site by mutation. This prevents the fusion protein from binding to endogenous Fc receptors.

[0603] The mask was generated as described herein. See the method of masking IFN of the present disclosure. The resulting construct, designated QXL138AM4.2-N297Q (anti-CD138-linker-IFNα2-cleavable linker-mask), contains a signal peptide (shown in purple) (SEQ ID NO:53), an IgG anti-CD138 heavy chain variable region (shown in orange) designated as (SEQ ID NO:7), a human IgG1 heavy chain constant region (shown in black) designated as (SEQ ID NO:54), an N297Q mutation (shown in bold black) (i.e., an asparagine to glutamine mutation at position 297), a fusion protein linker SGGAGGS (SEQ ID NO:5) (shown in black and underlined), an IFNα (shown in light blue) (SEQ ID NO:10), a second fusion protein linker SGGAGGS (SEQ ID NO:5), a ... [ka] (Cleavable linker shown in bold green and underlined) [ka] (SEQ ID NO: 16), and a mask of the present disclosure (D1 loop of IFNAR2) [ka] (See Figure 59 and SEQ ID NO: 49). The nucleic acid sequence of the QXL138AM4.2-N297Q. heavy chain is set forth in Figure 60 and is set forth as (SEQ ID NO: 50).

[0604] The sequence of the QXL138AM4.2-N297Q light chain is set forth in FIG. 24 and is disclosed as (SEQ ID NO: 8). The resulting construct contains a variable region (shown in red) disclosed as (SEQ ID NO: 9). The nucleic acid sequence of the QXL138AM4.2-N297Q light chain is set forth in FIG. 24 and is disclosed as (SEQ ID NO: 40).

[0605] The constructs described in Figures 24 and 59 are transiently expressed in CHO cells using methods known in the art. Further analysis of the heavy and light chains is assessed by mass spectrometry using standard methods.

[0606] Example 51: Methods for making masked fusion proteins. In this example, construct design and characterization of QXL138YNS4.2-N297Q are shown. Briefly, anti-CD138 IgG1 is generated using standard methods in the art. The heavy chain isotype is human gamma 1, and the light chain isotype is human kappa. The sequence of QXL138AM4.2-N297Q heavy chain is described and disclosed as (SEQ ID NO:6). For the purposes of this disclosure, the heavy chain further comprises an amino acid substitution at position 297 (N297Q) to remove the glycosylation site by mutation. This prevents the fusion protein from binding to endogenous Fc receptors.

[0607] The mask was generated as described herein. See the method of masking IFN of this disclosure. The resulting construct, designated QXL138YNS4.2-N297Q (anti-CD138-linker-IFNα2-cleavable linker-mask), contains a signal peptide (shown in purple) (SEQ ID NO:53), an IgG anti-CD138 heavy chain variable region (shown in orange) designated as (SEQ ID NO:7), a human IgG1 heavy chain constant region (shown in black) designated as (SEQ ID NO:55), an N297Q mutation (shown in bold black) (i.e., an asparagine to glutamine mutation at position 297), a fusion protein linker SGGAGGS (SEQ ID NO:5) (shown in black and underlined), an IFNα with a YNS mutation (shown in light blue) designated as bold blue (SEQ ID NO:56), a second fusion protein linker [ka] (Cleavable linker shown in bold green and underlined) [ka] (SEQ ID NO: 16), and a mask of the present disclosure (D1 loop of IFNAR2) [ka] (See Figure 61 and SEQ ID NO:51). The nucleic acid sequence of the QXL138YNS4.2-N297Q. heavy chain is set forth in Figure 62 and is set forth as (SEQ ID NO:52).

[0608] The sequence of the QXL138YNS4.2-N297Q light chain is set forth in FIG. 24 and is disclosed as (SEQ ID NO: 8). The resulting construct contains a variable region (shown in red) disclosed as (SEQ ID NO: 9). The nucleic acid sequence of the QXL138YNS4.2-N297Q light chain is set forth in FIG. 24 and is disclosed as (SEQ ID NO: 40).

[0609] The constructs described in Figures 24 and 61 are transiently expressed in CHO cells using methods known in the art. Further analysis of the heavy and light chains is assessed by mass spectrometry using standard methods.

[0610] Example 52: Human clinical trials to treat human carcinomas by using masked IFN fusion proteins that bind to specific TAAs. Masked IFN fusion proteins are synthesized according to the present invention that specifically accumulate in tumor cells and bind to specific TAAs used to treat certain tumors as well as other immune disorders and / or other diseases. Two clinical approaches have been successfully pursued in relation to each of these indications.

[0611] 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 drug or pharmaceutical drug or biologic drug or combination thereof. Protocol design is responsive to the effects assessed by the following examples, including but not limited to, reduction in tumor mass of primary or metastatic lesions, progression-free survival, prolonged overall survival, improved patient health, disease stabilization, and the ability to reduce the usual doses of standard chemotherapy and other biologics. These dose reductions allow additional and / or sustained treatment by reducing the toxicity associated with the administration of chemotherapeutic drugs or biologics.

[0612] II.) Monotherapy: In relation to the use of masked IFN fusion proteins that bind to specific TAAs in monotherapy against tumors, the masked IFN fusion proteins that bind to specific TAAs are administered to patients without chemotherapy or pharmaceuticals or biological agents. In one embodiment, monotherapy is clinically performed on terminal cancer patients with widespread metastatic disease. The protocol design corresponds to the effects assessed by the following examples, including but not limited to the reduction of tumor mass in primary or metastatic lesions, progression-free survival, prolonged overall survival, improved patient health, disease stabilization, and the ability to reduce the usual dose of standard chemotherapy and other biological agents.

[0613] Dosage Dosage regimen can be adjusted to provide the optimal desired response. For example, a single injection of the masked IFN fusion protein that binds to a specific TAA can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. "Unit dosage form" as used herein refers to a physically discrete unit suitable as a unit dosage for a mammalian subject to be treated, each unit containing a predetermined quantity of active compound calculated to produce a desired therapeutic effect, together with a necessary pharmaceutical carrier. The specifications of the unit dosage form of the present invention are determined 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 that is realized, and (b) the inherent limitations in the technical field of compounding such treatment compounds, such as hypersensitivity in individuals.

[0614] Clinical Development Plan (CDP) CDP advances and develops the treatment of cancer and / or immune disorders using the masked IFN fusion protein that binds to a specific TAA of the present disclosure in association with adjunctive or monotherapy. The trial first demonstrates safety, and then confirms efficacy with repeated dosing. The trial is an open-label comparison of standard chemotherapy and standard treatment plus masked IFN fusion protein that binds to a specific TAA. As can be understood, one non-limiting criterion that can be used in association with patient inclusion is the concentration of masked IFN fusion protein that binds to a specific TAA in tumors, as determined by standard detection methods known in the art.

[0615] The present invention is not limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual inventive aspects, and any functional equivalents are within the scope of the present invention. From the foregoing description and teachings, various modifications to the models, methods, and lifecycle methodologies of the present invention in addition to those described herein will be apparent to those skilled in the art and are also intended to be within the scope of the present invention. Such modifications or other embodiments can be made without departing from the true scope and spirit of the invention. [Table 1] [Table 2] [Table 3]

Claims

1. a. An antibody that specifically binds to the CD138 antigen, the antibody comprising a heavy chain variable region comprising SEQ ID NO: 7, a heavy chain constant region comprising SEQ ID NO: 54, and a variable light chain comprising SEQ ID NO: 9; b. A type I interferon, wherein the N-terminus of the type I interferon is fused to the C-terminus of the heavy chain and / or light chain of the antibody by a flexible peptide linker as set forth in SEQ ID NO: 37; and c. An interferon mask, the interferon mask comprising SEQ ID NO: 48, whereby the interferon mask is bound to the C-terminus of the type I interferon comprising a "masked targeted IFN".

2. The "masked targeted IFN" according to claim 1, wherein the interferon mask is bound to the C-terminus of the type I interferon further comprising a flexible peptide linker.

3. The masked targeted interferon according to claim 1, wherein the type I interferon or a functionally active variant thereof is as set forth in Table II.

4. The masked targeted IFN according to claim 1, wherein the functionally active variant is an IFNα2YNS variant.

5. The masked targeted interferon according to claim 1, wherein the antibody binds to CD138.

6. A method for producing the masked targeted interferon according to any one of claims 1.

7. A pharmaceutical composition comprising a therapeutically effective amount of the masked targeted IFN according to any one of claims 1, (i) optionally, the pharmaceutical composition is for use in a treatment including the treatment of cancer, 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.

8. A composition comprising the sequence set forth in SEQ ID NO:

49.

9. A kit comprising the composition according to claim 8.

10. a. An antibody that specifically binds to the CD138 antigen, the antibody comprising a heavy chain variable region comprising SEQ ID NO: 7, a heavy chain comprising SEQ ID NO: 6 and comprising an amino acid substitution at position 297 (N297Q), and a variable light chain comprising SEQ ID NO: 9; b. A type I interferon, wherein the N-terminus of the type I interferon is fused to the C-terminus of the heavy chain and / or light chain of the antibody by a flexible peptide linker set forth in SEQ ID NO: 38; and c. An interferon mask, comprising (SEQ ID NO: 34), whereby the interferon mask is bound to the C-terminus of the type I interferon, the interferon mask comprising a "masked targeted IFN".

11. The "masked targeted IFN" according to claim 10, wherein the interferon mask is bound to the C-terminus of the type I interferon further comprising a flexible peptide linker.

12. The masked targeted interferon according to claim 10, wherein the type I interferon or a functionally active variant thereof is set forth in Table II.

13. The masked targeted IFN according to claim 10, wherein the functionally active variant is an IFNα2YNS variant.

14. The masked targeted interferon according to claim 10, wherein the antibody binds to CD138.

15. A method for producing the masked targeted interferon according to claim 10.

16. A pharmaceutical composition comprising a therapeutically effective amount of the masked targeted IFN according to claim 10, wherein (i) optionally, the pharmaceutical composition is for use in a treatment 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-neoplastic agents, the pharmaceutical composition.

17. A composition comprising the sequence set forth in SEQ ID NO:

41.

18. A kit comprising the composition according to claim 17.

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