Fusion protein composition(s) comprising masked type i interferons (IFNα and IFNβ) for use in the treatment of cancer and methods thereof

Abstract

Fusion Protein compositions comprising masked IFNs and methods of making masked IFNs are disclosed herein. Consequently, the masked IFNs can be fused to a Mab or binding fragment thereof and be administered to patients as a therapeutic modality and provide a method of treating cancer, immunological disorders, and other diseases.

Description

[0001] Fusion Protein Composition(s) Comprising Masked Type i Interferons (IFNct and IFN|3) For Use in The Treatment of Cancer and Methods Thereof

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to United States Provisional Patent Application number 63 / 725,821 filed 27-November-2024, the contents of which are fully incorporated by reference herein,

[0004] SUBMISSION OF SEQUENCE LISTING ON PAPER COPY AND XML FILE (“SEQUENCE LISTING XML”)

[0005] The content(s) of the following submissions are fully incorporated by reference herein in their entirety: a paper copy of the Sequence Listing recorded November 26, 2025. Additionally, the content of a computer readable form (CRF) of the Sequence Listing entitled SEQ LIST XML (file name: 1441-20006.00 - SEQ LIST XML - 26-Nov-2025.xml), date recorded November 26, 2025.

[0006] STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH

[0007] Not applicable,

[0008] FIELD OF THE INVENTION

[0009] The invention described herein relates to the field of cancer therapy and therapy of other immunological disorders or diseases. Specifically, the invention relates to masked Type I interferon (IFN) compositions which can be fused to a 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 cancers and other immunological disorders and diseases.

[0010] BACKGROUND OF THE INVENTION

[0011] Cancer is the second leading cause of death next to coronary disease worldwide. Millions of people die from cancer every year and in the United States alone cancer killswell over a half-million people annually, with 1,688,780 new cancer cases diagnosed in 2017 (American Cancer Society). While deaths from heart disease have been declining significantly, those resulting from cancer generally are on the rise. In the early part of this century, cancer is predicted to become the leading cause of death unless medical developments change the current trend.

[0012] Several cancers stand out as having high rates of mortality. Hematological malignancies, including myeloma, cause 50,000 deaths annually in the United States alone (American Cancer Society, 2018). In addition, carcinomas of the lung (18.4% of all cancer deaths), breast (6.6% of all cancer deaths), colorectal (9.2% of all cancer deaths), liver (8.2% of all cancer deaths), and stomach (8.2% of all cancer deaths) represent major causes of cancer death for both sexes in all ages worldwide (GLOBOCAN 2018). These and virtually all other carcinomas share a common lethal feature in that they metastasize to sites distant from the primary tumor and with very few exceptions, metastatic disease is fatal Moreover, even for those cancer patients who initially survive their primary cancers, common experience has shown that their lives are dramatically altered. Many cancer patients experience strong anxieties driven by the awareness of the potential for recurrence or treatment failure. Many cancer patients also experience physical debilitations following treatment. Furthermore, many cancer patients experience a recurrence of their disease.

[0013] Although cancer therapy has improved over the past decades and survival rates have increased, the heterogeneity of cancer still demands new therapeutic strategies utilizing a plurality of treatment modalities. This is especially true in treating solid tumors at anatomical crucial sites (e.g.. glioblastoma, squamous carcinoma of the head and neck and lung adenocarcinoma) which are sometimes limited to standard radiotherapy and / or chemotherapy. Nonetheless, detrimental effects of these therapies are chemo- and radio resistance, which promote loco-regional recurrences, distant metastases and second primary tumors, in addition to severe side-effects that reduce the patients’ quality of life.

[0014] Furthermore, the therapeutic utility of monoclonal antibodies (mAbs) (G. Kohler and C. Milstein, Nature 256:495-497 (1975)) is being realized Monoclonal antibodies have now been approved as therapies in transplantation, cancer, infectious disease, cardiovascular disease, and inflammation. Different isotypes have different effector functions, Such differences in function are reflected in distinct 3-dimensional structures for the various immunoglobulin isotypes (P. M. ALZARI, et. a / ., Annual Rev. Immunol., 6:555-580 (1988)).

[0015] Additionally, the interferons, including IFNa and IFN|3 (type I) and IFNy (type II) are essential mediators of anti-cancer immunity having both direct anti-proliferative effects against many cancers as well as a multitude of anti-tumor immunotherapeutic effects. However, while IFNa has shown efficacy against multiple human cancers, its clinical utility to date has been limited by the inability to achieve effective concentrations of IFN at tumor sites without causing systemic toxicity.

[0016] Due to the systemic toxicity, several groups have approached this problem by using the tumor¬ targeting ability of monoclonal antibodies to carry IFNs directly to tumor sites. See, Huang, et al., J. Immunol. 179(10), pp 6881-6888 (2007) and Vasuthasawat, et. al., J. Immunol. 36(5), pp. 305-318 (2013). It is noted that the initial work has used anti-CD20-IFNa2 proteins to target IFNa to CD20 expressed on lymphomas and anti-CD138-IFNa2 fusion proteins to target CD138 expressed on

[0017] ' multiple myeloma. See, Vasuthasawat, et. al., MAbs 8(7), pp. 1386-1397 (2016). While these approaches have shown great therapeutic promise and are currently being assessed in human clinical trials and developed commercially, there are several deficiencies.

[0018] While it is noted that using the antibody binding specificity to target tumor-associated antigens delivers a greater percentage (%) of the IFN to the site of the tumor than is achieved when IFN is injected on its own, the attached interferon still is recognized and bound by interferon receptors expressed throughout the body that are not tumor associated. Thus, Mab-fused IFN may still induce toxicity and / or have increased clearance due to the systemic exposure and interaction with IFN receptors throughout the body.

[0019] From the aforementioned, it will be readily apparent to those skilled in the art that a new treatment paradigm is needed in the treatment of cancers and immunological diseases.

[0020] Given the current deficiencies associated with delivering IFN to a cancer cell, it is an object of the present invention to provide new and improved methods of treating cancer(s), immunological disorders, and other diseases utilizing a masked IFN that inhibits the activity of IFN until it reaches the tumor. Provided are compositions, kits and methods for use that meet such needs.

[0021] SUMMARY OF THE INVENTION

[0022] The invention provides for antibodies, antigen-binding fragments, and fusion protein compositions that bind to a full range of tumor associated antigens (TAAs). In a further embodiment, the fusion protein compositions comprise a type I Interferon, In a further embodiment, the IFN is masked so its activity is reduced or nullified until it reaches a tumor cell. In a further embodiment, the TAA is set forth 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 mesothelin. In a further embodiment, the TAA is CD20. In another embodiment, the TAA is 5T4. In another embodiment, the TAA is FAP, In yet another embodiment, the IFN or functionally active mutants are set forth in Table II. In a preferred embodiment, the IFN comprises IFNA2.

[0023] In a further embodiment, the invention comprises a targeted masked IFN. In a preferred embodiment, the targeted masked IFN comprises IFNA1.

[0024] In a further embodiment, the invention comprises a targeted masked IFN. In a preferred embodiment, the targeted masked IFN comprises I FNA14.

[0025] In a further embodiment, the invention comprises a targeted masked IFN. In a preferred embodiment, the targeted masked IFN comprises IFNB1.

[0026] In another embodiment, the present disclosure teaches methods of producing a targeted masked IFN. In another embodiment, the present disclosure teaches methods of treating cancer(s), immunological disorders, and other diseases in humans.

[0027] in a preferred embodiment, the present disclosure teaches methods of treating cancer with a masked I FN which is fused to a MAb which binds a TAA.

[0028] in some of any of the embodiments, the method(s) for treating a cancer involves administering to a subject, such as a human subject, a therapeutically effective amount of any of the compositions or any of the fusion proteins, such as any of the targeted Masked IFNs described herein.

[0029] Also provided are pharmaceutical compositions comprising a therapeutically effective amount of any of the compositions or any of the fusion proteins, such as any of the targeted Masked IFNs described herein. In some of the embodiments, the pharmaceutical composition is for use in therapy including treatment of cancer. In some of any of the embodiments, the cancer comprises a cancer found in a solid tumor; or the cancer arises in the hematopoietic system. In some of the embodiments, the pharmaceutical composition further comprises one or more anti-neoplastic agents,

[0030] Also provided are kits, such as kits comprising any of the compositions or any of the fusion proteins, such as any of the targeted Masked IFNs described herein.

[0031] BRIEF DESCRIPTION OF THE FIGURES

[0032] Figure 1. Amino Acid Sequence of IFNAR2. Signal sequence in grey. Peptides selected as masking peptides based on crystal structure data indicating that these are regions of interaction with type I interferons underlined.

[0033] Figure 2 IFNa Masking Peptide(s).

[0034] Figure 3. Additional IFNa Masking Peptide(s).

[0035] Figure 4. Description of Fusion Protein(s) and Masking Peptide(s).

[0036] Figure 5. Description of Additional Fusion Protein(s) and Masking Peptide(s)

[0037] Figure 6. Peptide Binding to anti-CD138 IFNaYNSCoated Wells.

[0038] Figure 7 Peptide Binding to anti-CD138 IFNaYNSCoated Wells in the Presence of Free anti-CD138 IFNaYNS,

[0039] Figure 8. Fusion Protein (FP) Binding to Immobilized Peptide.

[0040] Figure 9. Matriptase Treatment of Fusion Proteins.

[0041] Figure 10. Methods of Reducing and Restoring Masked IFNa Activity.

[0042] Figure 11. Methods of Reducing and Restoring Masked IFNa Activity.

[0043] Figure 12. Methods of Reducing and Restoring Masked IFNa Activity.

[0044] Figure 13. IFNaYNSFusion Proteins Binding to IFNAR2.

[0045] Figure 14. IFNaYNSFusion Proteins Binding to IFNAR2.

[0046] Figure 15. Reduction and Restoration of IP-10 Induction in PBMCs. Figure 16. Methods of Reducing and Restoring Masked IFNa / p Activity.

[0047] Figure 17. IP-10 Induction in Human Peripheral Blood Mononuclear Cells (“PMBC”) Using ELISA

[0048] Figure 18. Matriptase ST 14 Cleaves Mask 4 on QXL138AM4.2-N297Q from the Heavy Chain of an anti-CD138-IFNa Fusion Ab.

[0049] Figure 19. Methods of Reducing and Restoring Masked IFNa Activity.

[0050] Figure 20. Methods of Reducing and Restoring Masked IFNa Activity.

[0051] Figure 21. Reduction and Restoration of IP-10 Induction in PBMCs.

[0052] Figure 22. Methods of Reducing and Restoring Masked IFNa Activity.

[0053] Figure 23. Methods of Reducing and Restoring Masked IFNa Activity.

[0054] Figure 24. Methods of Reducing and Restoring Masked IFNa Activity.

[0055] Figure 25. Methods of Reducing and Restoring Masked IFNa Activity.

[0056] Figure 26. Methods of Reducing and Restoring Masked IFNa Activity.

[0057] Figure 27. Methods of Reducing and Restoring Masked IFNa Activity.

[0058] Figure 28. Reduction and Restoration of IP-10 Induction in Human PBMCs.

[0059] Figure 29. Reduction and Restoration of IP-10 Induction in Human PBMCs.

[0060] Figure 30. Proliferation Assay Evaluating Mask 4.2 Fusion Proteins in OVCAR3.

[0061] Figure 31. Characterization of QXL138AM4.2-N297Q (Lot 2) By Capillary Electrophoresis (CD- SDS). Figure 31(A). Shows CE-SDS in Non-Reducing Conditions. Figure 31(B). Shows CE-SDS in Reducing Conditions.

[0062] Figure 32. Characterization of QXL138YNSM4.2-N297Q (Lot 1) By Capillary Electrophoresis (CD-SDS). Figure 32(A). Shows CE-SDS in Non-Reducing Conditions. Figure 32(B). Shows CE-SDS in Reducing Conditions.

[0063] Figure 33. Characterization of QXL138AM4.3 (Q146 Lot 1) By Capillary Electrophoresis (CD- SDS). Figure 33(A). Shows CE-SDS in Non-Reducing Conditions. Figure 33(B). Shows CE-SDS in Reducing Conditions.

[0064] Figure 34. Characterization of QXL138YNSM4.3-N297Q (Q147 Lot 1) By Capillary Electrophoresis (CD-SDS). Figure 34(A). Shows CE-SDS in Non-Reducing Conditions. Figure 34(B). Shows CE-SDS in Reducing Conditions.

[0065] Figure 35. Matriptase Treatment of QXL138AM43-N297Q and QXL138YNSM4.3.

[0066] Figure 36. Methods of Reducing and Restoring Masked IFNa Activity.

[0067] Figure 37. Methods of Reducing and Restoring Masked IFNa Activity.

[0068] Figure 38. Tumor Inhibition of Multiple Fusion Proteins in H929 Tumor Cells In vivo.

[0069] Figure 39. Tumor Volumes of Individual Mice on Day 28 of Multiple Fusion Proteins in H929 Tumor Cells in vivo Figure 40. Body Weight Assessment of Individual Mice on Day 28 of Multiple Fusion Proteins in H929 Tumor Cells In vivo.

[0070] Figure 41. Characteristics and Sequence Information of Anti-CD138 Human Kappa Light Chain.

[0071] Figure 42. Characteristics and Sequence Information of QXL138AM4.2-N297Q (Anti-CD138- IFNa-Mask4.2-N297Q) Heavy Chain Amino Acid.

[0072] Figure 43. Characteristics and Sequence Information of QXL138AM4.2-N297Q Heavy Chain Nucleic Acid.

[0073] Figure 44. Characteristics and Sequence Information of QXL138YNS4.2-N297Q Heavy Chain Amino Acid.

[0074] Figure 45. Characteristics and Sequence Information of QXL138YNS4.2-N297Q Heavy Chain Nucleic Acid.

[0075] Figure 46. Characteristics and Sequence Information of QXL138AM4.3-N297Q Heavy Chain Amino Acid.

[0076] Figure 47. Characteristics and Sequence Information of QXL138YNS4.3-N297Q Heavy Chain Amino Acid.

[0077] Figure 48. Characteristics and Sequence Information of Anti-Mesothelin 6A4 Human Kappa Light Chain Amino Acid.

[0078] Figure 49. Characteristics and Sequence Information of Meso-YNS4.2-N297Q Heavy Chain Amino Acid.

[0079] Figure 50. Characteristics and Sequence Information of QXL138AM4.2 and QXL138YNS4.2-N297Q Human lgG1 Heavy Chain Constant Region.

[0080] Figure 51. Characteristics and Sequence Information of Meso-YNS4.2-N297Q Human lgG1 Heavy Chain Constant Region and the Meso-YNS4.2-N297Q Human lgG1 Heavy Chain Variable Region.

[0081] Figure 52. Proliferation Assay Evaluating Mask 5.2 Fusion Proteins in NCI-H1299, Figure 53. Proliferation Assay Evaluating Mask 5.2 Fusion Proteins in NCI-H2452.

[0082] Figure 54. Proliferation Assay Evaluating Mask 5.2 and Mask 4.2 Fusion Proteins in NCI- H596.

[0083] Figure 55. Proliferation Assay Evaluating QXL138A, QXL138YNS, and Meso-YNS Fusion Proteins in KLE.

[0084] Figure 56. Proliferation Assay Evaluating QXL138A, QXL138YNS, and Meso-YNS Fusion Proteins in NCI-N87.

[0085] Figure 57. Proliferation Assay Evaluating QXL138A, QXL138YNS, and Meso-YNS Fusion Proteins in NCI-N2052. Figure 58. Characteristics and Sequence Information of Meso-YNS.

[0086] Figure 59. Characterization of Meso-YNS (Q159) By Capillary Electrophoresis (CD-SDS). Figure 59(A). Shows CE-SDS in Reducing Conditions. Figure 59(B). Shows CE-SDS in Non-Reducing Conditions.

[0087] Figure 60. Tumor Inhibition of Multiple Fusion Proteins in Capan-1 Tumor Cells In vivo. Figure 61. Tumor Volumes of Individual Mice on Day 56 of Multiple Fusion Proteins in Capan- 1 Tumor Cells In vivo.

[0088] Figure 62. Tumor Inhibition of Q158 at Multiple Doses in Capan-1 Tumor Cells In vivo.

[0089] Figure 63. Tumor Volumes of Individual Mice on Day 51 of Q158 in Multiple Doses in Capan-1 Tumor Cells In vivo.

[0090] Figure 64. Body Weight Assessment of Individual Mice at Multiple Timepoints of Q158 in Multiple Doses in Capan-1 Tumor Cells In vivo.

[0091] DETAILED DESCRIPTION OF THE INVENTION

[0092] Provided herein are fusion proteins and compositions comprising an interferon (IFN), In some aspects, the provided fusion proteins and compositions comprise an IFN and an antibody or an antigen¬ binding fragment thereof, such as an antibody or antigen-binding fragment thereof that is specific for a tumor-associated antigen (TAA). In some embodiments, the interferon is a Type I IFN. In some aspects, the provided fusion proteins and compositions comprise an IFN and a mask, such as a polypeptide sequence that blocks the interaction between the IFN and its receptor, e.g., an IFN-o receptor (IFNAR). In some aspects, the provided fusion proteins or compositions comprise an IFN, an antibody or antigen-binding fragment thereof and a mask. In some of the embodiments provided, 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, the cleavage of the protease cleavage site, for example at or near the site of the tumor or in the tumor microenvironment (TME), can lead to an “unmasking” of the IFN and permit binding of the IFN to its receptor. In some aspects, the antibody or antigen-binding fragment thereof, e.g., that is specific for a AA, can target the fusion protein or composition, to particular sites or location of tumor or cancer. Accordingly, in some aspects, the provided for fusion proteins and compositions can be used for treating a disease or disorder, such as a cancer or a tumor. Also provided are methods of making such fusion proteins or compositions, methods related to using such fusion proteins or compositions, such as in a method of treatment or in a therapeutic method, and pharmaceutical compositions or kits comprising such fusion proteins or compositions. As described further herein, the provided embodiments, including the targeted masked IFNs, provide a unique advantage over available approaches for several reasons, including a masked IFN whose activity is significantly reduced and / or eliminated until it reaches the site of the tumor, so that non-specific activity is minimized, and the fusion protein is not trapped by interferon receptors that are not at the tumor site. At the site of the tumor, the mask can be removed, and the binding activity of the IFN is re-activated, which can maximize the efficacy in the tumor, and increase the effective concentration of the fusion protein without increasing the toxicity. In addition, by virtue of the antibody that is attached to the IFN, the fusion protein can be targeted to a specific tumor (e.g., a tumor that expresses the TAA specifically bound by the antibody). The specific targeting allows for greater opportunity that the IFN will be directed to the cancer of interest and avoid non-cancerous or non-tumorous tissue.

[0093] Outline of Sections

[0094] I.) Definitions

[0095] II.) Antibodies

[0096] HI.) Interferon(s)

[0097] IV.) Methods of Masking IFNs

[0098] a. Discussion of Available Approaches

[0099] b. Methods of Masking IFN of the Disclosure

[0100] i. IFNa Masking Peptides and Masking Constructs

[0101] V.) Treatment of Cancer(s) Expressing a Tumor Associates Antigen (TAA) VI.) Targeted Masked IFN Fusion Protein Cocktails

[0102] VII.) Combination Therapy

[0103] VIII.) KITS / Articles of Manufacture

[0104] All publications, including patent documents, scientific articles, and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference,

[0105] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0106] I.) Definitions: Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al,, Molecular Cloning: A Laboratory Manual 2nd. Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. As appropriate, procedures involving the use of commercially available kits and reagents are generally conducted in accordance with manufacturer defined protocols and / or parameters unless otherwise noted.

[0107] When a trade name is used herein, reference to the trade name also refers to the product formulation, the generic drug, and the active pharmaceutical ingredient(s) of the trade name product, unless otherwise indicated by context.

[0108] The terms “advanced cancer”, “locally advanced cancel-”, “advanced disease” and “locally advanced disease” mean cancers that have extended through the relevant tissue capsule and are meant to include stage C disease under the American Urological Association (AUA) system, stage C1-C2 disease under the Whitmore- Jewett system, and stage T3-T4 and N+ disease under the TNM (tumor, node, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease and these patients have substantially less favorable outcomes compared to patients having clinically localized (organ-confined) cancer.

[0109] “Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native antibody sequence (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and / or enzymatic means), and / or adding one or more glycosylation sites that are not present in the native antibody sequence, wherein the “native glycosylation pattern" refers to the natural post-translational glycosylation pattern resulting from a particular combination of an antibody sequence, cell type, and growth conditions used. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.

[0110] The term “analog" refers to a molecule which is structurally similar or shares similar or corresponding attributes with another molecule (e.g., a TAA-related protein). For example, an analog of a TAA protein can be specifically bound by an antibody or T cell that specifically binds to a TAA.

[0111] The term “antibody” is used in the broadest sense unless clearly indicated otherwise. Therefore, an “antibody” can be naturally occurring or synthetic such as monoclonal antibodies produced by conventional hybridoma or transgenic mice technology. Antibodies comprise 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 covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they specifically bind a TAA and / or exhibit the desired biological activity. Any specific antibody can be used in the methods and compositions provided herein. Thus, in one embodiment the term “antibody" encompasses a molecule comprising at least one variable region from a light chain immunoglobulin molecule and at least one variable region from a heavy chain molecule that in combination form a specific binding site for the target antigen. In one embodiment, the antibody is an IgG antibody. For example, the antibody is an lgG1, lgG2, lgG3, lgG4 antibody or any known antibody isotype. The antibodies useful in the present methods and compositions can be generated 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. Therefore, in one embodiment, an antibody of the present invention is a mammalian antibody. Phage techniques can be used to isolate an initial antibody or to generate 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, a recombinant antibody can be produced by transfecting a host cell with a vector comprising a DNA sequence encoding the antibody. One or more vectors can be used to transfect the DNA sequence expressing at least one VL and at least one VH region in the 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) An antibody of the present invention can be modified by recombinant means to increase efficacy of the antibody in mediating the desired function. Thus, it is within the scope of the invention that antibodies can be modified by substitutions using recombinant means. Typically, the substitutions will be conservative substitutions. For example, at least one amino acid in the constant region of the antibody can be replaced with a different residue. See, e.g., U. S. Pat. No. 5,624,821, U. S. Pat. No. 6,194.551, Application No. WO 9958572; and ANGAL, et al., Mol. Immunol. 30: 105-08 (1993). The modification in amino acids includes deletions, additions, and substitutions of amino acids. In some cases, such changes are made to reduce undesired activities, e.g., complement-dependent cytotoxicity. Frequently, the antibodies are labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. These antibodies can be screened for binding to normal or defective TAA. See e.g, ANTIBODY ENGINEERING: A PRACTICAL APPROACH (Oxford University Press, 1996). Suitable antibodies with the desired biologic activities can be identified using the following in vitro assays including but not limited to proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, transport, signal transduction, and the following in vivo assays such as the inhibition of tumor growth. The antibodies provided herein can also be useful in diagnostic applications. As capture or non-neutralizing antibodies, they can be screened for the ability to bind to the specific antigen without inhibiting the receptor-binding or biological activity of the antigen. As neutralizing antibodies, the antibodies can be useful in competitive binding assays. They can also be used to quantify the TAA or its receptor. In further embodiments, an antibody of the disclosure, comprises a sequence of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater amino acid homology to a TAA of the disclosure. In a further embodiment, an antibody of the disclosure comprises a sequence that has 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the TAAs of the disclosure.

[0112] The term “antigen-binding fragment” or “antibody fragment” of an antibody (or simply “antibody portion”), 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 within the term “antigen-binding fragment” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (ill) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarily determining region (CDR), Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (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 with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. The term “Fc,” as used herein, refers to a region comprising a hinge region, CH2 and / or CH3 domains.

[0113] As used herein, any form of the “antigen” can be used to generate an antibody that is specific for a TAA. Thus, the eliciting of antigen may 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 eliciting antigen may be an isolated full-length protein, a cell surface protein (e.g., immunizing with cells transfected with at least a portion of the antigen), or a soluble protein (e.g., immunizing with only the extracellular domain portion of the protein), The antigen may be produced in a genetically modified cell. The DNA encoding the antigen may be genomic or non-genomic (e.g., cDNA) and encodes at least a portion of the extracellular domain or intracellular domain. As used herein, the term “portion,” in the context of an antigen, refers to the minimal number of amino acids or nucleic acids, as appropriate, to constitute an immunogenic epitope of the antigen of interest. Any genetic vectors suitable for transformation of the cells of interest may be employed, including but not limited to adenoviral vectors, plasmids, and non-viral vectors, such as cationic lipids. In one embodiment, the antibody of the methods and compositions herein specifically bind at least a portion of the extracellular domain of the TAA of interest.

[0114] The antibodies or antigen binding fragments thereof provided 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 the antigen and / or enhances or mediates a desired biological effect to enhance cell-killing toxins 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 is capable of binding the desired antigen ic epitope and directly or indirectly exerting a biologic effect. Direct effects include, but are not limited to the modulation, stimulation, and / or inhibition of a growth signal, the modulation, stimulation, and / or inhibition of an anti-apoptotic signal, the modulation, stimulation, and / or inhibition of an apoptoticor necrotic signal, modulation, stimulation, and / or inhibition the ADCC cascade, and modulation, stimulation, and / or inhibition the CDC cascade.

[0115] As used herein, the term “conservative substitution" refers to substitutions of amino acids and / or amino acid sequences that are known to those of skill in this art and may be made generally without altering the biological activity of the resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson, et al., MOLECULAR BIOLOGY OF THE GENE, The Benjamin / Cummings Pub. Co., p. 224 (4th Edition 1987)). Such exemplary substitutions are preferably made in accordance with those amino acids set forth in Table(s) III. For example, such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa. Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three- dimensional structure of the protein. For example, glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered “conservative” in particular environments (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 may be determined empirically or in accord with known conservative substitutions The term “fusion protein” as used herein means a protein of the invention which is fused to an IFN of the invention at the C-terminus using the linkers and methods known in the art. See, for example, U. S. 9,803,021, which is incorporated by reference herein. Exemplary linkers which can be used to fuse an IFN to a protein of the invention include, but are not limited to: (I) GGGGSGGGGSGGGGS (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;

[0116] 1qo0EJ; lgG3 hinge; lgG3 hingeAcys; and / or lgG1 hingeAcys.

[0117] The terms “inhibit” or “inhibition of’ as used herein means to reduce by a measurable amount, or to prevent entirely.

[0118] The term “interferon” as used herein means a group of signaling proteins made and released by host cells in response to the presence of several viruses. In a typical scenario, a virus-infected cell will release interferons causing nearby cells to heighten their anti-viral defenses. IFNs belong to the large class of proteins known as cytokines; molecules used for communication between cells to trigger the protective defenses of the immune system that help eradicate pathogens.

[0119] The term “Type 1 interferon” or " Type I interferon” as used herein means 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-ci receptor (IFNAR) that consists of IFNAR1 and IFNAR.2 chains. An exemplary list of type I interferons of the present disclosure are set forth in Table II.

[0120] 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.

[0121] The term “mask" in referring to a masked IFN (also denoted as “masked" IFN) means for purposes of this invention, any peptide or protein that blocks cytokine interaction and / or activation of I FN AR. It is within the scope of the invention that “mask” can be modified by substitutions using recombinant means. The modification in amino acids includes deletions, additions, and substitutions of amino acids.

[0122] The term “targeted masked IFN” as used herein means a type I interferon in which a polypeptide is attached at the carboxy terminus of the IFN thereby reducing the ability to bind the IFNAR. The masked IFN further comprises attachment to the carboxy terminus of a targeted binding protein (i.e., antibody). It is within the scope of the invention that “targeted masked IFN(s) can be modified by substitutions using recombinant means, The modification in amino acids includes deletions, additions, and substitutions of amino acids. In further embodiments of the disclosure, a “targeted masked IFN” comprises a sequence with 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater amino acid homology to the “targeted masked IFN" compounds of the disclosure. In a further embodiment, a “targeted masked IFN” comprises a sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the “targeted masked IFN” compounds of the disclosure.

[0123] The terms “metastatic cancer” and “metastatic disease" mean cancers that have spread to regional lymph nodes or to distant sites and are meant to include stage D disease under the AUA system and stage T*N*M+ under the TNM system.

[0124] “Molecular recognition” means a chemical event in which a host molecule is able to form a complex with a second molecule (i.e., the guest). This process occurs through non-covalent chemical bonds, including but not limited to, hydrogen bonding, hydrophobic interactions, and ionic interaction.

[0125] The term “monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of antibodies directed against (or specific for) different epitopes. In one embodiment, the polyclonal antibody contains a plurality of monoclonal antibodies with different epitope specificities, affinities, or avidities within a single antigen that contains multiple antigenic epitopes. The modifier “monoclonal” indicates the character of the antibody as being 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 to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature 256: 495 (1975), or may be made by recombinant DNA methods (see, e.g., U. S. Pat. No. 4,816,567). The “monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222: 581-597 (1991), for example These monoclonal antibodies will usually bind with at least a Kd of about 1 pM, more usually with at least about 300 nM, typically at least about 30 nM, preferably at least about 10 nM, more preferably at least about 3 nM or better, usually determined by ELISA.

[0126] “Pharmaceutically acceptable” refers to a non-toxic, inert, and / or composition that is physiologically compatible with humans or other mammals.

[0127] As used herein, the term “single-chain Fvl!or “scFv” or “single chain” antibody refers to antibody fragments comprising the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the Vn an VL domains which enables the sFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun, THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer- Verlag, New York, pp. 269-315 (1994).

[0128] As used herein, the terms “specific,” “specifically binds” and “binds specifically” refer to the selective binding of the antibody to the target antigen epitope. Antibodies can be evaluated for specificity of binding by comparing binding to appropriate antigen to binding to irrelevant antigen or antigen mixture under a given set of conditions. If the antibody binds to the appropriate antigen at least 2, 5, 7, and preferably 10 times more than to an irrelevant antigen or antigen mixture then it is considered to be specific. In one embodiment, a specific antibody is one that only binds a TAA antigen but does not bind to the irrelevant antigen. In another embodiment, a specific antibody is one that binds human TAA antigen but does not bind a non-human TAA antigen with 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater amino acid homology with the TAA antigen. In another embodiment, a specific antibody is one that binds human TAA antigen but does not bind a non-human TAA antigen with 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater percent identity with the amino acid sequence of the TAA antigen. In another embodiment, a specific antibody is one that binds human TAA antigen and binds murine TAA antigen, but with a higher degree of binding the human antigen. In another embodiment, a specific antibody is one that binds human TAA antigen and binds primate TAA antigen, but with a higher degree of binding the human antigen. In another embodiment, the specific antibody binds to human TAA antigen and any non-human TAA antigen, but with a higher degree of binding the human antigen or any combination thereof.

[0129] As used herein “to treat” or “therapeutic” and grammatically related terms, refer to any improvement of any consequence of disease, such as prolonged survival, less morbidity, and / or a lessening of side effects which are the byproducts of an alternative therapeutic modality: as is readily appreciated in the art, full eradication of disease is a preferred but albeit not a requirement for a treatment act. II.) Antibodies

[0130] In some embodiments, the provided fusion proteins, such as a targeted masked interferon ( I FN ), and compositions, comprise an antibody or an antigen-binding fragment thereof. In some of the provided embodiments, the antibody binds, such as specifically binds, recognizes, targets an antigen that is an antigen that is associated with a disease or a disorder, such as a cancer or an immunological disorder or disease, such as a tumor associated antigen (TAA). In some aspects, by virtue of the binding of to the antigen (e.g., TAA), the provided fusion protein, such as the targeted masked IFN, can be targeted to the relevant physical location for the therapy, such as regions of the cancer or the tumor. In some aspects, the described antibody or antigen-binding fragment thereof can be used as the component for any of the fusion proteins provided herein, for example, in any of the targeted IFN, antibody-IFN fusion protein or targeted masked IFN, provided herein.

[0131] An aspect of the invention provides antibodies that bind to an antigen associated with a cancer or tumor, such as a tumor associated antigen (TAA) and TAA-related proteins (See, Table I). In one embodiment, the antibody that binds to a TAA-related protein is an antibody that specifically binds to a TAA protein comprising an amino acid of the proteins set forth in Table I. For example, antibodies that bind a TAA protein comprising the amino acid sequence of one of the proteins set forth in Table I can bind TAA-related proteins such as TAA variants and the homologs or analogs thereof,

[0132] In some aspects, antibodies that bind to a TAA or a TAA-related protein, such as anti-TAA antibodies of the provided embodiments are particularly useful in cancer, for prognostic assays, imaging, diagnostic, and therapeutic methodologies. In some aspects, the antibodies of the provided embodiments are therapeutic antibodies, e.g., therapeutic antibodies that specifically bind a TAA, such as a TAA set forth in Table I. Similarly, such antibodies are useful (e.g., when combined with a therapeutic agent, in a fusion protein, in the treatment, and / or prognosis of cancers, such as ovarian, head and neck, multiple myeloma, and other cancers, to the extent TAA is also expressed or overexpressed in these other cancers. Moreover, antibodies of the provided embodiments, including intracellularly expressed antibodies (e.g., single chain antibodies) are therapeutically useful in treating cancers in which the expression of TAA is involved, such as advanced or metastatic cancers in solid tumors or other advanced or metastatic cancers. In one embodiment is a TAA binding assay disclosed herein for use in detection of cancer, for example, in an immunoassay.

[0133] In some embodiments, the provided for fusion protein or composition comprises an antibody that binds a Tumor Associated Antigen (TAA), for example a TAA selected from exemplary TAAs set forth in Table I. In some embodiments, the TAA is an antigen expressed on the surface of a tumor, such as the surface of a tumor cell or a cancer cell. In some embodiments, the TAA includes any antigen associated with any of the disease or conditions described herein, such as any cancer described herein. In some embodiments, the TAA is an antigen that is expressed on the surface of a cell associated with a tumor, such as cells present in the tumor microenvironment (TME). In some embodiments, the TAA, is an antigen that is present in the TME.

[0134] In some embodiments, the provided for fusion protein or composition comprises an antibody which binds a Tumor Associated Antigen associated with tumors arising in the hematopoietic system, such as a hematological malignancy. In some embodiments, the provided for fusion protein or composition comprises an antibody that binds to a CD138 antigen. In some embodiments, the antibody binds to, specifically binds to, one of the TAAs set forth in Table I.

[0135] In some embodiments, the antibody comprises or is comprised in a fusion protein. In some embodiments, the antibody is comprised in any of the fusion proteins or compositions provided herein. In some embodiments, the antibody comprises a fusion protein which comprises a Type-1 IFN, such as a Type I IFN set forth in Table II. In some embodiments, the antibody comprises a fusion protein, further comprising targeted IFN-a / pha. In some embodiments, the antibody comprises a fusion protein, further comprising a targeted masked IFN-a / pha.

[0136] In some aspects, the antibody includes an antibody fragment, such as an antigen-binding antibody fragment. Examples of antibody fragments include but are not limited to, Fab fragments, Fab' fragments, F(ab)’z fragments, single-chain antibody molecules, e.g., single chain Fv proteins (“scFv”), disulfide stabilized Fv proteins (“dsFv”), Fv, Fab'-SH, diabodies, linear antibodies, and multi-specific antibodies formed from antibody fragments.

[0137] Various methods for the preparation of antibodies, such as monoclonal antibodies, are well known in the art. For example, antibodies can be prepared by immunizing a suitable mammalian host using 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 can also be used, such as a TAA GST-fusion protein. In a particular embodiment, a GST fusion protein comprising all or most of the amino acid sequence of Figure 1 is produced, and then used as an immunogen to generate appropriate antibodies. In another embodiment, a TAA-related protein is synthesized and used as an immunogen.

[0138] In addition, naked DNA immunization techniques known in the art are used (with or without purified TAA-related protein or TAA expressing cells) to generate an immune response to the encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).

[0139] The amino acid sequence of a TAA protein set forth in Table I can be analyzed to select specific regions of the TAA protein, for example as an immunogen or an epitope, for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of a TAA amino acid sequence are used to identify hydrophilic regions in the TAA structure. Regions of a TAA protein that show immunogenic structure, as well as other regions and domains, can readily be identified using various other methods known in the art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schuitz, or Jameson-Wolf analysis. Hydrophilicity profiles can be generated using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl. Acad. Sci. U. S. A. 78:3824-3828. Hydropathicity profiles can be generated using the method of Kyte, J and Doolittle, R. F., 1982, J. Mol. Biol 157:105-132. Percent (%) Accessible Residues 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 P. K., 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-turn profilescan be generated using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294. Thus, each region identified by any of these programs or methods is within the scope of the present invention, Preferred methods for the generation of TAA antibodies are further illustrated by way of the examples provided herein. Methods for preparing a protein or polypeptide for use as an immunogen are well known in the art. Also well known in the art are methods for preparing immunogenic conjugates of a protein with a carrier, such as BSA, KLH or other carrier protein In some circumstances, direct conjugation using, for example, carbodiimide reagents are used; in other instances, linking reagents such as those supplied by Pierce Chemical Co., Rockford, III., are effective. Administration of a TAA immunogen is often conducted by injection over a suitable time period and with use of a suitable adjuvant, as is understood in the art. During the immunization schedule, titers of antibodies can be taken to determine adequacy of antibody formation.

[0140] TAA monoclonal antibodies can be produced by various means well known in the art. For example, immortalized cell lines that secrete a desired monoclonal antibody are prepared using the standard hybridoma technology of Kohler and Milstein or modifications that immortalize antibodyproducing B cells, as is generally known. Immortalized cell lines that secrete the desired antibodies are screened by immunoassay in which the antigen is a TAA-related protein. When the appropriate immortalized cell culture is identified, the cells can be expanded, and antibodies produced either from in vitro cultures or from ascites fluid.

[0141] The antibodies or fragments of the invention can also be produced by recombinant means. Regions that bind specifically to the desired regions of a TAA protein can also be produced in the context of chimeric or complementarity-determining region (CDR) grafted antibodies of multiple species origin. Humanized or human TAA antibodies can also be produced and are preferred for use in therapeutic contexts. Methods for humanizing murine and other non-human antibodies, by substituting one or more of the non-human antibodies CDRs for corresponding human antibody sequences, are well known (see for example, Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen, et al., 1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151: 2296.

[0142] In one embodiment, human monoclonal antibodies of the invention can be prepared using Velocimmune mice into which genomic sequences bearing endogenous mouse variable segments at the immunoglobulin heavy chain (VH, DH, and JH segments) and / or kappa light chain (VK and JK) loci have been replaced, in whole or in part, with human genomic sequences bearing unrearranged germline variable segments of the human immunoglobulin heavy chain (VH, DH, and JH) and / or kappa light chain (VK and JK) loci (Regeneron, Tarrytown, N. Y.). See, for example, U. S, Pat, Nos. 6,586,251, 6,596,541, 7,105,348, 6,528,313, 6,638,768, and 6,528,314.

[0143] In addition, human antibodies of the invention can be generated using the HuMAb mouse (Medarex, Inc.) which contains human immunoglobulin gene miniloci that encode unrearranged human heavy (mu and gamma) and kappa light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous mu and kappa chain loci (see e.g., Lonberg, et al. (1994) Nature 368(6474): 856-859).

[0144] In another embodiment, fully human antibodies of the invention can be raised using a mouse that carries human immunoglobulin sequences on transgenes and transchomosomes, such as a mouse that carries a human heavy chain transgene and a human light chain transchromosome. Such mice, referred to herein as “KM mice,” such mice are described in Tomizuka, et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727 and PCT Publication WO 02 / 43478 to Tomizuka, et al.

[0145] Human monoclonal antibodies of the invention can also be prepared using phage display methods for screening libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies are established in the art. See for example: U. S. Pat. Nos. 5,223,409; 5,403,484; and U. S. Pat. No. 5,571,698 to Ladner et al.; U. S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et al.; U. S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U. S. Pat. Nos.

[0146] 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et ai.

[0147] Human monoclonal antibodies of the invention can also be prepared using SCID mice into which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization. Such mice are described in, for example, U. S. Pat. Nos. 5,476,996 and 5,698,767 to Wilson, et al.

[0148] Additionally, human antibodies of the present invention can be made with techniques using transgenic mice, inactivated for antibody production, engineered with human heavy and light chains loci referred to as Xenomouse (Amgen Fremont, Inc., formerly Abgenix, Inc.). An exemplary description of preparing transgenic mice that produce human antibodies can be found in U. S. Pat. No. 6,657,103. See, also, U. S. Pat. Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,545,806; and Mendez, et, al. Nature Genetics, 15: 146-156 (1998); Kellerman, S. A. & Green, L. L., Curr. Opin. Biotechnol 13, 593-597 (2002).

[0149] Any of the methods of production above result in antibodies that have a certain ability to bind TAA, or homologs or fragments or polypeptide sequences having 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to TAA. The binding affinity (KD) of the antibodies, binding fragments thereof, and antibody drug conjugates comprising the same for TAA may be 1 mM or less, 100 nM or less, 10 nM or less, 2 nM or less or 1 nM or less. Alternatively, the KD may be between 5 and 10 nM; or between 1 and 2 nM. The KD may be between 1 micromolar and 500 micromolar or between 500 micromolar and 1 nM.

[0150] The binding affinity of the antigen binding protein is determined by the association constant (Ka) and the dissociation constant (Kd) (KD=Kd / Ka). The binding affinity may be measured by BIACORE for example, by capture of the test antibody onto a protein-A coated sensor surface and flowing TAA over this surface. Alternatively, the binding affinity can be measured by FORTEBIO for example, with the test antibody receptor captured onto a protein-A coated needle and flowing TAA over this surface. One of skill in the art can identify other suitable assays known in the art to measure binding affinity.

[0151] The term "specifically binds”, as used herein in relation to TAA antigen binding, proteins means that the antigen binding protein binds to the TAA as well as a discrete domain, or discrete amino acid sequence, within a TAA with no or insignificant binding to other (for example, unrelated) proteins, This term, however, does not exclude the fact that the antibodies or binding fragments thereof may also be cross-reactive with closely related molecules. The antibodies and fragments thereof as well as fusion proteins comprising these described herein may specifically bind to a TAA, with at least 2, 5, 10, 50, 100, or 1000-fold greater affinity than they bind to closely related molecules,

[0152] In one aspect, the invention comprises an antibody which binds a Tumor Associated Antigen (TAA).

[0153] In another aspect, the invention comprises an antibody which binds a Tumor Associated Antigen (TAA) associated with a solid cancer tumor.

[0154] In another aspect, the invention comprises an antibody which binds a Tumor Associated Antigen associated with tumors arising in the hematopoietic system.

[0155] in another aspect, the invention comprises an antibody which binds to a CD138 antigen.

[0156] In another aspect, the invention comprises an antibody which binds to a CD20 antigen.

[0157] In another aspect, the invention comprises an antibody which binds to a mesothelin antigen. In another aspect, the invention comprises an antibody which binds to a 5T4 antigen.

[0158] In another aspect, the invention comprises an antibody which binds to a FAP antigen.

[0159] In another aspect, the invention comprises an antibody which binds to a PSCA antigen.

[0160] In another aspect, the antibody comprises a fusion protein

[0161] in another aspect, the antibody comprises a fusion protein which comprises a type 1 IFN set forth in Table II.

[0162] in another aspect, the antibody comprises a fusion protein, further comprising targeted IFN- alpha. In another aspect, the antibody comprises a fusion protein, further comprising a targeted masked IFN-a / pte.

[0163] in another aspect, the invention comprises an antibody which binds CD138 which further comprises a heavy chain shown in Figure 42.

[0164] in another aspect, the invention comprises an antibody which binds CD138 which further comprises a heavy chain with the following sequence:

[0165] MDPKGSLSWRILLFLSLAFELSYGQVQLQQSGSELMMPGASVKISCKATGYTFSNYWIEWVKQ RPGHGLEWIGEILPGTGRTIYNEKFKGKATFTADISSNTVQMQLSSLTSEDSAVYYCARRDYYG NFYYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYQSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 6)

[0166] In another aspect, the invention comprises an antibody which binds CD138 which further comprises a heavy chain variable region with the following sequence:

[0167] QVQLQQSGSELMMPGASVKISCKATGYTFSNYWIEVWKQRPGHGLEWIGEILPGTGRTIYNEK FKGKATFTADISSNTVQMQLSSLTSEDSAVYYCARRDYYGNFYYAMDYWGQGTSVTVSS

[0168] (SEQ ID NO: 7)

[0169] In another aspect, the invention comprises an antibody which binds CD138 which further comprises a heavy chain with an amino acid substitution at position 297 (N297Q).

[0170] In another aspect, the invention comprises an antibody which binds CD138 which further comprises a heavy chain set forth in (SEQ ID NO: 6) with an amino acid substitution at position 297 (N297Q).

[0171] In another aspect, the invention comprises an antibody which binds CD138 which further comprises a heavy chain variable region set forth in (SEQ ID NO: 7) with an amino acid substitution at position 297 (N297Q).

[0172] In another aspect, the invention comprises an antibody which binds CD138 which further comprises a heavy chain variable region set forth in (SEQ ID NO: 7), which further comprises a flexible linker set forth in (SEQ ID NO: 5), which further comprises IFNa2 set forth in (SEQ ID NO: 10), which further comprises a protease cleavable linker set forth in (SEQ ID NO: 35), which further comprises a mask sequence set forth in (SEQ ID NO: 36), which further comprises a an amino acid substitution at position 297 (N297Q). See, Figure 42 (SEQ ID NO: 39).

[0173] In another aspect, the invention comprises an antibody which binds CD138 which further comprises a light chain shown in Figure 41.

[0174] In another aspect, the invention comprises an antibody which binds CD138 which further comprises a light chain with the following sequence:

[0175] METDTLLLWVLLLWVPGSTGDIQMTQSTSSLSASLGDRVTISCSASQGINNYLNWYQQKPDGT VELLIYYTSTLQSGVPSRFSGSGSGTDYSLTISNLEPEDIGTYYCQQYSKLPRTFGGGTKLEIKR TVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 8)

[0176] In another aspect, the invention comprises an antibody which binds CD138 which further comprises a light chain variable region with the following sequence:

[0177] DIQMTQSTSSLSASLGDRVTISCSASQGINNYLNWYQQKPDGTVELLIYYTSTLQSGVPSRFSG SGSGTDYSLTISNLEPEDIGTYYCQQYSKLPRTFGGGTKLEIK (SEQ ID NO: 9)

[0178] In another aspect, the invention comprises an antibody which binds CD138 which further comprises a light chain variable region (SEQ ID NO: 9) and a heavy chain variable region (SEQ ID NO: 7).

[0179] in another aspect, the invention comprises an antibody which binds CD138 which further comprises a light chain variable region (SEQ ID NO: 9) and a heavy chain variable region (SEQ ID NO: 7) and further comprises an amino acid substitution at position 297 (N297Q).

[0180] In another aspect, the invention comprises an antibody which binds GDI 38 which further comprises a light chain (SEQ ID NO: 8) and a heavy chain (SEQ ID NO: 6) and further comprises an amino acid substitution at position 297 (N297Q).

[0181] In another aspect, the invention comprises an antibody which binds CD138 which further comprises a heavy chain variable region set forth in (SEQ ID NO: 7), which further comprises a flexible linker set forth in (SEQ ID NO: 5), which further comprises IFNa2 set forth in (SEQ ID NO: 10), which further comprises a protease cleavable linker set forth in (SEQ ID NO: 35), which further comprises a mask sequence set forth in (SEQ ID NO: 36) or (SEQ ID NO: 37), which further comprises a an amino acid substitution at position 297 (N297Q). See, Figure 44 (SEQ ID NO: 41).

[0182] In another aspect, the invention comprises an antibody which binds GDI 38 which further comprises a heavy chain variable region set forth in (SEQ ID NO: 7), which further comprises a flexible linker set forth in (SEQ ID NO: 5), which further comprises IFNa2 set forth in (SEQ ID NO: 10), which further comprises a protease cleavable linker set forth in (SEQ ID NO: 35), which further comprises a mask sequence set forth in (SEQ ID NO: 36) or (SEQ ID NO: 37), which further comprises a an amino acid substitution at position 297 (N297Q). See, Figure 47 (SEQ ID NO: 44),

[0183] in another aspect, the invention comprises an antibody which binds mesothelia which further comprises a heavy chain shown in Figure 49.

[0184] In another aspect, the invention comprises an antibody which binds mesothelin which further comprises a heavy chain with the following sequence:

[0185] MDPKGSLSWRILLFLSLAFELSYGQVHLVESGGGWQPGRSLRLSCVASGITFRIYGMHVWRQ APGKGLEWVAVLWYDGSHEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARDGD YYDSGSPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYQSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 17)

[0186] In another aspect, the invention comprises an antibody which binds mesothelin which further comprises a heavy chain variable region with the following sequence:

[0187] QVHLVESGGGWQPGRSLRLSCVASGITFRIYGMHWVRQAPGKGLEWVAVLWYDGSHEYYA DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARDGDYYDSGSPLDYWGQGTLVTVSS

[0188] (SEQ ID NO: 18)

[0189] In another aspect, the invention comprises an antibody which binds mesothelin which further comprises a heavy chain with an amino acid substitution at position 297 (N297Q).

[0190] In another aspect, the invention comprises an antibody which binds mesothelin which further comprises a heavy chain set forth in (SEQ ID NO: 17) with an amino acid substitution at position 297 (N297Q).

[0191] in another aspect, the invention comprises an antibody which binds mesothelin which further comprises a heavy chain variable region set forth in (SEQ ID NO: 18) with an amino acid substitution at position 297 (N297Q).

[0192] In another aspect, the invention comprises an antibody which binds mesothelin which further comprises a heavy chain variable region set forth in (SEQ ID NO: 18), which further comprises a flexible linker set forth in (SEQ ID NO: 5), which further comprises IFNa2 set forth in (SEQ ID NO: 10), which further comprises a protease cleavable linker set forth in (SEQ ID NO: 35), which further comprises a mask sequence set forth in (SEQ ID NO: 36) or (SEQ ID NO: 37), which further comprises a an amino acid substitution at position 297 (N297Q). See, Figure 49 (SEQ ID NO: 46),

[0193] In another aspect, the invention comprises an antibody which binds mesothelin which further comprises a light chain shown in Figure 48 (SEQ ID NO: 45).

[0194] In another aspect, the invention comprises an antibody which binds mesothelin which further comprises a light chain with the following sequence:

[0195] METDTLLLWVLLLWVPGSTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQA PRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 19)

[0196] In another aspect, the invention comprises an antibody which binds mesothelin which further comprises a light chain variable region with the following sequence:

[0197] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG SGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK (SEQ ID NO: 20)

[0198] In another aspect, the invention comprises an antibody which binds mesothelin which further comprises a light chain variable region (SEQ ID NO: 20) and a heavy chain variable region (SEQ ID NO: 18).

[0199] In another aspect, the invention comprises an antibody which binds mesothelin which further comprises a light chain variable region (SEQ ID NO: 20) and a heavy chain variable region (SEQ ID NO: 18) and further comprises an amino acid substitution at position 297 (N297Q).

[0200] In another aspect, the invention comprises an antibody which binds mesothelin which further comprises a heavy chain variable region set forth in (SEQ ID NO: 53).

[0201] In another aspect, the invention comprises an antibody which binds mesothelin which further comprises a heavy chain constant region set forth in (SEQ ID NO: 52), which further comprises an amino acid substitution at position 297 (N297Q). See. Figure 51.

[0202] In another aspect, the invention comprises an antibody which binds mesothelin which further comprises a heavy chain variable region set forth in (SEQ ID NO: 53), which further comprises a flexible linker set forth in (SEQ ID NO: 5), which further comprises IFNa2 set forth in (SEQ ID NO: 10), further comprising a YNS Mutation (H57Y, E58N, and Q61S), which further comprises a heavy chain constant region (SEQ ID NO: 52). with an amino acid substitution at position 297 (N297Q). See, Figure 58 (SEQ ID NO: 54).

[0203] in another aspect, the invention comprises an antibody which binds mesothelin which further comprises a heavy chain variable region set forth in (SEQ ID NO: 53), which further comprises the following CDR Sequences:

[0204] Mesothelin Heavy CDR Sequence(s):

[0205] IYGMH (SEQ ID NO: 56) VLWYDGSHEYYADSVKG (SEQ ID NO: 57) DGDYYDSGSPLDY (SEQ ID NO: 58)

[0206] In another aspect, the invention comprises an antibody which binds mesothelin which further comprises a light chain variable region (SEQ ID NO: 20), which further comprises the following CDR Sequences:

[0207] Mesothelin Light CDR Sequence(s):

[0208] RASQSVSSYLA (SEQ ID NO: 59) DASNRAT (SEQ ID NO: 60) QQRSNWPLT (SEQ ID NO: 61)

[0209]

[0210] In another aspect, the invention comprises an antibody which binds mesothelin which further comprises a heavy chain set forth in (SEQ ID NO: 54) and a light chain set forth in (SEQ ID NO: 55). See, Figure 58.

[0211] III.) Interferon(s)

[0212] in some embodiments, the provided for fusion proteins, such as a targeted interferon (IFN), e.g., a targeted masked IFN, and compositions, comprise a component that is an interferon (IFN) or a variant thereof. In some aspects, also provided are targeted masked IFNs, such as a Type I IFN that is fused with an antibody, a fragment, or a chain thereof, and an “interferon mask.”

[0213] In some embodiments, provided are fusions proteins comprising an interferon (IFN) or a variant thereof, and an antibody or antigen-binding fragment thereof that specifically binds a tumor associated antigen (TAA), such as an antibody-IFN fusion protein or a targeted IFN. In some aspects, the IFN is any type of IFN described herein, in particular aspects, exemplary of IFN in the provided embodiments include Type I IFNs, including any known Type I IFNs and any described herein, such as those set forth in Table IL Exemplary antibodies in the fusion protein include any described herein, for example, in Section li or Table I. In some of any of the embodiments, provided are interferons (IFNs) or a variant thereof that is attached to or connected to a “mask”, such as a peptide or protein that blocks cytokine interaction and / or activation of interferon a receptor (IFNAR); also, in some cases referred to as an interferon mask, in some aspects, provided are masked IFNs. in some aspects, exemplary of IFN in the provided masked IFN include Type I IFNs, including any known Type I IFNs and any described herein, such as those set forth in Table II. Exemplary masks and methods for masking an interferon include any described herein, e.g., in Section IV. In some embodiments, the antibody-IFN fusion protein comprises a “masked” IFN that comprises an IFN component that is selected from the IFN in Table I.

[0214] In some aspects, IFNs are proteins used for therapy or treatment of a disease or disorder. In some aspects, the provided for fusion proteins and compositions contain the IFN component that can be effective in treating a disease or disorder, such as a cancer or a tumor, and / or can be used to increase the effectiveness of a therapeutic agent, such as an anti-cancer or anti-neoplastic agent.

[0215] IFNs are a group of signaling proteins made and released by the cells of a subject or host, such as host cells, in response to the presence of foreign entities in the body, such as a pathogen, including several viruses In a typical scenario, a virus-infected cell will release interferons causing nearby cells to heighten their anti-viral defenses.

[0216] IFNs belong to the large class of proteins known as cytokines; molecules used for communication between cells to trigger the protective defenses of the immune system that help eradicate pathogens. Interferons are named for their ability to "interfere" with viral replication by protecting cells from virus infections. In some aspects, IFNs also have various other functions: (i) they activate immune cells, such as natural killer cells and macrophages; and (ii) they increase host defenses by up-regulating antigen presentation by virtue of increasing the expression of major histocompatibility complex ( HC) antigens.

[0217] More than twenty (20) distinct IFN genes and proteins have been identified in animals, including humans. They are typically divided among three classes: Type I IFN, Type II IFN, and Type III IFN. IFNs belonging to all three classes are important for fighting viral infections and for the regulation of the immune system.

[0218] Interferon Type I: All type I IFNs bind to a specific cell surface receptor complex known as the IFN-a / p receptor (IFNAR) that consists of IFNAR1 and IFNAR2 chains. Notably, there is only one amino acid difference between IFNa2a and I FNa2b at position 23. IFNa2a has a lysine, while I FNa2b has an arginine. See, GABAIN, et. al., Eur. J. Biochem. 190, pp. 257-261 (1990). There are thirteen (13) type i interferons present in humans including, but not limited to IFN-a, IFN-p, IFN-E, IFN- K and IFN-w. In general, type I interferons are produced when the body recognizes a virus that has invaded it. They are produced by fibroblasts and monocytes. However, the production of type I IFN-a is prohibited by another cytokine known as Interleukin-10. Once released, type I interferons bind to specific receptors on target cells, which leads to expression of proteins that will prevent the virus from producing and replicating its RNA and DNA.

[0219] Interferon type II (IFN-v in humans): This is also known as immune interferon and is activated

[0220]

[0221] by Interleukin-12. Furthermore, type II interferons are released by cytotoxic T cells and T helper cells, Type I specifically. However, they block the proliferation of T helper cells type two. The previous, results in an inhibition of T h2 immune response and a further induction of T 1 immune response, which leads to the development of debilitating diseases, such as multiple sclerosis. IFN Type II binds to IFNGR, which consists of IFNGR1 and IFNGR2 chains.

[0222] Interferon type III: Signal through a receptor complex consisting of IL10R2 (also called CRF2-4)

[0223]

[0224] and IFNLR1 (also called CRF2-12). Current information demonstrates the importance of Type III IFNs in some types of virus or fungal infections.

[0225] In general, type I and II interferons are responsible for regulating and activating the immune response. Expression of type I and 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 interferon is induced by cytokines such as IL-12, and its expression is restricted to immune cells such as T cells and NK cells.

[0226] In some aspects, IFNs and proteins containing or derived from IFNs can be used as a therapeutic agent. In some of the provided embodiments, the IFN component of the fusion protein, e.g., targeted masked IFN, is used as a therapeutic agent for treatment of the disease or disorder, such as a cancer.

[0227] In some aspects, Interferon therapy is used (in combination with chemotherapy and radiation) as a treatment for some cancers. This treatment can be used in hematological malignancy; leukemia and lymphomas including hairy cell leukemia, chronic myeloid leukemia, nodular lymphoma, and cutaneous T-cell lymphoma In some instances, patients with recurrent melanomas receive recombinant IFN-a2b

[0228] A major limitation with using available IFN in cancer therapy has been the inability to achieve effective concentrations of IFN at tumor sites without causing systemic toxicity. In order to overcome this limitation, several attempts have been made to solve this problem, by using the tumor targeting ability of monoclonal antibodies to carry IFNs directly to the tumor sites. See, Huang, eta / ., J. Immunol.

[0229] 179(10), pp. 6881-6888 (2007) and Vasuthasawat, et. al., J. Immunol. 36(5), pp. 305-318 (2013). It is noted that the initial work has used anti-CD20-IFNa2 proteins to target IFNa to CD20 expressed on lymphomas and anti-CD138-IFNa2 fusion proteins to target CD138 expressed on multiple myeloma. See, Vasuthasawat, et. al., MAbs 8(7), pp. 1386-1397 (2016). While these approaches have shown great therapeutic promise and are currently being tested in human clinical trials and developed commercially, there are several deficiencies of these available approaches.

[0230] Although using the antibody binding specificity to target tumor-associated antigens delivers a greater percentage of the IFN to the site of the tumor than is achieved when IFN is injected by itself, the attached interferon still can be recognized and bound by interferon receptors expressed by cells

[0231] 97 throughout the body, such as cells that are not tumor associated or normal cells. This binding can result both in less IFN reaching the tumor and in unwanted off-target toxicity. Provided are embodiments that overcome such limitations and deficiencies

[0232] Accordingly, it is an object of the present invention to overcome these limitations by providing a mechanism of “masking" the function or the activity of the IFN until at which time the IFN reaches the location or region that is relevant for the treatment of the disease or disorder, such as the tumor. At that time, the IFN is “unmasked” and the activity and function is effectively switched back on. As such, in some embodiments, provided are fusion proteins, e.g., a targeted masked IFN, that is “unmasked” or “activated" only at the location of interest for the therapeutic effect, such as the tumor. In some aspects, masking of the function or activity of the IFN in the rest of the body, e.g., the systemic circulation in general, can reduce or prevent non-specific activity of the IFN and also reduce or prevent the therapeutic agent, e.g., the IFN, from being trapped in or soaked up by non-cancerous or non-tumorous cells in the body. In some aspects, masking of the function or activity of the IFN and targeting the IFN to location of interest, e.g., to a tumor, by virtue of the antibodies present in the provided embodiments, can effectively increase the concentration of the therapeutic agent (e g., the IFN), for example by preventing the IFN from being bound to and / or trapped in by specific targeting of the agent by the antibody.

[0233] Accordingly, in some embodiments, the invention comprises an antibody-IFN fusion protein in which the IFN will selectively bind IFN receptors once it reaches the tumor.

[0234] In another embodiment, the antibody-IFN fusion protein comprises an IFN that is separated by a peptide linker that is a site for proteolytic cleavage.

[0235] In another embodiment, the antibody-IFN fusion protein comprises a “masked" IFN.

[0236] In another embodiment, the antibody-IFN fusion protein comprises a “masked" IFN that is separated by a peptide linker that is a site for proteolytic cleavage.

[0237] In another embodiment, the antibody-IFN fusion protein comprises a “masked” IFNA1.

[0238] In another embodiment, the antibody-IFN fusion protein comprises a “masked" IFNA1 that is separated by a peptide linker that is a site for proteolytic cleavage.

[0239] In another embodiment, the antibody-IFN fusion protein comprises a “masked” IFNA2.

[0240] In another embodiment, the antibody-IFN fusion protein comprises a “masked” IFNA2 that is separated by a peptide linker that is a site for proteolytic cleavage.

[0241] In another embodiment, the antibody-IFN fusion protein comprises a “masked" IFNB1.

[0242] In another embodiment, the antibody-IFN fusion protein comprises a “masked" IFNB1 that is separated by a peptide linker that is a site for proteolytic cleavage.

[0243] In another embodiment, the antibody-IFN fusion protein comprises a “masked” IFN selected from the IFN set forth in Table II. In another embodiment, the antibody-IFN fusion protein comprises a “masked” IFN that is separated by a peptide linker that is a site for proteolytic cleavage and is selected from the IFN set forth in Table II.

[0244] in another aspect, the antibody-IFN fusion protein comprises IFNa comprising the following:

[0245] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIF NLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLY LKEKKYSPCAWEWRAEIMRSFSLSTNLQESLRSKE (SEQ ID NO: 10)

[0246] In another aspect of the disclosure, it is understood that increasing the binding affinity will specifically increase the antiproliferative potency of IFNa. 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.0103079 (July 9, 2015). Accordingly, in one aspect of the disclosure, the antibody-IFN fusion protein comprises IFNa (SEQ ID NO: 10), further comprising a YNS Mutation (H57Y, E58N. and Q61S).

[0247] In another aspect, the antibody-IFN fusion protein comprises IFNa comprising the following:

[0248] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLYNMISQIF NLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEWRAEIMRSFSLSTNLQESLRSKE (SEQ ID NO: 21).

[0249] See for example, Figure 44 (QXL138YNS4.2-N297Q), Figure 47 (QXL138YNS4.3-N297Q), Figure 49 (Meso-YNS4.2-N297Q), and Figure 58 (Meso-YNS).

[0250] IV.) Methods of Masking IFNs

[0251] As previously noted, it is an object of the present invention to provide for targeted masked IFN compositions whereby the activity of an IFN of the present invention (See, Table II) is inhibited until at which time it reaches a tumor and the mask is removed by proteases, such as tumor-associated proteases. In some aspects, the provided masked IFNs, such as targeted masked IFNs, are “unmasked” at or near the location of the disease or disorder to be treated, e.g., at or near a tumor, such as a tumor microenvironment, and become capable of binding to and / or activating the interferon receptor (e.g., IFNAR). In some aspects, the unmasking or activating of the provided fusion proteins occurs by virtue of cleavage of a component, such as a peptide linker, by a protein in the environment of the tumor, such as a tumor associated protease.

[0252] (a) Discussion of Available Approaches. Available approaches that are related to this endeavor are limited. The disclosure presents an explanation of the available approaches to further demonstrate and show the technical advantages of the present invention. A prior approach to improving tumor specific delivery of therapeutics was to create a therapeutic which is activated by tumor associated proteases. An example of this approach has been to take antibodies that recognize tumor associated antigens but are limited in their efficacy because they also recognize antigens present on normal cells and modify them so that they only bind antigen when localized in the target tumor. See, U. S. 8,563,269 (CytomX Therapeutics, San Francisco, CA), The so-called “Probodies” have an associated peptide that blocks the antibody binding site joined by a linker that is cleavable by proteases present in the tumor microenvironment. In one case the antibody, CETUXIMAB, specific for epidermal growth factor receptor (EGFR) that was only activated to bind when localized to the tumors was produced. See, Desnoyers, et. al., Sci. Transl. Med., 16:5(207) pp 207ra144 (2013). In this " Probody”, a mask sequence that binds the variable region of CETUXIMAB followed by a GS-linker and then the sequence LSGRSDNHGSSGT (SEQ ID NO: 11) was attached to the amino terminus of the heavy chain of the antibody. In that case, the underlined sequence is a substrate for UFA and matriptase, proteases known to be up regulated in a variety of human carcinomas with minimal activity in normal tissues. This probody demonstrated improved safety and increased half-life in nonhuman primates.

[0253] In addition to producing Probodies with antibody binding activated in the tumor microenvironment, it has been possible to produce a protease activated interferon alpha proprotein. See, U. S. 8,399,219 (CytomX Therapeutics, San Francisco, CA). In that case a peptide mask for IFN-a TDVDYYREWSWTQVS (SEQ ID NO: 12) was placed at the amino terminus of single chain recombinant IFNa separated from the IFNa by a cleavable sequence. The resulting construct:

[0254] GQSGQ TDVDYYREWSETQVS GSSGGS VHMPLGFLGP GGS (SEQ ID NO: 13) IFNa contained IFNa that was selectively activated in the tumor microenvironment. It is taught that VHMPLGFLGP (SEQ ID NO: 14) is a substrate for MMP-9.

[0255] (b) Methods of Masking IFN of the Disclosure.

[0256] Previously, we have disclosed alternative approaches to generating masked IFNs that can be fused to antibodies which bind TAAs. See, for example, United States Patent Application Publication No.: US2020 / 0331966, published 22-October-2020 (Qwixel Therapeutics, Inc., Los Angeles, CA.). The rnask(s) disclosed herein are derived from a peptide identified through phage display and is not a native human sequence. The subject of the present disclosure teaches novel alternatives to generating masked IFNs. The disclosed peptide masks are capable of being unmasked at or near the location of the disease or disorder. From the aforementioned, the methods of masking IFNs of the present disclosure are clearly distinguishable and provide an advantage over any available approaches. Some advantages include, but are not limited to, (i) the ability to mask substantially type I interferons, (ii) superior masking properties based on higher affinity, and (iii) reduced risk of immunogenicity.

[0257] Furthermore, as described above, the provided embodiments include a mechanism of “masking” the function or the activity of the IFN until at which time the IFN reaches the location or region that is relevant for the treatment of the disease or disorder, such as the tumor, and specific physical targeting of the fusion protein at the location of the tumor by virtue of the TAA-specific antibody that is fused to the IFN. Accordingly, the fusion proteins described herein provide multiple advantages, including, but not limited to, that the IFN is “unmasked” or “activated” only at the location of interest for the therapeutic effect, such as the tumor; non-specific activity of the IFN is reduced; that the IFN is prevented from being trapped in or soaked up by non-cancerous or non-tumorous cells in the body; and / or effectively increasing the concentration of the therapeutic agent (e.g., the IFN) without an increase in toxicity.

[0258] In some of the embodiments, the provided for fusion protein, e.g., a targeted masked IFN, is only unmasked at or near the site of the disease or disorder, e.g., the tumor. Accordingly, in some embodiments, the invention comprises an antibody-IFN fusion protein in which the IFN will selectively bind IFN receptors once it reaches the location or region of the tumor.

[0259] In some embodiments, the antibody-IFN fusion protein comprises an IFN that is separated by a peptide linker that is a site for proteolytic cleavage. In some aspects, the proteolytic cleavage of the peptide linker can “unmask” or activate the IFN, for example, at or near a tumor.

[0260] As described in the disclosure, the provided embodiments include an antibody-IFN in which the IFN, such as those described in Section III or Table II herein, is fused to the antibody as described herein, for example in Section II or Table I. The IFN is then “masked” so that it will only become active and bind its receptors at the site of the tumor. In an uncleaved state the ideal peptide mask inhibits binding of the protein (e.g., the IFN) to its binding partner (e.g., interferon receptor, such as IFNAR), and following cleavage the peptide mask does not inhibit binding of the protein to its binding partners. In the embodiments provided herein, the cleavage of the “mask,” for example, at or near the site of the tumor, allows the Type I IFN to bind to its receptor (e.g., IFNAR), and exert its therapeutic effect.

[0261] The following describes an exemplary embodiment.

[0262] (i) IFNa Masking Peptides and Masking Constructs

[0263] Disclosed herein are novel peptide masks that are designed based on crystal structure models of IFNa binding to IFNAR2. As a result of these studies, regions of IFNAR2 that interact with IFNa were synthesized as peptides and tested for binding to IFNa as well as for competitive inhibition of IFNa- IFNAR2.

[0264] Development of the peptides disclosed herein is based on the rational that all type I IFNs share the same receptor-binding mode and form structurally highly similar signaling complexes. See, PI EH LER. et. at., Immunol Rev.. 250(1): 317-334 (Nov. 2012). Furthermore, it has been shown that Receptor-ligand cross-reactivity of I FNa2 and IFNAR2 is enabled by conserved receptor-ligand "anchorpoints" interspersed amongst ligand-specific interactions that ‘tune’ the relative IFN binding affinities, in an apparent extracellular igand proofreading’ mechanism that modulates biological activity.

[0265] Additionally, functional differences between IFNs are linked to their respective receptor recognition chemistries, in concert with a ligand-induced conformational change in IFNAR1, that collectively controls signal initiation and complex stability, ultimately regulating differential STAT phosphorylation profiles, receptor internalization rates, and downstream gene expression patterns. See, THOMAS, et, al., Cell, 146(4): 621-632 (Aug. 2011). In addition, comparison of the mutual binding sites of IFNa2 and IFNAR2 suggests that I FNa2 interacts with both domains of IFNAR2 and exposes several “hot spot” residues. See, ROISMAN, et. al., PNAS, vol. 98, no. 23, 13231-13236 (Nov. 2001) and Figure 1 (SEQ ID NO: 25). Based on the foregoing, several peptides were generated and are set forth in Figure 2 and Figure 3.

[0266] IFNa2 is used along with a protease cleavage site (e.g., LSGRSDNH (SEQ ID NO: 15) or KQSRVVNH (SEQ ID NO: 16)) and a peptide “mask” (Figure 2 and Figure 3) that inhibits IFN binding is placed on the 3’ terminus fused to CH3. It is contemplated by the disclosure that at the site of the tumor, proteases within the tumor microenvironment will cleave the protease cleavage site releasing the mask and freeing the IFN so that it can bind to its receptor.

[0267] In another aspect, the protease cleavage recognition site KQSRVVNH has been replaced with the sequence GSGGSGGS to prevent protease cleavage of the peptide “mask.” The control protein resulting from this change is contemplated to provide data on the fusion protein efficacy when IFNa2 remains “masked”.

[0268] The nucleic acids for the construction of a recombinant heavy chain with a protease cleavage site and IFN inhibitory mask were obtained (ATUM, Newark, California) and used to modify the heavy chain (H chain) of anti-TAA-IFNa2 by producing the following fusion at its 3’ end:

[0269] WRAEIMRSFSLSTNLQESLRSKEqssqKQSRWNHassaasaasgqsa ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSiyPTHYTLLYTIMSKPEDLKVVKNCANTTRSrcDLTD i^RSTH^TVLE^NTTLFS^FMDMS (SlQ ID NO: 22).

[0270] For the purposes of this disclosure, (SEQ ID NO: 22) comprises the amino acid reside (glutathione disulfide) GSSG (SEQ ID NO: 62) at residues 25 thru 28 and further comprises gssggsggsggsg (SEQ ID NO: 63) at residues 37 thru 49. Single underline indicates the carboxy-terminus of IFNa2, double underline represents the sequence for the protease cleavage site, and dashed underline represents the IFNci mask (SEQ ID NO: 36). Linker sequences are shown as lower case.

[0271] In another embodiment, the construct comprises:

[0272] WRAEIMRSFSLSTNLQESLRSKEqssqKQSRWNHqssqqsqqsqqsq ISYDSPDYTDESCTFKISLRNFRSILSWELKNHLIVPIHYTLLYTIMSKPEDLKVyKNCANTKRSFCDLTD EWRSTHEAWTV^ ID NO: 23).

[0273] For the purposes of this disclosure, (SEQ ID NO: 23) comprises the amino acid reside (glutathione disulfide) GSSG (SEQ ID NO: 62) at residues 25 thru 28 and further comprises gssggsggsggsg (SEQ ID NO: 63) at residues 37 thru 49.

[0274] Single underline indicates the carboxy-terminus of IFNa2, double underline represents the sequence for the protease cleavage site, and dashed underline represents the IFNo mask (SEQ ID NO: 37). Linker sequences are shown as lower case.

[0275] In another embodiment, the construct comprises:

[0276] WRAEIMRSFSLSTNLQESLRSKEgssggsggsggsgssggsggsggslSYDSPDYTDESCTFKISLRNFRSILSWE LKNHSIVPTHYTLL WSKPEDLKWKNCANTTRSFCDLTDE F LAID_MS (SEQ ID NO: 24).

[0277] For the purposes of this disclosure, (SEQ ID NO: 24) comprises gssggsggsggsgssggsggsggs (SEQ ID NO: 64) at residues 25 thru 48.

[0278] Single underline indicates the carboxy-terminus of IFNa2. linker sequences are shown in lower case, and dashed underline represents the IFNo mask (SEQ ID NO: 36). Linker sequences are shown as lower case.

[0279] In one embodiment, the “masked” IFN comprises:

[0280] IS^DSPDWESCTFKJSLRNFRS^

[0281]

[0282] EWRSJK^YV]VLEGF_SGNTTLFSC^ ID NO: 36).

[0283] In one embodiment, the “masked” IFN comprises:

[0284] ISYDSPDYJlDESCTFKJSLRffi

[0285]

[0286] EWRSLHEAtYTVLEGFSGNTTLF (SEQ ID NO: 36) and further comprises IFNal.

[0287] In one embodiment, the “masked” IFN comprises:

[0288] ISYDSPDYJ1DESCTFKJSLR®

[0289]

[0290] EWRSJE^YWL^ (SEQ ID NO: 36) and further comprises IFNa2.

[0291] In one embodiment, the “masked" IFN comprises:

[0292] ISYDSPD^fTDESCTFKJSLRNFRJSILSW^

[0293]

[0294] EWRSJK^Yy]VLEGF_SGNTTLFSC^^^^^ (SEQ ID NO: 36) and further comprises IFNa4. In one embodiment, the “masked” IFN comprises:

[0295] ISYDSPDWESCTFKJSLRiTO

[0296]

[0297] EWRSWAYW^ (SEQ ID NO: 36) and further comprises IFNo5.

[0298] In one embodiment, the “masked” IFN comprises:

[0299] ISYDSPDYTJDESCTFKJSLRNFRSJLSy^

[0300]

[0301] EWRSJLEAYWLEGFS_GJ (SEQ ID NO: 36) and further comprises IFNct 1 fused to an antibody which binds CD138.

[0302] In one embodiment, the “masked” IFN comprises:

[0303]

[0304] ISYDSPPWESCTFKJS^ EWRSTAEAYyTyLEGFSA TTLFSCAHAFWPAPM. S (SEQ ID NO: 36) and further comprises IFNol fused to an antibody which binds CD20.

[0305] In one embodiment, the “masked” IFN comprises:

[0306] ISYDSPDYADESCTFKJSLRN^^

[0307]

[0308] EWRSJHEWWLEGFSGNTTLFSC^ (SEQ ID NO: 36) and further comprises IFNcd fused to an antibody which binds Her2.

[0309] In one embodiment, the “masked” IFN comprises:

[0310] ISADSPDYTOESCTFKJSLRNFRM^

[0311]

[0312] -EWRSTOWTVLEGFSGNTTLFSCSH^ (SEQ ID NO: 36) and further comprises IFNol fused to an antibody which binds CSPG4.

[0313] In one embodiment, the “masked” IFN comprises:

[0314] ISYDLPPYTOESCTFKJSLRNFRSJLLVVELKLtHSm^^ EWRSLHEAYVTVLEGFSGNTTLFSCSHAF (SEQ ID NO: 36) and further comprises IFNol fused to an antibody which binds PSCA.

[0315] In one embodiment, the “masked” IFN comprises: ISADSPDYTAESCTFKJSLRNFRSJLSWELK^^^. EWRSTAEAYVTVLEGFSGNTTLFSCSHAF (SEQ ID NO: 36) and further comprises IFNol fused to an antibody which binds CEA.

[0316] In one embodiment, the “masked” IFN comprises: ISYDSPPXTDESCTFKJSLRNFRAILSWELKNHSjyPTH^LPYTJMSXPEDLKyyANAANTTRSACDLTD

[0317]

[0318] EWRSJEEAYYWLEGFSGNTTipSCSH^^ (SEQ ID NO: 36) and further comprises IFNol fused to an antibody which binds RCC.

[0319] In one embodiment, the “masked” IFN comprises:

[0320]

[0321] ISYDSPPXTDESCTFKJSLRNFR^^^ EWRSTKEAYYTVLEGFSG TTLFSCSHAFWLADMS. (SEQ ID NO: 36) and further comprises IFNol fused to an antibody which binds 5T4. In one embodiment, the “masked” IFN comprises:

[0322] ISYDSPDWESCTFKJSLRiTO

[0323]

[0324] EWRSWAYVTVLEGFS1GNTTLFS (SEQ ID NO: 36) and further comprises IFNal fused to an antibody which binds mesothelin,

[0325] in one embodiment, the “masked” IFN comprises:

[0326] ISYDSPDYTDESCTFKISLRNFRSILSWELKNHLIVPTHYTLLYTIMSKPEDLKWKNCANTKRSFCDLTD EWRSTHEAYVTVLEGFSGNTLLFSCSHNFWLAIDMS (SEQ ID NO: 37).

[0327] In one embodiment, the “masked” IFN comprises:

[0328] ISYDSPDYTDESCTFKISLRNFRSILSWELKNHLIVPTHYTLLYTIMSKPEDLKWKNCANTKRSFCDLTD EWRSTHEAYVTVLEGFSGNTLLFSCSHNFWLAIDMS (SEQ ID NO: 37) and further comprises IFNal.

[0329] In one embodiment, the “masked” IFN comprises:

[0330] ISYDSPDYTDESCTFKISLRNFRSILSWELKNHLIVPTHYTLLYTIMSKPEDLKWKNCANTKRSFCDLTD EWRSTHEAYVTVLEGFSGNTLLFSCSHNFWLAIDMS (SEQ ID NO: 37) and further comprises IFNa2.

[0331] In one embodiment, the “masked” IFN comprises:

[0332] ISYDSPDYTDESCTFKISLRNFRSILSWELKNHLIVPTHYTLLYTIMSKPEDLKWKNCANTKRSFCDLTD EWRSTHEAYVTVLEGFSGNTLLFSCSHNFWLAIDMS (SEQ ID NO: 37) and further comprises IFNci4.

[0333] In one embodiment, the “masked” IFN comprises:

[0334] ISYDSPDYTDESCTFKISLRNFRSILSWELKNHLIVPTHYTLLYTIMSKPEDLKWKNCANTKRSFCDLTD EWRSTHEAYVTVLEGFSGNTLLFSCSHNFWLAIDMS (SEQ ID NO: 37) and further comprises IFNo5.

[0335] In one embodiment, the “masked” IFN comprises:

[0336] ISYDSPDYTDESCTFKISLRNFRSILSWELKNHLIVPTHYTLLYTIMSKPEDLKWKNCANTKRSFCDLTD EWRSTHEAYVTVLEGFSGNTLLFSCSHNFWLAIDMS (SEQ ID NO: 37) and further comprises IFNal fused to an antibody which binds CD138.

[0337] In one embodiment, the “masked” IFN comprises:

[0338] ISYDSPDYTDESCTFKISLRNFRSILSWELKNHLIVPTHYTLLYTIMSKPEDLKVVKNCANTKRSFCDLTD EWRSTHEAYVTVLEGFSGNTLLFSCSHNFWLAIDMS (SEQ ID NO: 37) and further comprises IFNal fused to an antibody which binds CD20.

[0339] In one embodiment, the “masked” IFN comprises:

[0340] ISYDSPDYTDESCTFKISLRNFRSILSWELKNHLIVPTHYTLLYTIMSKPEDLKWKNCANTKRSFCDLTD EWRSTHEAYVTVLEGFSGNTLLFSCSHNFWLAIDMS (SEQ ID NO: 37) and further comprises IFNal fused to an antibody which binds Her2.

[0341] In one embodiment, the “masked” IFN comprises:

[0342] ISYDSPDYTDESCTFKISLRNFRSILSWELKNHLIVPTHYTLLYTIMSKPEDLKWKNCANTKRSFCDLTD EWRSTHEAYVTVLEGFSGNTLLFSCSHNFWLAIDMS (SEQ ID NO: 37) and further comprises IFNal fused to an antibody which binds CSPG4. In one embodiment, the “masked” IFN comprises:

[0343] ISYDSPDYTDESCTFKISLRNFRSILSWELKNHLIVPTHYTLLYTIMSKPEDLKWKNCANTKRSFCDLTD EWRSTHEAYVTVLEGFSGNTLLFSCSHNFWLAIDMS (SEQ ID NO: 37) and further comprises IFNal fused to an antibody which binds PSCA.

[0344] in one embodiment, the “masked” IFN comprises:

[0345] ISYDSPDYTDESCTFKISLRNFRSILSWELKNHLIVPTHYTLLYTIMSKPEDLKWKNCANTKRSFCDLTD EWRSTHEAYVTVLEGFSGNTLLFSCSHNFWLAIDMS (SEQ ID NO: 37) and further comprises IFNal fused to an antibody which binds CEA.

[0346] In one embodiment, the “masked” IFN comprises:

[0347] ISYDSPDYTDESCTFKISLRNFRSILSWELKNHLIVPTHYTLLYTIMSKPEDLKWKNCANTKRSFCDLTD EWRSTHEAYVTVLEGFSGNTLLFSCSHNFWLAIDMS (SEQ ID NO: 37) and further comprises IFNal fused to an antibody which binds ROC.

[0348] in one embodiment, the “masked” IFN comprises:

[0349] ISYDSPDYTDESCTFKISLRNFRSILSWELKNHLIVPTHYTLLYTIMSKPEDLKWKNCANTKRSFCDLTD EWRSTHEAYVTVLEGFSGNTLLFSCSHNFWLAIDMS (SEQ ID NO: 37) and further comprises IFNal fused to an antibody which binds 5T4.

[0350] In one embodiment, the “masked” IFN comprises:

[0351] ISYDSPDYTDESCTFKISLRNFRSILSWELKNHLIVPTHYTLLYTIMSKPEDLKWKNCANTKRSFCDLTD EWRSTHEAYVTVLEGFSGNTLLFSCSHNFWLAIDMS (SEQ ID NO: 37) and further comprises IFNal fused to an antibody which binds mesothelin.

[0352] in one embodiment, the “masked” IFN comprises a neutralizing scFv which also acts as a mask, (as defined within the context of the disclosure) to provide a synergistic ability to mask the IFNAR.

[0353] The resulting embodiments, such as the targeted masked IFN provide a unique advantage over the prior approaches for several reasons. First, the IFN is masked so that its activity is significantly reduced and / or eliminated until it reaches the tumor. At which time, the mask is removed, and the activity is re-activated, which maximizes the efficacy in the tumor. Second, by attaching the C-terminal linked masked IFN to the C-terminal of the antibody the masked IFN can be targeted to a specific TAA. The specific targeting allows for greater opportunity that the IFN will be directed to the cancer of interest and avoid normal tissue. Third, since the masking sequence is derived from the extracellular sequence of a human protein (IFNAR2), they have a lower potential to generate an immunogenic response Fourth, the masks provided here offer potential to mask a broader range of type I interferons and with higher affinity may provide more effective masking.

[0354] The disclosure contemplates general embodiments of the resulting Targeted Masked IFN, including but not limited to the following:

[0355] First, a composition as shown herein: (i) Antibody-linker-cytokine (e.g., IFN)-linker-Protease cleavage (PC) site-iinker-mask; and Second, a composition as shown herein:

[0356] (ii) Antibody-linker-PC cleavage site-linker-cytokine (e.g., IFN)

[0357] It will be appreciated that one of skill in the art will appreciate and understand that the composition set forth in (ii) has an additional property of the antibody acting as a partial mask through steric hindrance of the cytokine presented in the composition.

[0358] In some embodiments, the protease cleavage site is a tumor-associated protease cleavage site. A "tumor-associated protease cleavage site" as provided herein is an amino acid sequence recognized by a protease, whose expression is specific for a tumor cell or tumor cell environment thereof. In some embodiments, exemplary protease cleavage site include a tumor-associated protease cleave site, such as a matrix metalloprotease (MMP) cleavage site, a disintegrin and metalloprotease domain-containing (ADAM) metal loprotease cleavage site, a prostate specific antigen (PSA) protease cleavage site, a urokinase-type plasminogen activator (uPA) protease cleavage site, a membrane type serine protease 1 (MT-SP1) protease cleavage site, a atriptase protease cleavage site (ST14) or a legumain protease cleavage site. In some aspects protease cleavage sites may be designated by a specific amino acid sequence. In one aspect of the disclosure the protease cleavage site is LSGRSDNH (SEQ ID NO: 15). In one aspect of the disclosure the protease cleavage site is KQSRWNH (SEQ ID NO: 16).

[0359] V.) T reatment of Cancer(s) Expressing a T umor Associates Antigen (TAA)

[0360] Also provided herein are fusion proteins, such as targeted masked IFNs, or compositions, which are useful in a variety of therapeutic, diagnostic, and prophylactic methods and uses. For example, the fusion proteins and compositions are useful in treating a variety of diseases and disorders in a subject, such as a cancer or a tumor. Such methods and uses include therapeutic methods and use, for example, involving administration of the fusion protein or compositions, to a subject having a disease or disorder, such as a tumor or a cancer. In some embodiments, the fusion protein or compositions are administered in an effective amount to effect treatment of the disease or disorder. Uses include uses of the fusion proteins or compositions in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. In some embodiments, the fusion proteins or compositions are for use in treating a variety of diseases and disorders in a subject, for example, in accordance with the therapeutic methods. In some embodiments, the methods are carried out by administering the fusion proteins or compositions, to the subject having or suspected of having the disease or disorder, such as a tumor or a cancer. In some embodiments, the methods thereby treat the disease or disorder in the subject. In some aspects, the provided for fusion proteins, e.g., targeted masked IFNs, are employed in methods, or uses for treatment of a disease or disorder such as a tumor or a cancer, including those expressing a tumor-associated antigen (TAA). The identification of a TAA of the disclosure as a protein that is normally expressed in a restricted set of tissues or cells, but which is also expressed in cancers, for example, solid tumor cancer, opens a number of therapeutic approaches to the treatment of such cancers utilizing masked fusion proteins disclosed herein.

[0361] Of note, targeted antitumor therapies have been useful even when the targeted protein is expressed on normal tissues or cells, even vital normal organ tissues. A vital organ is one that is necessary to sustain life, such as the heart or colon. A non-vital organ is one that can be removed whereupon the individual is still able to survive. Examples of non-vital organs are ovary, breast, and prostate.

[0362] Expression of a target protein in normal tissue, even vital normal tissue, does not defeat the utility of a targeting agent for the protein as a therapeutic for certain tumors in which the protein is also overexpressed. For example, expression in vital organs is not in and of itself detrimental. In addition, organs regarded as dispensable, such as the prostate and ovary, can be removed without affecting mortality Finally, some vital organs are not affected by normal organ expression because of an immunoprivilege. Immunoprivileged organs are organs that are protected from blood by a blood-organ barrier and thus are not accessible to immunotherapy. Examples of immunoprivileged organs are the brain and testis.

[0363] Accordingly, therapeutic approaches that inhibit the activity of a TAA protein, comprising a targeted masked IFN fusion proteins of the invention are useful for patients suffering from a cancer that expresses a TAA (such as, for example, solid tumor cancers in the lung, kidney, prostate, ovary, breast, and other types of cancer known in the art). The therapeutic approach involves IFNA induced killing (e.g., when the “mask” is removed and the IFN is reactivated in the tumor of interest), ADCC, CDC, and / or immune modulation. In addition, the antibody which binds the TAA may also synergistically modulate the function of a cancer cell. In addition, further, the “unmasked” or activated IFN can modulate activity of immune cells involved in anti-tumor or anti-cancer immunity, by virtue of binding of the IFN to the interferon receptor (e.g., IFNAR). The modulation of the antibody which binds the TAA generally falls into two classes. The first class modulates TAA function as it relates to tumor cell growth leading to inhibition or retardation of tumor cell growth or inducing its killing. The second class comprises various methods for inhibiting the binding or association of a TAA protein with its binding partner or with other proteins.

[0364] Accordingly, Cancer patients can be evaluated for the presence and level of TAA expression, preferably using immunohistochemical assessments of tumor tissue, quantitative TAA imaging, or other techniques that reliably indicate the presence and degree of TAA expression. Immunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this purpose, if applicable. Methods for immunohistochemical analysis of tumor tissues are well known in the art.

[0365] VI.) Targeted Masked IFN Fusion Protein Cocktails

[0366] Therapeutic methods of the invention contemplate the administration of single targeted masked IFN fusion protein as well as combinations, or cocktails, of different MAbs (i.e., naked MAbs that bind the same TAA as the masked IFN fusion protein or MAbs that bind another protein or another targeted masked IFN fusion protein which binds another TAA altogether). Such MAb cocktails can have certain advantages inasmuch as they contain MAbs that target different epitopes, exploit different effector mechanisms, or combine directly cytotoxic MAbs with MAbs that rely on immune effector functionality. Such MAbs in combination can exhibit synergistic therapeutic effects. In addition, targeted masked IFN fusion proteins can be administered concomitantly with other therapeutic modalities, including but not limited to various chemotherapeutic and biologic agents, androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery or radiation. In a preferred embodiment, the targeted masked IFNs are administered in fusion protein form.

[0367] Targeted masked IFN fusion protein formulations are administered via any route capable of delivering the antibodies to a tumor cell. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumor, intradermal, and the like. Treatment generally involves repeated administration of the targeted masked IFN fusion protein preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range, including but not limited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg / kg body weight, In general, doses in the range of 10-1000 mg targeted masked IFN fusion protein per week are effective and well tolerated.

[0368] Based on clinical experience with the Herceptin® (Trastuzumab) in the treatment of metastatic breast cancer, an initial loading dose of approximately 4 mg / kg patient body weight IV, followed by weekly doses of about 2 mg / kg IV of the MAb preparation represents an acceptable dosing regimen. Preferably, the initial loading dose is administered as a 90-minute or longer infusion. The periodic maintenance dose is administered as a 30 minute or longer infusion, provided the initial dose was well tolerated. As appreciated by those of skill in the art, a range of factors can influence the ideal dose regimen in a particular case. Such factors include, for example, the binding affinity and half-life of the TAA MAbs used, the degree of TAA expression in the patient, the extent of circulating shed TAA antigen, the desired steady-state antibody concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention (i.e., targeted masked IFN), as well as the health status of a particular patient. Optionally, patients should be evaluated for the levels of TAA in a given sample (e.g,, the levels of circulating TAA and / or TAA expressing ceils) in order to assist in the determination of the most effective dosing regimen, etc. Such evaluations are also used for monitoring purposes throughout therapy and are useful to gauge therapeutic success in combination with the evaluation of other parameters (for example, urine cytology and / or ImmunoCyt levels in bladder cancer therapy, or by analogy, serum PSA levels in prostate cancer therapy).

[0369] An object of the present invention is to provide targeted masked IFN fusion proteins, which inhibit or retard the growth of tumor cells expressing a specific TAA to which the fusion protein binds. A further object of this invention is to provide methods to inhibit angiogenesis and other biological functions and thereby reduce tumor growth in mammals, preferably humans, using such targeted masked IFN comprising fusion proteins which bind TAAs, and in particular using such targeted masked IFN comprising fusion proteins which find a specific TAA combined with other drugs or immunologically active treatments.

[0370] VII.) Combination Therapy

[0371] In some embodiments, also provided are methods and uses that involve a combination therapy, for example, which involves the use of any of the provided for fusion proteins or compositions, and an additional therapeutic agent, such as a chemotherapeutic agent or radiation. In some embodiments, the provided fusion proteins or compositions can be used in combination with an additional therapeutic agent for treatment of the disease or disorder, such as an anti-cancer or anti-tumor agent.

[0372] In some embodiments, there is synergy when tumors, including human tumors, are treated with targeted masked IFN fusion proteins which bind a specific TAA in conjunction with an additional therapeutic agent, such as, chemotherapeutic agents, radiation, an immunomoduiating therapy, or any combinations thereof. In other words, the inhibition of tumor growth by a targeted masked IFN fusion protein which binds a specific TAA is enhanced more than expected when combined with chemotherapeutic agents or radiation or combinations thereof. Synergy may be shown, for example, by greater inhibition of tumor growth with combined treatment than would be expected from a treatment of only targeted masked IFN fusion proteins which bind a specific TAA or the additive effect of treatment with a targeted masked IFN fusion proteins which bind a specific TAA and a chemotherapeutic agent or radiation Preferably, synergy is demonstrated by remission of the cancer where remission is not expected from treatment either from a targeted masked IFN fusion proteins which bind a specific TAA or with treatment using an additive combination of a targeted masked IFN fusion proteins which bind a specific TAA and a chemotherapeutic agent or radiation.

[0373] The method for inhibiting growth of tumor cells using a targeted masked IFN fusion proteins which bind a specific TAA and a combination of chemotherapy, radiation, or immunomoduiating therapies, or a combination of any one, two, or three comprises administering the targeted masked IFN fusion proteins which bind a specific TAA before, during, or after commencing chemotherapy or radiation therapy, as well as any combination thereof (i.e. before and during, before and after, during and after, or before, during, and after commencing the chemotherapy and / or radiation therapy). For example, the targeted masked IFN fusion proteins which bind a specific TAA is typically administered between 1 and 60 days, preferably between 3 and 40 days, more preferably between 5 and 12 days before commencing radiation therapy and / or chemotherapy. However, depending on the treatment protocol and the specific patient needs, the method is performed in a manner that will provide the most efficacious treatment and ultimately prolong the life of the patient.

[0374] The administration of chemotherapeutic agents can be accomplished in a variety of ways including systemically by the parenteral and enteral routes. In one embodiment, the targeted masked IFN fusion proteins which bind a specific TAA, and the chemotherapeutic agent are administered as separate molecules, Particular examples of chemotherapeutic agents or chemotherapy include bortezomib, carfilzomib, lenalidomide, pomalidomide, cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere), aldesleukin, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon 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.

[0375] The source of radiation, used in combination with a targeted masked IFN fusion proteins which bind a specific TAA, can be either external or internal to the patient being treated. When the source is external to the patient, the therapy is known as external beam radiation therapy (EBRT). When the source of radiation is internal to the patient, the treatment is called brachytherapy (BT).

[0376] The above-described therapeutic regimens may be further combined with additional cancer treating agents and / or regimes, for example, bortezomib, pomalidomide, and / or additional chemotherapy, cancer vaccines, signal transduction inhibitors, agents useful in treating abnormal cell growth or cancer, antibodies (e.g., Anti-CTLA-4 antibodies as described in WO / 2005 / 092380 (Pfizer)) or other ligands that inhibit tumor growth by binding to IGF-1 R, and cytokines.

[0377] Examples of immunomodulating 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.

[0378] When the mammal is subjected to additional chemotherapy, chemotherapeutic agents described above may be used. Additionally, growth factor inhibitors, biological response modifiers, anti- hormonal therapy, selective estrogen receptor modulators (SERMs), angiogenesis inhibitors, and anti¬ androgens may be used. For example, anti-hormones, for example anti-estrogens such as Nolvadex (tamoxifen) or, anti-androgens such as Casodex (4'-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3-'-(trifluoromethyl)propionanilide) may be used.

[0379] The above therapeutic approaches can be combined with any one of a wide variety of surgical, chemotherapy or radiation therapy regimens. The therapeutic approaches of the invention can enable the use of reduced dosages of chemotherapy (or other therapies) and / or less frequent administration, an advantage for all patients and particularly for those that do not tolerate the toxicity of the chemotherapeutic agent well.

[0380] VIII.) Kits / Articles of Manufacture

[0381] For use in the laboratory, prognostic, prophylactic, diagnostic, and therapeutic applications described herein, kits, article of manufacture, systems, and apparatuses are within the scope of the invention. Such kits can comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method, along with a label or insert comprising instructions for use, such as a use described herein. For example, the container(s) can comprise a targeted masked IFN fusion protein which bind a specific TAA or several TAAs of the disclosure. Kits can comprise a container comprising a targeted masked IFN. The kit can include ail or part of the targeted masked IFN fusion protein which binds a specific TAA and / or diagnostic assays for detecting cancer and / or other immunological disorders.

[0382] The kit of the invention will typically comprise the container described above, and one or more other containers associated therewith that comprise materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes; carrier, package, container, vial and / or tube labels listing contents and / or instructions for use, and package inserts with instructions for use.

[0383] A label can be present on or with the container to indicate that the composition is used for a specific therapy or non -therapeutic application, such as a prognostic, prophylactic, diagnostic, or laboratory application, and can also indicate directions for either in vivo or in vitro use, such as those described herein. Directions and / or other information can also be included on an insert(s) or label(s) which is included with or on the kit. The label can be on or associated with the container A label can be on a container when letters, numbers or other characters forming the label are molded or etched into the container itself; a label can be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. The label can indicate that the composition is used for diagnosing, treating, prophylaxing, or prognosing a condition, such as a cancer or other immunological disorder. The terms “kit” and “article of manufacture” can be used as synonyms.

[0384] In another embodiment of the invention, an article(s) of manufacture containing compositions, such as targeted masked IFN fusion protein which binds a specific TAA of the disclosure. The article of manufacture typically comprises at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass, metal, or plastic. The container can hold one or several targeted masked IFN fusion proteins which bind a specific TAAs and / or one or more therapeutics doses of targeted masked IFNs,

[0385] The container can alternatively hold a composition that is effective for treating, diagnosis, prognosing or prophylaxing a condition and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agents in the composition can be a targeted masked IFN fusion protein which bind a specific TAA of the present disclosure.

[0386] The article of manufacture can further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and / or dextrose solution. It can 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.

[0387] EXEMPLARY EMBODIMENTS

[0388] 1) A composition, comprising ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYTIMSKPEDLKWKNCANTT RSFCDLTDEWRSTHEAYVTVLEGFSGNTTLFSCSHNFWLAIDMS (SEQ ID NO.: 36), wherein said composition masks the activity of a Type-1 interferon and wherein said composition further comprises a fusion protein which is fused to an antibody that binds to a Tumor Associated Antigen (“TAA”).

[0389] 2) A composition, comprising ISYDSPDYTDESCTFKISLRNFRSILSWELKNHLIVPTHYTLLYTIMSKPEDLKVVKNCANTK RSFCDLTDEWRSTHEAYVTVLEGFSGNTLLFSCSHNFWLAIDMS (SEQ ID NO.: 37), wherein said composition masks the activity of a Type-1 interferon and wherein said composition further comprises a fusion protein which is fused to an antibody that binds to a tumor associated antigen.

[0390] 3) The composition of any of claims 1-2, further comprising a flexible peptide linker.

[0391] 4) The composition of claim 3, further comprising a tumor associated protease cleavage site.

[0392] 5) The composition of any of claims 1-2, wherein the Type-1 interferon comprises IFNcd, 6) The composition of any of claims 1-2, wherein the Type-1 interferon comprises IFNa2. 7) The composition of any of claims 1-2, wherein the Type-1 interferon comprises IFNo4. 8) The composition of any of claims 1-2, wherein the Type-1 interferon comprises IFNa5. 9) The composition of any of claims 1-2, wherein the Type-1 interferon comprises IFNaS. 10) The composition of any of claims 1-2, wherein the Type-1 interferon comprises IFNa14.

[0393] 11) The composition of any of claims 1-2, wherein the Type-1 interferon comprises IFN|31. 12) The composition of any of claims 1-2, wherein the Type-1 interferon or functionally active mutant is selected from a Type-1 interferon as shown in Table il,

[0394] 13) The composition of any of claims 1-2, wherein the TAA comprises CD138.

[0395] 14) The composition of any of claims 1-2, wherein the TAA comprises Mesothelin.

[0396] 15) The composition of any of claims 1-2, wherein the TAA, comprises PSCA.

[0397] 16) The composition of any of claims 1-2, wherein the TAA, comprises CD20.

[0398] 17) The composition of any of claims 1-2, wherein the TAA is selected from a tumor associated antigen as shown in Table I.

[0399] 18) A “Targeted Masked IFN” comprising:

[0400] a. an antibody which specifically binds to a tumor associated antigen;

[0401] b. a type-1 interferon, wherein the N-terminal of said Type-1 interferon is fused to the C-terminal of said antibody heavy and / or light chains; and

[0402] c. c. an interferon mask which comprises (SEQ ID NO: 36) whereby said interferon mask is attached at the C-terminal of said type-1 interferon.

[0403] 19) A “Targeted Masked IFN” comprising:

[0404] a. an antibody which specifically binds to a tumor associated antigen;

[0405] b. a type-1 interferon, wherein the N-terminal of said Type-1 interferon is fused to the C-terminal of said antibody heavy and / or light chains; and

[0406] c. an interferon mask which comprises (SEQ ID NO: 37) whereby said interferon mask is attached at the C-terminal of said type-1 interferon.

[0407] 20) The “Targeted Masked IFN" of any of claims 18-19, wherein the N-terminal of said Type-1 interferon is fused to the C-terminal of said antibody heavy and / or light chain further comprising a flexible peptide linker.

[0408] 21) The “Targeted Masked IFN” of any of claims 18-19, wherein the interferon mask is attached to the C-terminal of said Type-1 interferon further comprising a flexible peptide linker.

[0409] 22) The “Targeted Masked IFN” of claim 20, further comprising a tumor associated protease cleavage site inserted between said antibody and said flexible peptide linker.

[0410] 23) The " Targeted Masked IFN of claim 21, further comprising a tumor associated protease cleavage site inserted between said IFN and said IFN mask. 24) The Targeted Masked Interferon of any of ciaim(s) 18-23, wherein said antibody binds a tumor associated antigen set forth in Table I.

[0411] 25) The Targeted Masked Interferon of any of claim(s) 18-23, wherein said type-1 interferon or functionally active mutant is set forth in Table II.

[0412] 26) The Targeted Masked Interferon of any of ciaim(s) 18-23, wherein the functionally active mutant is a YNS mutant.

[0413] 27) The Targeted Masked Interferon of any of claim(s) 18-26, wherein said antibody binds CD138.

[0414] 28) The Targeted Masked Interferon of any of claim(s) 18-26, wherein said antibody binds Mesothelia.

[0415] 29) A method of making a Targeted Masked Interferon of any of claim(s) 18-28.

[0416] 30) A pharmaceutical composition comprising a therapeutically effective amount of the Targeted Masked I FN of any of claim(s) 18-28, (i) wherein, optionally, the pharmaceutical composition is for use in therapy including treatment of cancer, wherein, optionally, (a) the cancer comprises a cancer found in a solid tumor; or (b) the cancer arises in the hematopoietic system, and (ii) wherein, optionally, the pharmaceutical composition further comprises one or more anti-neoplastic agents;

[0417] 31) A kit comprising a Targeted Masked IFN of any ofclaim(s) 18-28.

[0418] 32) A method of treating cancer in a subject, comprising administering to said subject a therapeutically effective amount of the Targeted Masked IFN according to claim(s) 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 wherein, optionally, the subject is a human subject,

[0419] 33) A composition, comprising ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYTIMSKPEDLKWKNCANTT RSFCDLTDEWRSTHEAYVTVLEGFSGNTTLFSCSHNFWLAIDMS (SEQ ID NO.: 36), wherein said composition masks the activity of a Type-1 interferon and wherein said composition further comprises a fusion protein comprising a peptide linker (SEQ ID NO: 5) which is fused to an antibody that binds to CD138 and wherein the antibody comprises the heavy chain set forth in (SEQ ID NO: 6) and further comprises the light chain set forth in (SEQ ID NO: 8).

[0420] 34) The composition of any of claim 33, further comprising a flexible peptide linker set forth as GSSGKQSRWNHGSSGGSGGSGGS (SEQ ID NO: 35), wherein said flexible peptide linker further comprises a cleavable linker set forth as KQSRWNH (SEQ ID NO: 16). 35) The composition of any of claim 33, where in the antibody comprises a variable heavy chain set forth in (SEQ ID NO: 7). 36) The composition of any of claim 33, where in the antibody comprises a variable light chain set forth in (SEQ ID NO: 9).

[0421] 37) The composition of any of claims 33-36, wherein the Type-1 interferon comprises IFNal.

[0422] 38) The composition of any of claims 33-36, wherein the Type-1 interferon comprises IFNo2, 39) The composition of any of claims 33-36, wherein the Type-1 interferon comprises IFNo4.

[0423] 40) The composition of any of claims 33-36, wherein the Type-1 interferon comprises IFNo5.

[0424] 41) The composition of any of claims 33-36, wherein the Type-1 interferon comprises IFNo6.

[0425] 42) The composition of any of claims 33-36, wherein the Type-1 interferon comprises IFNal 4.

[0426] 43) The composition of any of claims 33-36, wherein the Type-1 interferon comprises IFNpi.

[0427] 44) The composition of any of claims 33-36, wherein the Type-1 interferon or functionally active mutant is selected from a Type-1 interferon as shown in Table II.

[0428] 45) The composition of any of claims 33-36, wherein the Type-1 interferon comprises a YNS mutation.

[0429] 46) A “Targeted Masked IFN” comprising:

[0430] a. an antibody which specifically binds to a CD138 antigen, wherein said antibody comprises a variable heavy chain set forth in (SEQ ID NO: 7) and a variable light chain set forth in (SEQ ID NO: 9);

[0431] b. a type-1 interferon, wherein the N-terminal of said Type-1 interferon is fused to the C- terminal of said antibody heavy and / or light chains; and

[0432] c. an interferon mask which comprises (SEQ ID NO: 36) whereby said interferon mask is attached at the C-terminal of said type-1 interferon.

[0433] 47) The “Targeted Masked IFN” of claim 46, wherein the N-terminal of said Type-1 interferon is fused to the C-terminal of said antibody heavy and / or light chain further comprising a flexible peptide linker set forth in (SEQ ID NO: 35).

[0434] 48) The " Targeted Masked IFN” of claim 46, wherein the N-terminal of said Type-1 interferon is fused to the C-terminal of said antibody heavy and / or light chain further comprising a flexible peptide linker set forth in (SEQ ID NO: 47).

[0435] 49) The “Targeted Masked IFN” of claim 47, wherein the interferon mask is attached to the C- terminal of said Type-1 interferon further comprising a flexible peptide linker.

[0436] 50) The “Targeted Masked IFN” of claim 47, further comprising a tumor associated protease cleavage site inserted between said antibody and said flexible peptide linker.

[0437] 51) The “Targeted Masked IFN of claim 47, further comprising a tumor associated protease cleavage site inserted between said IFN and said IFN mask.

[0438] 52) The Targeted Masked Interferon of claim 47, wherein said type-1 interferon or functionally active mutant is set forth in Table II. 53) The Targeted Masked IFN of claim 47, wherein the functionally active mutant is a YNS mutant.

[0439] 54) The Targeted Masked Interferon of claim 47, wherein said antibody binds CD138.

[0440] 55) A method of making a Targeted Masked Interferon of any of claim(s) 47,

[0441] 56) The “Targeted Masked IFN” of claim 48, wherein the interferon mask is attached to the C-terminal of said Type-1 interferon further comprising a flexible peptide linker.

[0442] 57) The " Targeted Masked IFN” of claim 48, further comprising a tumor associated protease cleavage site inserted between said antibody and said flexible peptide linker,

[0443] 58) The “Targeted Masked IFN of claim 48. further comprising a tumor associated protease cleavage site inserted between said IFN and said IFN mask.

[0444] 56) The Targeted Masked Interferon of claim 48, wherein said type-1 interferon or functionally active mutant is set forth in Table II.

[0445] 57) The Targeted Masked IFN of claim 48, wherein the functionally active mutant is a YNS mutant.

[0446] 58) The Targeted Masked Interferon of claim 48, wherein said antibody binds GDI 38.

[0447] 59) A method of making a Targeted Masked Interferon of any of claim(s) 48.

[0448] 60) A pharmaceutical composition comprising a therapeutically effective amount of the Targeted Masked IFN of any of claim(s) 46-59, (i) wherein, optionally, the pharmaceutical composition is for use in therapy including treatment of cancer, wherein, optionally, (a) the cancer comprises a cancer found in a solid tumor; or (b) the cancer arises in the hematopoietic system, and (ii) wherein, optionally, the pharmaceutical composition further comprises one or more anti-neoplastic agents,

[0449] 61) A kit comprising a Targeted Masked IFN of any of claim(s) 60.

[0450] 62) A method of treating cancer in a subject, comprising administering to said subject a therapeutically effective amount of the Targeted Masked IFN according to claim(s) 60, wherein, optionally, the subject is a human subject.

[0451] 63) A composition comprising the sequence set forth in Figure 41 (SEQ ID NO: 38).

[0452] 64) A composition comprising the sequence set forth in Figure 42 (SEQ ID NO: 39).

[0453] 65) A composition comprising the sequence set forth in Figure 43 (SEQ ID NO: 40).

[0454] 66) A composition comprising the sequence set forth in Figure 44 (SEQ ID NO: 41).

[0455] 67) A composition comprising the sequence set forth in Figure 45 (SEQ ID NO: 42).

[0456] 68) A composition comprising the sequence set forth in Figure 46 (SEQ ID NO: 43).

[0457] 69) A composition comprising the sequence set forth in Figure 47 (SEQ ID NO: 44).

[0458] 70) A kit comprising the composition of any of daim(s) 63-69. 71) A method of treating cancer in a subject, comprising administering to said subject a therapeutically effective amount of the composition according to any of claim(s) 63-69, wherein, optionally, the subject is a human subject.

[0459] 72) A “Targeted Masked IFN” comprising:

[0460] a. an antibody which specifically binds to a Mesothelin antigen, wherein said antibody comprises a heavy chain variable region (SEQ ID NO: 53) and a heavy chain constant region (SEQ ID NO: 52), and a variable light chain set forth in (SEQ ID NO: 20); b. a type-1 interferon, wherein the N-terminal of said Type-1 interferon is fused to the C- terminal of said antibody heavy and / or light chains; and

[0461] c. an interferon mask which comprises (SEQ ID NO: 36) whereby said interferon mask is attached at the C-terminal of said type-1 interferon.

[0462] 73) The “Targeted Masked IFN” of claim 72, wherein the N-terminal of said Type-1 interferon is fused to the C-terminal of said antibody heavy and / or light chain further comprising a flexible peptide linker set forth in (SEQ ID NO: 35).

[0463] 74) The " Targeted Masked IFN” of claim 73, wherein the interferon mask is attached to the C-terminal of said Type-1 interferon further comprising a flexible peptide linker.

[0464] 75) The “Targeted Masked IFN” of claim 72, further comprising a tumor associated protease cleavage site inserted between said antibody and said flexible peptide linker.

[0465] 76) The “Targeted Masked IFN of claim 72. further comprising a tumor associated protease cleavage site inserted between said IFN and said IFN mask.

[0466] 77) The Targeted Masked Interferon of claim 72, wherein said type-1 interferon or functionally active mutant is set forth in Table II.

[0467] 78) The Targeted Masked IFN of claim 72, wherein the functionally active mutant is a YNS mutant

[0468] 79) The Targeted Masked Interferon of claim 72, wherein said antibody binds mesothelin. 80) A method of making a Targeted Masked Interferon of any of claim(s) 72.

[0469] 81) A pharmaceutical composition comprising a therapeutically effective amount of the Targeted Masked IFN of any of claim(s) 72-80, (i) wherein, optionally, the pharmaceutical composition is for use in therapy including treatment of cancer, wherein, optionally, (a) the cancer comprises a cancer found in a solid tumor; or (b) the cancer arises in the hematopoietic system, and (ii) wherein, optionally, the pharmaceutical composition further comprises one or more anti-neoplastic agents;

[0470] 82) A kit comprising a Targeted Masked IFN of any of claim(s) 81. 83) A method of treating cancer in a subject, comprising administering to said subject a therapeutically effective amount of the Targeted Masked IFN according to claim(s) 81, wherein, optionally, the subject is a human subject.

[0471] 84) A composition comprising the sequence set forth in Figure 48 (SEQ ID NO: 45).

[0472] 85) A composition comprising the sequence set forth in Figure 49 (SEQ ID NO: 46).

[0473] 86) A composition comprising the sequence set forth in Figure 58 (SEQ ID NO: 54).

[0474] 87) A composition comprising the sequence set forth in Figure 58 (SEQ ID NO: 55).

[0475] 88) A composition comprising the CDR sequences set forth in (SEQ ID NO: 56), (SEQ ID NO:

[0476] 57), and (SEQ ID NO: 58).

[0477] 89) A composition comprising the CDR sequences set forth in (SEQ ID NO: 59), (SEQ ID NO:

[0478] 60), and (SEQ ID NO: 61).

[0479] 90) A kit comprising the composition of claim(s) 84, 85, 86, 87, 88, or 89.

[0480] 91) A method of treating cancer in a subject, comprising administering to said subject a therapeutically effective amount of the composition according to claims 84, 85, 86, 87, 88, or 89, wherein, optionally, the subject is a human subject.

[0481] EXAMPLES:

[0482] Various aspects of the invention are further described and illustrated by way of the several examples that follow, none of which is intended to limit the scope of the invention.

[0483] Example 1: Characterization and Peptide Binding to anti-CD138 IFNaYNS.

[0484] In this example, several masking peptides were evaluated for binding affinity. Briefly, Immulon 2 HB plates (Thermofisher) were coated with 10 pg / mL anti-CD138 IFNaYNSfusion protein overnight at 4 °C and blocked with 2% BSA (Fisher) for a minimum of 2 hours at room temperature. Weils were washed three (3) times with PBS + 0.05% Tween (Sigma). Indicated peptide (Thermofisher) concentrations were overlayed overnight at 4 °C. Wells were washed 3 times with PBS + 0,05% Tween. Bound peptides were detected with Streptavidin- HRP (Pierce) diluted 1:5000 in PBS + 1% BSA. After wells were washed three (3) times with PBS + 0.05% Tween. HRP substrate TMB (Fisher) was added to the wells. Reaction was terminated by addition of 0.1 M sulfuric acid (Fisher) and absorbance changes were detected at 450 nm using a Biotek EPOCH ELISA reader.

[0485] The resulting analysis shows that Peptide(s) 22 and 23 bind the strongest relative to Peptide 21 and 22 to the immobilized fusion protein. (See, Figure 6).

[0486] Example 2: Methods of Binding Fusion Proteins to Masking Peptides. In this example, it is shown that a plurality of Fusion Abs specifically binds to additional peptide masks of the disclosure, For reference, the peptide sequences of PEP1, PEP21, PEP22, PEP23, and PEP24 are set forth in Figure 3.

[0487] Briefly, Immulon 2 HB plates (Thermofisher) were coated with 10 ug / mL anti-CD138 IFNaYNSfusion protein overnight at 4 deg. C and blocked with 2% BSA (Fisher) for a minimum of 2 hrs. at RT. Wells were washed 3x with PBS +0.05% Tween (Sigma). Indicated peptide (Thermofisher) concentrations were overlayed O / N at 4 deg. C. Wells were washed 3x with PBS + 0.05% Tween. Bound peptides were detected with Streptavidin-HRP (Pierce) diluted 1:5000 in PBS + 1% BSA. After wells were washed 3x with PBS + 0.05% Tween, HRP substrate TMB (Fisher) was added to the wells. Reaction was terminated by addition of 0.1 M sulfuric acid (Fisher) and absorbance changes were assayed at 450 nm using a Biotek EPOCH ELISA reader.

[0488] The results show that free fusion protein is able to compete off binding of Peptide 22 and Peptide 23 to immobilized fusion protein in a dose dependent manner. (See, Figure 7).

[0489] Example 3: Methods of Binding Fusion Proteins to Masking Peptides.

[0490] In this example, it is shown that a plurality of Fusion Abs specifically binds to additional peptide masks of the disclosure, For reference, the peptide sequences of PEP22 and PEP23 are set forth in Figure 3.

[0491] Briefly, Streptavidin coated plates (Pierce) were overlayed with 50 pM of each indicated peptide (Thermofisher) for a minimum of 2 hours at room temperature. Wells were washed three (3) times with PBS + 0.05% Tween, Abs at the indicated concentrations were then allowed to bind overnight at 4 °C. Wells were washed 3x with PBS + 0.05% Tween, Bound Abs were detected with anti-human Kappa-AP (Southern Biotech) diluted 1:3000 in PBS + 1% BSA. Absorbance changes after the addition of AP substrate (Sigma) were detected at 410 nm using a Biotek EPOCH ELISA reader.

[0492] The results show that free Fusion Protein is able to bind to immobilized PEP23, but not PEP 21 or PEP22. (See, Figure 8).

[0493] Example 4: Matriptase Treatment of Fusion Proteins.

[0494] In this example, it is shown that a mask on the YNS mutant can be cleaved with MST. Briefly, for samples treated with MST14 (R& D Systems), 50 pg of Ab was incubated with 0.5 pg MST14 for 1 hour at 37 °C. 1 pg of each purified Ab was denatured by heating to 95 °C, reduced with '2% betamercaptoethanol (Thermofisher), and was run on 4-12% Bis-Tris SDS-PAGE gels (Invitrogen). Gel was stained with EZ Stain (Fisher) according to the manufacturer’s protocol The results show that The mask on the masked YNS mutant can be cleaved with MST. MST treatment also results in partial cleavage of the IFN / mask moiety as observed by the increase in a band the size of unfused Ab. (See, Figure 9).

[0495] Example 5: Methods of Reducing and Restoring masked IFNa Activity.

[0496] In this example, further studies were performed to show that fusion proteins of the disclosure (QXL138YNS, QXL138YNSM1.2-N297Q, and QXL138YNS 1.2-N297Q w / MST) can reduce and restore IFNa activity.

[0497] Briefly, HEK Blue IFNa / b cells (Invivogen) were seeded into 96-well tissue culture plates (Fisher) at a density of 1x10e4 cells / well (100 uL / well). 100 uL / well of recombinant IFNa (Novus Biologicals) or indicated Abs were incubated with the cells at concentrations sufficient to generate the final indicated concentrations O / N at 37 deg. C. Abs cleaved with MST14 (R& D Systems) were prepared by incubating 50 ug of Ab with 0,5 ug of MST 14 for 1 hr. At 37 deg. C, 20 uL of supernatant was then added to a plate containing 180 uL / well Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader.

[0498] The results show the YNS mutant is active, but the mask (based on PEP23) on the YNS mutant did appear to be active. (See, Figure 10).

[0499] Example 6: Methods of Reducing and Restoring masked IFNa Activity.

[0500] In this example, further studies were performed to show that fusion proteins of the disclosure (QXL138YNS and QXL138YNSM2.2-N297Q) can reduce and restore IFNa activity.

[0501] Briefly, HEK Blue IFNa / b cells (Invivogen) were seeded into 96-well tissue culture plates (Fisher) at a density of 1x10e4 cells / well (100 uL / well). 100 uL / well of recombinant IFNa (Novus Biologicals) or indicated Abs were incubated with the cells at concentrations sufficient to generate the final indicated concentrations O / N at 37 deg. C. Abs cleaved with MST14 (R& D Systems) were prepared by incubating 50 ug of Ab with 0.5 ug of MST 14 for 1 hr. At 37 deg. C. 20 uL of supernatant was then added to a plate containing 180 uL / well Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader.

[0502] The results show that QXL138YNSM2.2-N297Q (based on a combination of PEP2 and PEP23) appears to show masking effects, with the EC50 ~3X higher than the never masked Fusion Protein. (See, Figure 11).

[0503] Example 7: Methods of Reducing and Restoring masked IFNa Activity. In this example, further studies were performed to show that fusion proteins of the disclosure (QXL138YNS, QXL138YNSM2.2-N297Q, and QXL138YNSM2.2-N297Q w / MST) can reduce and restore IFNa activity.

[0504] Briefly, HEK Blue IFNa / b cells (Invivogen) were seeded into 96-well tissue culture plates (Fisher) at a density of 1x10e4 cells / well (100 uL / well). 100 uL / well of recombinant IFNa (Novus Biologicals) or indicated Abs were incubated with the cells at concentrations sufficient to generate the final indicated concentrations O / N at 37 deg. C. Abs cleaved with MST14 (R& D Systems) were prepared by incubating 50 ug of Ab with 0.5 ug of MST14 for 1 hr, At 37 deg. C. 20 uL of supernatant was then added to a plate containing 180 uL / well Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader.

[0505] The results show that QXL138YNSM2.2-N297Q treated w / MST does not appear to restore the IFN activity, despite SDS-PAGE showing a majority of the mask has been cleaved. (See, Figure 12).

[0506] Example 8: Methods of Reducing and Restoring masked IFNa Activity.

[0507] In this example, further studies were performed to show that fusion proteins of the disclosure (QXL138YNS and QXL138YNSM2.2-N297Q) can reduce and restore IFNa activity.

[0508] Briefly, Immulon 2 HB plates (Thermofisher) were coated with 10 ug / mL IFNaR2 (R& D Systems) overnight at 4 deg. C and blocked with 2% BSA (Fisher) for a minimum of 2 hrs. at RT. Wells were washed 3x with PBS +0.05% Tween (Sigma). Indicated Ab concentrations were overlayed O / N at 4 deg. C. Wells were washed 3x with PBS + 0.05% Tween. Bound Abs were detected with anti-human Kappa-AP (Southern Biotech) diluted 1:3000 in PBS + 1% BSA. Absorbance changes after addition of AP substrate (Sigma) were assayed at 410 nm using a Biotek EPOCH ELISA reader,

[0509] The results show that QXL138YNSM2.2-N297Q appears to bind IFNAR2 less than QXL138YNS, with the EC50 ~ 15X higher than the never masked FP. (See, Figure 13).

[0510] Example 9: Methods of Reducing and Restoring masked IFNa Activity.

[0511] In this example, further studies were performed to show that fusion proteins of the disclosure (QXL138YNS and QXL138YNSM2.2-N297Q) can reduce and restore binding to IFNAR2.

[0512] Briefly, immulon 2 HB plates (Thermofisher) were coated with 10 g / mL IFN AR2 / Fc Chimera (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 3 times with PBS +0.05% Tween-20 (Sigma) immediately prior to use. Indicated Ab concentrations were overlayed overnight at 4 °C. Wells were washed 3 times with PBS +0.05% Tween-20, and bound Abs were detected with anti-human Kappa-AP (Southern Biotech) diluted 1:3000 in PBS + 1% BSA. Absorbance changes after the addition of AP substrate (Sigma) were detected at 410 nm using a Biotek EPOCH ELISA reader. The results show IFNAR2 binding with Kd of masked fusion proteins (QXL138YNSM2.2-N297Q (based on a combination of PEP2 and PEP23)) ~6X higher than demasked Fusion Protein. (See, Figure 14).

[0513] Example 10: Methods of Reducing and Restoring IP-10 Induction in PBMCs.

[0514] In this example, QXL138YNS and QXL138YNSM2.2-N297Q were assessed for the reduction and restoration of IP-10 induction.

[0515] Briefly, freshly thawed human PBMCs (HumanCells Biosciences) were washed once with cold RPMI + 10% FBS (Invitrogen) and seeded into a 12-well plate (Themofisher) at a density of -1x1 Oe3cells / well (1 mL / well). Any Fc receptor and / or CD138 antigen expression was blocked / reduced with addition of 1 uM human IgG (Fisher) to the cells for 1 hr. before proceeding with the experiment. Recombinant human I FNa (Novus Biologicals) or indicated Abs were added to the cells and allowed to incubate at 37 deg, C for an additional 7 hrs. Abs cleaved with MST14 (R& D Systems) were prepared by incubating 50 ug of Ab with 0.5 ug of MST14 for 1 hr. At 37 deg. C. After the seven (7) hr. incubation, cells were spun down for 3 min. at 500xg and 20 uL of supernatant from each sample was assayed for IP-10 (Abeam) by ELISA according to the manufacturers protocol.

[0516] The results show that comparing the levels of IP-10 induction produced by 0.01 nM QXL138YNS versus 1 nM QXL138YNSM2.2-N297Q, more than 100x masked FP is needed to achieve the same level of induction as never masked FP. (Figure 15).

[0517] Example 11: Methods of Reducing and Restoring masked IFNa Activity.

[0518] In this example, further studies were performed to show that fusion proteins of the disclosure (QXL138YNS-N297Q, QXL138YNSM1.2-N297Q, QXL138YNSM1.2-N297Q w / MST, QXL138YNSM2.2-N297Q, and QXL138YNSM2.2-N297Q w / MST) can reduce and restore IFNa activity.

[0519] Briefly, HEK Blue IFNa / b cells (Invivogen) were seeded into 96-weli tissue culture plates (Fisher) at a density of 1x10e4 cells / well (100 uL / well). 100 uL / well of recombinant IFNa (Novus Biologicals) or indicated Abs were incubated with the cells at concentrations sufficient to generate the final indicated concentrations O / N at 37 deg. C. Abs cleaved with MST14 (R& D Systems) were prepared by incubating 50 ug of Ab with 0.5 ug of MST 14 for 1 hr. At 37 deg. C. 20 uL of supernatant was then added to a plate containing 180 uL / well Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader.

[0520] The results show that QXL138YNSM1.2-N297Q (based on PEP23) does not appear to have meaningful masking activity compared to never masked fusion protein. QXL138YNS2.2-N297Q (based on PEP23 and PEP2) only shows modest masking activity compared to never masked fusion protein. (See, Figure 16).

[0521] Example 12: Methods of Reducing IP-10 Induction in PBMCs.

[0522] In this example, it is shown that a plurality of Masked Fusion Abs (anti-5T4 IFNa mask 1 and anti-mesothelin IFNa mask 1) of the disclosure can reduce IP-10 induction. Briefly, freshly thawed human PBMCs (Human Cells Biosciences) were washed once with cold RPM I + 10% FBS (Invitrogen) and seeded into a 12-well plate (Themofisher) at a density of ~1x10e6 cells / well (1 mL / well). Any Fc receptor and / or CD138 antigen expression was blocked / reduced with addition of 300 nM unfused anti-CD138 lgG1 to the ceils for one (1) hr. before proceeding with the described experiment. Then, recombinant human IFNa (Novus Biologicals) or indicated Abs (5T4 or mesothelin) were added to the cells and allowed to incubate at 37 deg. C for an additional seven (7) hrs. Abs cleaved with MST14 (R& D Systems) were prepared by incubating 50 ug of Ab with 0.5 ug of MST 14 for 1 hr. At 37 deg. C. After the seven (7) hr. incubation, cells were spun down for 3 min. at 500xg and 20 uL of supernatant from each sample and were assayed for IP-10 (Abeam) by ELISA according to the manufacturer's protocol.

[0523] The results show that the mask reduces IP-10 induction in both anti-5T4 and anti-mesothelin fusion Abs (See, Figure 17).

[0524] Example 13: Characterization of QXL138AM4.2-N297Q.

[0525] In this example, it is shown that QXL138AM4.2-N297Q can be cleaved from the H chain using Matripase ST 14 (“MST 14”). Briefly, for samples treated with MST14 (R& D Systems), 50 ug of Ab was incubated with 0.5 ug MST 14 for 1 hr. At 37 deg. C. 1 ug of each purified Ab was denatured by heating to 95 deg. C, reduced with ~2% beta-mercaptoethanol (Thermofisher), and run on 4-12% Bis-Tris SDS-PAGE gels (Invitrogen). Gel was stained with EZ Stain (Fisher) according to the manufacturer’s protocol.

[0526] The resulting analysis shows that Mask 4 on QXL138AM4.2-N297Q can be cleaved with MST. (See, Figure 18).

[0527] Example 14: Methods of Reducing and Restoring masked IFNa Activity.

[0528] In this example, further studies were performed to show that fusion proteins of the 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 IFNa activity. Briefly, HEK Blue IFNa / b cells (Invivogen) were seeded into 96-well tissue culture plates (Fisher) at a density of 1 x10e4 cells / well (100 uL / well). 100 uL / well of recombinant IFNa (Novus Biologicals) or indicated Abs were incubated with the cells at concentrations sufficient to generate the final indicated concentrations O / N at 37 deg. C. Abs cleaved with MST14 (R& D Systems) were prepared by incubating 50 ug of Ab with 0.5 ug of MST14 for 1 hr. At 37 deg. C. 20 uL of supernatant was then added to a plate containing 180 uL / well Quanti-Blue substrate (Invivogen). Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader.

[0529] The results show that the EC50 of QXL138AM4.2-N297Q (based on D1 loop of IFNAR2) appears to be ~50x to 100x higher than when the mask is removed indicating a significant masking effect compared to QXL138AM2.2-N297Q. Furthermore, the EC50 for QXL138AM4.2-N297Q is ~20x higher than the EC50 for QXL138AM2.2 indicating an improvement over Mask2.2. (See, Figure 19).

[0530] Example 15: Methods of Reducing and Restoring masked IFNa Activity.

[0531] in this example, further studies were performed to show that fusion proteins of the disclosure from multiple manufacturing lots (QXL138AM2.2-N297Q (Lot 10), QXL138AM2.2-N297Q (Lot 10) + MST, QXL138AM4.2-N297Q, and QXL138AM4.2-N297Q + MST can bind to IFNAR2.

[0532] Briefly, Immulon 2 HB plates (Thermofisher) were coated with 10 pg / mL IFNAR2 / Fc Chimera (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 3 times with PBS + 0.05% Tween-20 (Sigma) immediately prior to use. Indicated Ab concentrations were overlayed overnight at 4 °C. Wells were washed 3 times with PBS +0.05% Tween-20, and bound Abs were detected with anti-human Kappa-AP (Southern Biotech) diluted 1:3000 in PBS + 1% BSA, Absorbance changes after the addition of AP substrate (Sigma) were detected at 410 nm using a Biotek EPOCH ELISA reader.

[0533] The results show that the Kd of QXL138AM4.2-N297Q is ~9x higher than the Kd of QXL138AM2.2-N297Q when they are masked suggesting that Mask 4.2 performs better to prevent interaction with IFNAR2. When the masks are removed, the Kd of the FPs is restored to the level of unmasked FP (QXL138A-N297). (See, Figure 20).

[0534] Example 16: Methods of Reducing and Restoring IP-10 Induction in PBMCs.

[0535] in this example, QXL138A-N297Q, QXL138AM2.2-N297Q, QXL138AM2.2-N297Q+MST, QXL138AM4.2-N297Q, QXL138AM4.2-N297Q+MST were tested for the reduction and restoration of IP- 10 induction.

[0536] Briefly, freshly thawed human PBMCs (HumanCells Biosciences) were washed once with cold RPMI + 10% FBS (Invitrogen) and seeded into a 12-well plate (Themofisher) at a density of ~1x10e6 cells / well (1 mL / well), Any Fc receptor and / or CD138 antigen expression was blocked / reduced with addition of 1 uM human IgG (Fisher) to the cells for 1 hr, before proceeding with the experiment.

[0537] Recombinant human I FNa (Novus Biologicals) or indicated Abs were added to the cells and allowed to incubate at 37 deg. C for an additional 7 hrs. Abs cleaved with MST14 (R& D Systems) were prepared by incubating 50 ug of Ab with 0.5 ug of MST14 for 1 hr, At 37 deg. C. After the seven (7) hr. incubation, cells were spun down for 3 min. at 500xg and 20 uL of supernatant from each sample was assayed for IP-10 (Abeam) by ELISA according to the manufacturer's protocol.

[0538] The results show that the EC50 for IP-10 induction in PBMCs of QXL138AM42-N297Q is ~30x higher than the EC50 for never masked QXL138A-N297Q and ~3x higher for QXL138AM2.2-N297Q showing an improvement over Mask2.2. (Figure 21).

[0539] Example 17: Methods of Reducing and Restoring masked IFNa Activity.

[0540] In this example, QXL138AM4.2-N297Q, QXL138YNS4.2-N297Q, QXL138YNS4.2-N297Q+MST were evaluated for masking and restoration activity.

[0541] Briefly, HEK Blue I FNa / b reporter cell (Invivogen) activity showing IFNAR activation in response to QXL138YNS-N297Q, QXL138YNSM4.2-N297Q, QXL138AM4.2-N297Q, and QXL138YNSM4.2-N297Q+MST (matriptase). HEK Blue IFNa / b cells were seeded into 96-well tissue culture plates (Fisher) at a density of 5x104cells / well (150 pL / well). 50 pL / well of indicated Abs were incubated with the cells at the indicated concentrations overnight at 37 °C in a tissue culture incubator. Abs cleaved with MST14 (R& D Systems) were prepared by incubating 44 ng MST14 / pg QXL138YNSM4.2-N297Q for 1.5 hours at 37 °C. 10 pL of supernatant was then added to a plate containing 90 pL / well Quanti- Biue substrate (Invivogen). Absorbance changes were detected at 630 nm using a Biotek EPOCH ELISA reader.

[0542] The results show that Mask 4.2 increased the EC50 of QXL138YNSM4.2 ~30-fold compared to unmasked QXL138YNS-N297Q but decreased ~4-fold compared to QXL138AM4.2-N297Q suggesting Mask 4.2 is able to interfere with wild-type IFNa activation of IFNAR in these cells better than the YNS mutant of IFNa When the mask is removed, activity is restored to the level of unmasked fusion protein. (Figure 22).

[0543] Exampie 18: Methods of Reducing and Restoring masked IFNa Activity.

[0544] In this example, QXL138YNS-N297Q, QXL138YNS4.2-N297Q, QXL138YNS4.2-N297Q+MST, and QXL138AM4.2-N297Q were assessed for masking and restoration activity.

[0545] Briefly, IFNaYNSfusion proteins bound to IFNAR2 / Fc Chimera. Immulon 2 HB plates (Thermofisher) were coated with 10 pg / mL IFNAR2 / Fc Chimera (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 3 times with PBS +0.05% Tween-20 (Sigma) immediately prior to use. Abs cleaved with MST14 (R& D Systems) were prepared by incubating 44 ng MST14 / pg QXL138YNSM4.2-N297Q for 1.5 hours at 37 °C. Indicated Ab concentrations were overlayed overnight at 4 °C. Wells were washed 3 times with PBS + 0.05% Tween-20, and bound Abs were detected with anti-human Kappa-AP (Southern Biotech) diluted 1:3000 in PBS + 1% BSA, Absorbance changes after the addition of AP substrate (Sigma) were detected at 410 nm using a Biotek EPOCH ELISA reader.

[0546] The results show that Mask 4.2 increases the EC50 of QXL138AM4.2-N297Q and QXL138YNSM4.2-N297Q ~40-fold and ~20-fold respectively, compared to unmasked fusion protein (QXL138YNS-N297Q). When the mask is removed, the activity of QXL138YNSM4.2-N297Q is restored to the level of unmasked fusion protein. (Figure 23).

[0547] Example 19: Methods of Reducing and Restoring masked IFNa Activity.

[0548] In this example, several fusion proteins consisting of several types of masks were assessed for masking and restoration activity.

[0549] Briefly, HEK Blue I FNa / b reporter cell (Invivogen) activity showing IFNAR activation in response to QXL138AM2.2-N297Q, QXL138AM4.2-N297Q, and QXL138YNSM4.2-N297Q all with and without MST (matriptase). HEK Blue IFNa / b cells were seeded into 96-well tissue culture plates (Fisher) at a density of 5x104cells / well (150 pL / well). 50 pL / well of indicated Abs were incubated with the cells at the indicated concentrations overnight at 37 °C in a tissue culture incubator. Abs cleaved with MST14 (R& D Systems) were prepared by incubating 5 ng MST 14 / pg fusion protein for 1.5 hours at 37 °C. 20 pL of supernatant was then added to a plate containing 180 pL / well Quanti-Blue substrate (Invivogen). Absorbance changes were detected at 630 nm using a Biotek EPOCH ELISA reader.

[0550] The results show that the EC50 increase seen when Mask 4.2 is used in QXL138AM4.2-N297Q compared to when Mask 2.2 is used suggested Mask 4.2 bound IFNa more tightly thus interfering with IFNAR activation more than Mask 2.2. This effect was seen to a lesser degree when Mask 4.2 was used in QXL138YNSM4.2-N297Q possibly due to the IFNaYNSmutant having a higher affinity for IFNAR compared to IFNa. (See, Figure(s) 24).

[0551] In another assay, Wild-type IFNa fusion proteins and mutant IFNaYNSfusion proteins bound to IFNAR2 / Fc Chimera. Immulon 2 HB plates (Thermofisher) were coated with 10 pg / mL IFNAR2 / Fc Chimera (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 3 times with PBS + 0.05% Tween-20 (Sigma) immediately prior to use. Abs cleaved with MST14 (R& D Systems) were prepared by incubating 5 ng MST 14 / pg fusion protein for 1.5 hours at 37 °C. Indicated Ab concentrations were overlayed overnight at 4 °C. Wells were washed 3 times with PBS + 0.05% Tween-20, and bound Abs were detected with anti¬ human Kappa-AP (Southern Biotech) diluted 1:3000 in PBS + 1% BSA. Absorbance changes after the addition of AP substrate (Sigma) were detected at 410 nm using a Biotek EPOCH ELISA reader. The results show, the increased Kd (lower affinity) seen when Mask 4.2 is used in QXL138AM4.2-N297Q compared to when Mask 2.2 is used suggested Mask 4.2 bound more tightly to IFNa thus interfering with IFNAR2 binding more than Mask 2.2. This effect was seen to a lesser degree when Mask 4.2 was used in QXL138YNSM4.2-N297Q possibly due to the IFNaYNSmutant having a higher affinity for IFNAR compared to wild-type IFNa. (See. Figure(s) 25).

[0552] Example 20: Methods of Reducing and Restoring masked IFNa Activity.

[0553] In this example, another assay was performed on several fusion proteins consisting of several types of masks for masking and restoration activity.

[0554] Briefly, HEK Blue IFNa / b reporter cell (Invivogen) activity showing IFNAR activation in response to QXL138AM2.2-N297Q, QXL138AM4.2-N297Q, and QXL138YNSM4.2-N297Q all with and without MST (matriptase). HEK Blue IFNa / b cells were seeded into 96-well tissue culture plates (Fisher) at a density of 2.5x104cells / well (150 pL / well), 50 pL / well of indicated Abs were incubated with the cells at the indicated concentrations overnight at 37 °C in a tissue culture incubator. Abs cleaved with MST14 (R& D Systems) were prepared by incubating 5 ng MST 14 / pg fusion protein for 1.5 hours at 37 °C. 20 pL of supernatant was then added to a plate containing 180 pL / well Quanti-Blue substrate (Invivogen). Absorbance changes were detected at 630 nm using a Biotek EPOCH ELISA reader.

[0555] The results show that The EC50 increase seen when Mask 4.2 is used in QXL138AM4.2- N297Q compared to when Mask 2.2 is used suggested Mask 4.2 bound IFNa more tightly thus interfering with IFNAR activation more than Mask 2.2. This effect was seen to a lesser degree when Mask 4.2 was used in QXL138YNSM4.2-N297Q possibly due to the IFNaYNSmutant having a higher affinity for IFNAR compared to IFNa. (See, Figure(s) 26),

[0556] In another assay, HEK Blue IFNa / b reporter cell (Invivogen) activity showing IFNAR activation in response to QXL138AM2.2-N297Q, QXL138AM4.2-N297Q, and QXL138YNSM4.2-N297Q all with and without MST (matriptase). This was done with antigen (Ag) blocking by QXL138-N297Q to understand the IFNa / IFNAR interaction independent of Ag targeting. HEK Blue IFNa / b cells were seeded into 96-well tissue culture plates (Fisher) at a density of 2.5x104cells / well (150 pL / well) in the presence of 0.5 pM QXL138-N297Q for 6 hours prior to addition of treatment Abs. 50 pL / well of indicated Abs were incubated with the cells at the indicated concentrations overnight at 37 °C in a tissue culture incubator. Abs cleaved with MST14 (R& D Systems) were prepared by incubating 5 ng MST14 / pg fusion protein for 1.5 hours at 37 °C. 20 pL of supernatant was then added to a plate containing 180 pL / well Quanti-Blue substrate (Invivogen). Absorbance changes were detected at 630 nm using a Biotek EPOCH ELISA reader.

[0557] The results show that without antigen targeting, the EC50 for all fusion proteins assayed was greatly increased reinforcing the importance of antigen targeting for potency. The EC50 increase seen when Mask 4.2 is used in QXL138AM4.2-N297Q compared to when Mask 2.2 is used suggested Mask 4.2 bound IFNa more tightly thus interfering with IFNAR activation more than Mask 2.2. This effect was seen to a lesser degree when Mask 4.2 was used in QXL138YNSM4.2-N297Q possibly due to the IFNaYNSmutant having a higher affinity for IFNAR compared to I FNa. (See, Figure(s) 27),

[0558] Example 21: IP-10 Induction of PBMCs by IFNa fusion proteins.

[0559] In this example, the IP-10 Induction of PBMCs by IFNa fusion proteins was performed to evaluate effectiveness of Mask 4.2 and Mask 22. Briefly, freshly thawed human PBMCs (HumanCells Biosciences) were washed once with cold RPMI + 10% FBS (Invitrogen) and seeded into a 12-well plate (Themofisher) at a density of ~5x105cells / well (1 mL / well), Recombinant human IFNa (Reprokine) or indicated Abs at indicated concentrations were added to the cells and allowed to incubate at 37 °C in a tissue culture incubator for 7 hours. After the 7-hour incubation, ceils were spun down for 3 minutes at 500xg and 20 pL of supernatant from each sample was assayed for IP-10 (Abeam) by ELISA according to the manufacturer’s protocol.

[0560] The results show that Mask 4.2 increases the EC50 of QXL138AM4.2-N297Q more than Mask 2.2 (QXL138AM2.2-N297Q) indicating at matched concentrations, Mask 4.2 is better than Mask 2.2 at reducing IP-10 production, Mask 4.2 is also effective at masking the IFNaYNSmutant, showing an -187-fold EC50 increase over unmasked QXL138YNS-N297Q. (Figure(s) 28).

[0561] Example 22: IP-10 Induction of PBMCs by IFNa fusion proteins.

[0562] In this example, the IP-10 Induction of PBMCs by IFNa fusion proteins was performed to evaluate effectiveness of Mask 4.2 and Mask 2.2 with and without demasking, Briefly, Freshly thawed human PBMCs (HumanCells Biosciences) were washed once with cold RPMI + 10% FBS (Invitrogen) and seeded into a 12-well plate (Themofisher) at a density of -5x105cells / well (1 mL / well) Indicated Abs at indicated concentrations were added to the cells and allowed to incubate at 37 °C in a tissue culture incubator for 7 hours. Abs cleaved with IMST14 (R& D Systems) were prepared by incubating 5 ng MST 14 / pg Ab for 1.5 hours at 37 °C. After the 7-hour incubation, cells were spun down for 3 minutes at 500xg and 20 pL of supernatant from each sample was assayed for IP-10 (Abeam) by ELISA according to the manufacturer’s protocol.

[0563] The results show that Mask 4.2 increases the EC50 of QXL138AM4.2-N297Q more than Mask 2.2 (QXL138AM2.2-N297Q) indicating at matched concentrations, Mask 4.2 is better than Mask 2.2 at reducing IP-10 production. Mask 4.2 is also effective at masking the IFNaYNSmutant, showing an -176- fold EC50 increase over demasked fusion protein. As expected, demasking the fusion proteins decreases the EC50. (Figure(s) 29). Example 23: Proliferation Assays to Evaluate Fusion Proteins.

[0564] In this example, an 0VCAR3 proliferation assay evaluating Mask 4.2 fusion proteins compared to Mask 2.2 fusion protein using MTS was performed. Briefly, approximately 1 x104cells / well (150 L / well) were seeded into 96-well tissue culture plates (Becton Dickinson). Demasked Abs were made by preincubating samples with MST 14 (R& D Systems), 5 ng MST 14 / pg Ab for 1.5 hours at 37 °C. 50 pL / well of each indicated Ab at the indicated concentrations were added to the plates. 3 days later, 20 pL / well MTS reagent (Promega) was added to the plates. Absorbance changes are detected at 490 nm with a Biotek EPOCH reader.

[0565] The results show the potency of QXL138AM2.2-N297Q was greater than QXL138AM4.2-N297Q suggesting Mask 4.2 was better at masking the cytotoxic effects of the IFNa fusion. Demasked QXL138YNSM4.2-N297Q had greater potency than either demasked QXL138AM2.2-N297Q or demasked QXL138AM4.2-N297Q suggesting the mutant IFNaYN£moiety is more cytotoxic than the wild-type IFNa moiety in OVCAR3 ceils. (Figure(s) 30).

[0566] Example 24: Characterization of QXL138AM4.2-N297Q (Lot 2) by CE-SDS.

[0567] In this example, characterization of QXL138AM4.2-N297Q (Lot 2) was performed using Capillary Electrophoresis (CE-SDS). The data was generated by Curia using the manufacturer’s protocols.

[0568] The results in Figure 31(A) show that under non-reducing conditions, the bulk of the protein can be separated into 2 peaks (sizes) with sizes -221 kDa and -228 kDa suggesting some heterogeneity in protein size. Under reducing conditions (Figure 31(B)), the protein is separated into an expected light chain peak -29 kDa and 2 heavy chain peaks at -102 kDa and -112 kDa. The 2 heavy chain peaks suggest possible glycosylation differences in the mask region.

[0569] Example 25: Characterization of QXL138YNS4.2-N297Q (Q1 3 Lot 1) by CE-SDS.

[0570] In this example, the characterization of QXL138YNS4.2-N297Q (Q143 Lot 1) was performed using Capillary Electrophoresis (CE-SDS). The data was generated by Curia using the manufacturer's protocols.

[0571] The results in Figure 32(A) show that Under non-reducing conditions, the bulk of the protein can be separated into 3 peaks (sizes) with sizes -204 kDa, -217 kDa and -225 kDa suggesting some heterogeneity in protein size. Under reducing conditions (Figure 32(B)), the protein is separated into an expected light chain peak -28 kDa and 2 predominate heavy chain peaks at -101 kDa and -111 kDa. The 2 predominate heavy chain peaks along with 2 minor heavy chain peaks suggest possible glycosylation differences in the mask region and / or partial demasking of the protein. Example 26: Characterization of QXL138AM4.3-N297Q (Lot 1) by CE-SDS.

[0572] In this example, the characterization of QXL138YNS4.3-N297Q (Lot 1) was performed using Capillary Electrophoresis (CE-SDS). The data was generated by Curia using the manufacturer's protocols.

[0573] The results in Figure 33(A) show that under non-reducing conditions, the bulk of the protein can be separated into 3 peaks (sizes) with sizes -207 kDa, -217 kDa and -224 kDa suggesting some heterogeneity in protein size. Under reducing conditions (Figure 33(B)), the protein is separated into an expected light chain peak -28 kDa and 2 predominate heavy chain peaks at -105 kDa and -115 kDa. The 2 predominate heavy chain peaks along with 2 minor heavy chain peaks suggest possible glycosylation differences in the mask region and / or partial demasking of the protein.

[0574] Example 27: Characterization of QXL138YNS4.3-N297Q (Lot 1) by CE-SDS.

[0575] In this example, the characterization of QXL138YNS4.3-N297Q (Lot 1) was performed using Capillary Electrophoresis (CE-SDS). The data was generated by Curia using the manufacturer’s protocols.

[0576] The results in Figure 34(A) show that under non-reducing conditions, the bulk of the protein can be separated into 2 peaks (sizes) with sizes -217 kDa and -223 kDa suggesting some heterogeneity in protein size, Under reducing conditions (Figure 34(B)), the protein is separated into an expected light chain peak -27 kDa and 2 predominate heavy chain peaks at -101 kDa and -111 kDa. The 2 predominate heavy chain peaks along with 2 minor heavy chain peaks suggest possible glycosylation differences in the mask region and / or partial demasking of the protein.

[0577] Example 28: Characterization of QXL138AM4.3-N297Q and QXL138YNS4.3-N297Q by SDS-PAGE.

[0578] In this example, Reduced SDS-PAGE showing QXL138AM4.3-N297Q and QXL138YNSM4.3-N297Q with and without matriptase was performed. Briefly, Samples were incubated with and without 5 ng MST / pg fusion protein at 37 °C for 1.5 hours prior to being denatured by heating to 70 °C for 10 minutes and reduced by incubating at 37 °C for 20 minutes in the presence of 5% beta¬ mercaptoethanol. 2 pg of each sample was run a 12% Bis-Tris gel (Thermofisher). Gel was stained with EZ Stain (Fisher) according to the manufacturer’s protocol.

[0579] The results showed Mask 4.3 cannot be cleaved by matriptase under the same conditions used to cleave Mask 4.2. (Figure 35).

[0580] Example 29: Methods of Reducing and Restoring masked IFNa Activity. In this example, another assay was performed on several fusion proteins consisting of several types of masks for masking and restoration activity.

[0581] Briefly, Briefly, HEK Blue IFNa / b cells (Invivogen) were seeded into 96-weil tissue culture plates (Fisher) at a density of 1x10e4 cells / well (100 uL / well). 100 uL / well of recombinant IFNa (Novus Biologicals) or indicated Abs were incubated with the cells at concentrations sufficient to generate the final indicated concentrations O / N at 37 deg. C. Abs cleaved with MST14 (R& D Systems) were prepared by incubating 50 ug of Ab with 0.5 ug of MST14 for 1 hr. At 37 deg. C. 20 uL of supernatant was then added to a plate containing 180 uL / well Quanti-Blue substrate (Invivogen), Absorbance changes were read at 630 nm using a Biotek EPOCH ELISA reader.

[0582] The results show that The EC50 of QXL138AM4.3-N297Q +1- MST is largely unchanged, suggesting the mask in QXL138AM4.3-N297Q was not cleaved due to the lack of an MST recognition site. (Figure 36).

[0583] Additionally, The EC50 for QXL138YNSM4.3-N297Q + / - MST is largely unchanged, suggesting the mask was not cleaved due to the lack of an MST recognition site. (Figure 37).

[0584] Example 30: Methods of Evaluating Activity of Fusion Proteins In vivo.

[0585] In another experiment, 1x106H929 cells were injected in 8 weeks old female NSG mice sc in the back. Mice were then treated iv bi-weekly for 4 weeks starting on day 14 with 0.1mg / kg of PBS, Q139, Q143, or Q147. Tumor volumes were measured over time, when mice reached terminal tumor volumes (>2000mm3) values were kept to calculate the mean until all mice in the group reached terminal size.

[0586] The results show that the masked version Q143 was not significantly different that the unmasked version or uncleaveable masked version. Notably, having the mask on at this dosing schedule does not increase efficacy against H929 tumors. Moreover, if the mask is in fact staying intact, the YNS IFNa is still just as active. (See, Figure 38). in addition, measuring tumor volumes of individual mice on Day 28, the results show the first control mouse reached terminal size. 2 mice from non-tumor related deaths were excluded from the data set in the Q147 treated group. (See, Figure 39). Finally, during the entirety of the treatment period, the results show no significant body weight change at 0.1 mg / kg dose level as well as no signs of toxicity. (See, Figure 40).

[0587] Example 31: Methods of Making Mask Fusion Proteins.

[0588] In this example, the sequence of an Anti-CD138 Human Kappa Light Chain is developed and set forth in Figure 41 (SEQ ID NO: 38). The signal peptide is set forth in blue. The red represents the light chain variable region, and the black is the kappa light chains. The constructs set forth in Figure 41 are transiently expressed in CHO cells using methods known in the art. Further analysis of the heavy and light chain is assessed by mass spectrometer using standard methods.

[0589] Example 32: Methods of Making Mask Fusion Proteins.

[0590] In this example, the construct design and characterization of QXL138AM4.2-N297Q is shown. Briefly, an anti-CD138 lgG1 is made using standard methods in the art. The heavy chain isotype is a human gamma 1 and the light chain isotype is human kappa. The sequence of the QXL138AM4.2-N297Q heavy chain is set forth and is disclosed as (SEQ ID NO: 6). For purposes of this disclosure, the heavy chain further comprises an amino acid substitution at position 297 (N297Q) in order to mutate out the glycosylation site. This prevents the fusion protein from binding to the endogenous Fc receptor.

[0591] The mask was generated as described in the instant specification, See, Methods of Masking IFNs of the Disclosure. The resulting construct denoted QXL138AM4.2-N297Q (Anti-CD138-linker-IFNo2-cleavable linker-mask) comprises a signal peptide (set forth in blue) (SEQ ID NO: 48), an IgG anti-CD138 heavy chain variable region (set forth in red) and is described as (SEQ ID NO: 7), a human lgG1 heavy chain constant region (set forth in black) and is described as (SEQ ID NO: 49), a N297Q mutation (set forth in bold black) (i.e. asparagine to glutamine mutation at position 297), a fusion protein linker SGGAGGS (SEQ ID NO: 5) (set forth in green), IFNa (set forth in gray) (SEQ ID NO: 10), a second fusion protein linker GSSGKQSRWNHGSSGGSGGSGGS (set forth in black underline) (SEQ ID NO: 35), which comprises a cleavable linker KQSRWNH set forth in black bold underline (SEQ ID NO: 16) and a mask of the disclosure (D1 loop of IFNAR2) ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYTIMSKPEDLKWKNCANTTRSFCDLTD EWRSTHEAYVTVLEGFSGNTTLFSCSHNFWLAIDMS (set forth in orange) (SEQ ID NO: 36) (See, Figure 42 and SEQ ID NO: 39). The nucleic acid sequence of the QXL138AM4.2-N297Q. heavy chain is set forth in Figure 43 and is described as (SEQ ID NO: 40).

[0592] In one embodiment, the sequence of the QXL138AM4.2-N297Q light chain is set forth in Figure 41 and is disclosed as (SEQ ID NO: 8) and / or (SEQ ID NO: 38).

[0593] The constructs set forth in Figure 41 and Figure 42 are transiently expressed in CHO cells using methods known in the art. Further analysis of the heavy and light chain is assessed by mass spectrometer using standard methods.

[0594] Example 33: Methods of Making Mask Fusion Proteins.

[0595] In this example, the construct design and characterization of QXL138YNS4.2-N297Q is shown. Briefly, an anti-CD138 lgG1 is made using standard methods in the art. The heavy chain isotype is a human gamma 1 and the light chain isotype is human kappa. The sequence of the QXL138YNS4.2-N297Q heavy chain is set forth and is disclosed as (SEQ ID NO: 6). For purposes of this disclosure, the heavy chain further comprises an amino acid substitution at position 297 (N297Q) in order to mutate out the glycosylation site. This prevents the fusion protein from binding to the endogenous Fc receptor.

[0596] The mask was generated as described in the instant specification, See, Methods of Masking IFNs of the Disclosure. The resulting construct denoted QXL138YNS4.2-N297Q (Anti-CD138-linker-IFNo2-cleavable linker-mask) comprises a signal peptide (set forth in blue) (SEQ ID NO: 53), an IgG anti-CD138 heavy chain variable region (set forth in red) and is described as (SEQ ID NO: 7), a human lgG1 heavy chain constant region (set forth in black) and is described as (SEQ ID NO: 50), a N297Q mutation (set forth in bold black) (i.e. asparagine to glutamine mutation at position 297), a fusion protein linker SGGAGGS (SEQ ID NO: 5) (set forth in green), IFNa (set forth in gray) further comprising a YNS Mutation (H57Y, E58N, and Q61S) (SEQ ID NO: 21), a second fusion protein linker GSSGKQSRWNHGSSGGSGGSGGS (set forth in black underline) (SEQ ID NO: 38), which comprises a cleavable linker KQSRWNH set forth in black bold underline (SEQ ID NO: 16) and a mask of the disclosure (D1 loop of IFNAR2) ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYTIMSKPEDLKWKNCANTTRSFCDLTD EWRSTHEAYVTVLEGFSGNTTLFSCSHNFWLAIDMS (set forth in orange) (SEQ ID NO: 36) (See, Figure 44 and SEQ ID NO: 41). The nucleic acid sequence of the QXL138YNS4.2-N297Q. heavy chain is set forth in Figure 45 and is described as (SEQ ID NO: 42).

[0597] The sequence of the QXL138YNS4.2-N297Q light chain is set forth in Figure 41 and is disclosed as (SEQ ID NO: 8) and / or (SEQ ID NO: 38). The resulting construct comprises a variable region (set forth in red) and is disclosed as (SEQ ID NO: 9).

[0598] The constructs set forth in Figure 41 and Figure 44 are transiently expressed in CHO cells using methods known in the art. Further analysis of the heavy and light chain is assessed by mass spectrometer using standard methods.

[0599] Example 34: Methods of Making Mask Fusion Proteins.

[0600] In this example, the construct design and characterization of QXL138AM4.3-N297Q is shown. Briefly, an anti-CD138 lgG1 is made using standard methods in the art. The heavy chain isotype is a human gamma 1 and the light chain isotype is human kappa. The sequence of the QXL138AM4.3-N297Q heavy chain is set forth and is disclosed as (SEQ ID NO: 6). For purposes of this disclosure, the heavy chain further comprises an amino acid substitution at position 297 (N297Q) in order to mutate out the glycosylation site. This prevents the fusion protein from binding to the endogenous Fc receptor.

[0601] The mask was generated as described in the instant specification, See. Methods of Masking IFNs of the Disclosure. The resulting construct denoted QXL138AM4.3-N297Q (Anti-CD138-linker-IFNa2-cleavable linker-mask) comprises a signal peptide (set forth in blue) (SEQ ID NO: 53), an IgG anti-CD138 heavy chain variable region (set forth in red) and is described as (SEQ ID NO: 7), a human lgG1 heavy chain constant region (set forth in black) and is described as (SEQ ID NO: 49) or (SEQ ID NO: 50), a N297Q mutation (set forth in bold black) (i,e. asparagine to glutamine mutation at position 297), a fusion protein linker SGGAGGS (SEQ ID NO: 5) (set forth in green), IFNa (set forth in gray) (SEQ ID NO: 10), a second fusion protein linker GSSGGSGGSGGSGSSGGSGGSGGS (set forth in black underline) (SEQ ID NO: 47) and a mask of the disclosure (D1 loop of IFNAR2) ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYTIMSKPEDLKWKNCANTTRSFCDLTD EWRSTHEAYVTVLEGFSGNTTLFSCSHNFWLAIDMS (set forth in orange) (SEQ ID NO: 36) (See, Figure 46 and SEQ ID NO: 43).

[0602] In one embodiment, the sequence of the QXL138AM4.3-N297Q light chain is set forth in Figure 41 and is disclosed as (SEQ ID NO: 8) and / or (SEQ ID NO: 38). The resulting construct comprises a variable region (set forth in red) and is disclosed as (SEQ ID NO: 9).

[0603] The constructs set forth in Figure 41 and Figure 46 are transiently expressed in CHO ceils using methods known in the art. Further analysis of the heavy and light chain is assessed by mass spectrometer using standard methods.

[0604] Example 35: Methods of Making Mask Fusion Proteins.

[0605] In this example, the construct design and characterization of QXL138YNS4.3-N297Q is shown, Briefly, an anti-CD138 lgG1 is made using standard methods in the art. The heavy chain isotype is a human gamma 1 and the light chain isotype is human kappa. The sequence of the QXL138YNS4.3- N297Q heavy chain is set forth and is disclosed as (SEQ ID NO: 6). For purposes of this disclosure, the heavy chain further comprises an amino acid substitution at position 297 (N297Q) in order to mutate out the glycosylation site. This prevents the fusion protein from binding to the endogenous Fc receptor.

[0606] The mask was generated as described in the instant specification, See. Methods of Masking IFNs of the Disclosure. The resulting construct denoted QXL138YNS4.3-N297Q (Anti-CD138-linker-IFNa2-cleavable linker-mask) comprises a signal peptide (set forth in blue) (SEQ ID NO: 53), an IgG anti-CD138 heavy chain variable region (set forth in red) and is described as (SEQ ID NO: 7), a human lgG1 heavy chain constant region (set forth in black) and is described as (SEQ ID NO: 50) further comprising a N297Q mutation (set forth in bold black) (i.e. asparagine to glutamine mutation at position 297), a fusion protein linker SGGAGGS (SEQ ID NO: 5) (set forth in green), IFNa (set forth in gray) further comprising a YNS Mutation (H57Y, E58N, and Q61S) (SEQ ID NO: 21), a second fusion protein linker GSSGGSGGSGGSGSSGGSGGSGGS (set forth in black underline) (SEQ ID NO: 47), and a mask of the disclosure (D1 loop of IFNAR2) ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYTIMSKPEDLKWKNCANTTRSFCDLTD EWRSTHEAYVTVLEGFSGNTTLFSCSHNFWLAIDMS (set forth in orange) (SEQ ID NO: 36) (See, Figure 47 and SEQ ID NO: 44). The sequence of the QXL138YNS4.3-N297Q light chain is set forth in Figure 41 and is disclosed as (SEQ ID NO: 8) and / or (SEQ ID NO: 38). The resulting construct comprises a variable region (set forth in red) and is disclosed as (SEQ ID NO: 9).

[0607] The constructs set forth in Figure 41 and Figure 47 are transiently expressed in CHO cells using methods known in the art. Further analysis of the heavy and light chain is assessed by mass spectrometer using standard methods.

[0608] Example 36: Methods of Making Mask Fusion Proteins.

[0609] In this example, the sequence of an Anti-Mesothelin 6A4 Human Kappa Light Chain is developed and set forth in Figure 48 (SEQ ID NO: 45). The signal peptide is set forth in red. The blue represents the light chain variable region, and the black is the kappa light chains. In one embodiment, the mesotheiin light chain comprises (SEQ ID NO: 19). In a further embodiment, the mesotheiin light chain comprises a variable region (SEQ ID NO: 20). The constructs set forth in Figure 48 are transiently expressed in CHO cells using methods known in the art. Further analysis of the heavy and light chain is assessed by mass spectrometer using standard methods.

[0610] Example 37: Methods of Making Mask Fusion Proteins.

[0611] In this example, the construct design and characterization of Meso-YNS4.2-N297Q is shown. Briefly, an anti-mesotheiin lgG1 is made using standard methods in the art. The heavy chain isotype is a human gamma 1. The sequence of the Meso-YNS4.2-N297Q heavy chain is set forth and is disclosed as (SEQ ID NO: 17). For purposes of this disclosure, the heavy chain further comprises an amino acid substitution at position 297 (N297Q) in order to mutate out the glycosylation site. This prevents the fusion protein from binding to the endogenous Fc receptor.

[0612] The mask was generated as described in the instant specification, See, Methods of Masking IFNs of the Disclosure. The resulting construct denoted Meso-YNS4.2-N297Q (Anti-Meso-linker-l FNa2-cleavable linker-mask) comprises a signal peptide (set forth in red) (SEQ ID NO: 51), an IgG anti-meso heavy chain variable region (set forth in blue) and is described as (SEQ ID NO: 53), a human lgG1 heavy chain constant region (set forth in black) and is described as (SEQ ID NO: 52), further comprising a N297Q mutation (set forth in bold black) (i.e. asparagine to glutamine mutation at position 297), a fusion protein linker SGGAGGS (SEQ ID NO: 5) (set forth in green), IFNa (set forth in violet) further comprising a YNS Mutation (H57Y, E58N, and Q61S) (SEQ ID NO: 21), a second fusion protein linker GSSGKQSRWNHGSSGGSGGSGGS (set forth in black underline) (SEQ ID NO: 38), which comprises a cleavable linker KQSRWNH set forth in black bold underline (SEQ ID NO: 16) and a mask of the disclosure (D1 loop of IFNAR2)

[0613] ISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYTIMSKPEDLKWKNCANTTRSFCDLTD EWRSTHEAYVTVLEGFSGNTTLFSCSHNFWLAIDMS (set forth in orange) (SEQ ID NO: 36) (See, Figure 49 and SEQ ID NO: 46).

[0614] The sequence of the Meso-YNS4.2-N297Q light chain is set forth in Figure 48 and is disclosed as (SEQ ID NO: 45) and / or (SEQ ID NO: 19). The resulting construct comprises a variable region (set forth in red) and is disclosed as (SEQ ID NO: 20).

[0615] The constructs set forth in Figure 48 and Figure 49 are transiently expressed in CHO cells using methods known in the art. Further analysis of the heavy and light chain is assessed by mass spectrometer using standard methods.

[0616] Example 38: Proliferation Assay to Evaluate Fusion Proteins Using NCI-H1299.

[0617] In this example, a NCI-H 1299 proliferation assay evaluating MESO-YNSM5.2 + / -MST compared to QXL138AM4.2 + / -MST was performed. Briefly, Approximately 3000 cells / well (150 pL / well) were seeded into 96-well tissue culture plates (Becton Dickinson). Demasked Abs were made by preincubating samples with MST 14 (R& D Systems), 5 ng MST 14 / pg Ab for 1.5 hours at 37 °C. 50 pL / well of each indicated Ab at the indicated concentrations were added to the plates. Three (3) days later, 20 pL / well MTS reagent (Promega) was added to the plates. Absorbance changes are detected at 490 nm with a Biotek EPOCH reader.

[0618] The results show the potency of Meso-YNS5,2 was slightly more potent when compared to QXL138AM4.2 in NCI-H 1299 cells. Notably, NCI-H 1299 is not sensitive to the proteins assessed. These cells express low levels of CD138 and Mesothelin, which may explain the low sensitivity.

[0619] (Figure(s) 52).

[0620] Example 39: Proliferation Assay to Evaluate Fusion Proteins Using NCI-H2452.

[0621] In this example, a NCI-H2452 proliferation assay evaluating MESO-YNSM5.2 + / -MST compared to QXL138AM4.2 + / -MST was performed. Briefly, Approximately 2000 cells / well (150 pL / well) were seeded into 96-well tissue culture plates (Becton Dickinson). Demasked Abs were made by preincubating samples with MST14 (R& D Systems), 5 ng MST 14 / pg Ab for 1.5 hours at 37 °C. 50 pL / well of each indicated Ab at the indicated concentrations were added to the plates. Four (4) days later, 20 pL / well MTS reagent (Promega) was added to the plates. Absorbance changes are detected at 490 nm with a Biotek EPOCH reader.

[0622] The results show the potency of Meso-YNS5.2 was significantly more potent when compared to QXL138AM4.2 in NCI-H2452 cells. Notably, NCI-H2452 are sensitive to the proteins assessed. These cells moderately express CD138 and Mesothelin. (Figure(s) 53).

[0623] Example 40: Proliferation Assay to Evaluate Fusion Proteins Using NCI-H596. In this example, a NCI-H596 proliferation assay evaluating MESO-YNSM5.2 + / -MST compared to QXL138AM4.2 + / -MST was performed. Briefly, Approximately 3500 cells / well (150 pL / well) were seeded into 96-well tissue culture plates (Becton Dickinson). Demasked Abs were made by preincubating samples with MST14 (R& D Systems), 5 ng MST14 / pg Ab for 1.5 hours at 37 °C. 50 pL / well of each indicated Ab at the indicated concentrations were added to the plates. Four (4) days later, 20 pL / well MTS reagent (Promega) was added to the plates. Absorbance changes are detected at 490 nm with a Biotek EPOCH reader.

[0624] The results show the potency of Meso-YNS5.2 was moderately more potent when compared to QXL138AM4.2 in NCI-H596 cells. Notably, NCI-H596 are moderately sensitive to the proteins assessed. These cells express prominent levels of CD138 and Mesothelin. (Figure(s) 54).

[0625] Example 41: Proliferation Assay to Evaluate Fusion Proteins Using KLE.

[0626] In this example, a KLE proliferation assay evaluating QXL138A, QXL138YNS, and MESO-YNS was performed. Briefly, Approximately 2500 cells / well (150 L / well) were seeded into 96-well tissue culture plates (Becton Dickinson). Demasked Abs were made by preincubating samples with MST 14 (R& D Systems), 5 ng MST14 / pg Ab for 1.5 hours at 37 °C. 50 pL / well of each indicated Ab at the indicated concentrations were added to the plates. Four (4) days later, 20 pL / well MTS reagent (Promega) was added to the plates. Absorbance changes are detected at 490 nm with a Biotek EPOCH reader.

[0627] The results show the KLE cells were sensitive to all three (3) Fusion Proteins to varying degrees. MESO-YNS was more potent than QXL138YNS, which in turn, was more potent than QXL138A. The cells express mesothelin, but not CD138 suggesting the higher overall potency of MESO-YNS was a result of a combination of Ag targeting and better potency of mutant IFNYNScompared to wild-type IFNa. (Figure(s) 55).

[0628] Example 42: Proliferation Assay to Evaluate Fusion Proteins Using NCI-N87.

[0629] In this example, a NCI-N87 proliferation assay evaluating QXL138A, QXL138YNS, and MESO- YNS was performed. Briefly, Approximately 2500 cells / well (150 pL / well) were seeded into 96-well tissue culture plates (Becton Dickinson). Demasked Abs were made by preincubating samples with MST14 (R& D Systems), 5 ng MST14 / pg Ab for 1.5 hours at 37 °C. 50 pL / well of each indicated Ab at the indicated concentrations were added to the plates. Four (4) days later, 20 pL / well MTS reagent (Promega) was added to the plates. Absorbance changes are detected at 490 nm with a Biotek EPOCH reader.

[0630] The results show the NCI-N87 cells were sensitive to all three (3) Fusion Proteins to varying degrees. Notably, MESO-YNS and QXL138YNS were equally potent and in turn more potent than QXL138A. The similar potencies of MESO-YNS and QXL138YNS were likely due to these cells expressing both mesothelin and CD138. (Figure(s) 56).

[0631] Example 43: Proliferation Assay to Evaluate Fusion Proteins Using NCI-N2052.

[0632] In this example, a NCI-N2052 proliferation assay evaluating QXL138A, QXL138YNS, and MESO-YNS was performed, Briefly, Approximately 2000 cells / well (150 L / well) were seeded into 96- well tissue culture plates (Becton Dickinson). Demasked Abs were made by preincubating samples with MST14 (R& D Systems), 5 ng MST14 / pg Ab for 1.5 hours at 37 °C. 50 pL / well of each indicated Ab at the indicated concentrations were added to the plates. Six (6) days later, 20 pL / well MTS reagent (Promega) was added to the plates. Absorbance changes are detected at 490 nm with a Biotek EPOCH reader.

[0633] The results show the NCI-N2052 cells were sensitive to all three (3) Fusion Proteins to varying degrees. MESO-YNS and QXL138YNS were equally potent and in turn more potent than QXL138A. The similar potencies of MESO-YNS and QXL138YNS were likely due to these cells expressing both mesothelin and CD138. (Figure(s) 57).

[0634] Example 44: Methods of Making Fusion Proteins.

[0635] In this example, the construct design and characterization of Meso-YNS is shown. Briefly, an anti-mesothelin lgG1 is made using standard methods in the art. The heavy chain isotype is a human gamma 1. The sequence of the Meso-YNS heavy chain is set forth and is disclosed as (SEQ ID NO: 54). The heavy chain variable region is set forth in (SEQ ID NO: 53) and contains the following CDR Sequences:

[0636] Mesothelin Heavy CDR Sequence(s):

[0637] IYGMH (SEQ ID NO: 56) VLWYDGSHEYYADSVKG (SEQ ID NO: 57) DGDYYDSGSPLDY (SEQ ID NO: 58) The heavy chain constant region set forth in (SEQ ID NO: 52), which further comprises an amino acid substitution at position 297 (N297Q) in order to mutate out the glycosylation site. This prevents the fusion protein from binding to the endogenous Fc receptor.

[0638] The Meso-YNS a light chain (SEQ ID NO: 55) which comprises a variable region (SEQ ID NO: 20), which further comprises the following CDR Sequences:

[0639] Mesothelin Light CDR Sequence(s):

[0640] RASQSVSSYLA (SEQ ID NO: 59) DASNRAT (SEQ ID NO: 60)

[0641]

[0642] QQRSNWPLT (SEQ ID NO: 61)

[0643]

[0644] The mask was generated as described in the instant specification, See, Methods of Masking IFNs of the Disclosure. The resulting construct denoted Meso-YNS comprises a signal peptide (set forth in red) (SEQ ID NO: 51), an IgG anti-meso heavy chain variable region (set forth in blue) and is described as (SEQ ID NO: 53), a human lgG1 heavy chain constant region (set forth in black) and is described as (SEQ ID NO: 52), further comprising a N297Q mutation (set forth in bold black) (i.e. asparagine to glutamine mutation at position 297), a fusion protein linker SGGAGGS (SEQ ID NO: 5) (set forth in green), IFNa (set forth in violet) further comprising a YNS Mutation (H57Y, E58N, and Q61S) (SEQ ID NO: 21).

[0645] The sequence of the Meso-YNS light chain is set forth in Figure 58 and is disclosed as (SEQ ID NO: 55).

[0646] The constructs set forth in Figure 58 are transiently expressed in CHO cells using methods known in the art. Further analysis of the heavy and light chain is assessed by mass spectrometer using standard methods.

[0647] Example 45: Characterization of Meso-YNS (Q159 Lot 1) by CE-SDS.

[0648] In this example, the characterization of Meso-YNS (Q159 Lot 1) was performed using Capillary Electrophoresis (CE-SDS). Samples were denatured by heating in the presence of SDS with or without reducing agent.

[0649] The results show that under reduced conditions, the protein separates into two peaks. One peak corresponding to the light chain had a measured mass of 26 kDa, while the peak corresponding to the heavy chain had a measured mass of 76 kDa. Under unreduced conditions the protein runs predominately as a single peak of approximately 178 kDa. See, Figure 59(A) and Figure 59(B).

[0650] Example 46: Methods of Evaluating Activity of Fusion Proteins In vivo.

[0651] In another experiment, 1x106Capan-1 cells were injected s.c into NCG mice in a 200ul volume 1:1 PBS Matrigel. Mice were treated twice a week with 5mg / kg of either QXL138AM (Q119), MESO- YNS (Q159) or MESO-YNS5.2 (Q158) on day 11, 14, 17, 21, 24, and 28 Tumor volumes were measured over time, when mice reached terminal tumor volumes (>1000mm3) values were kept to calculate the mean until all mice in the group reached terminal size.

[0652] The results show that while there were no complete responses (CRs) at this dose level, there is shown an elevated level of efficacy in Capan-1. (See, Figure 60).

[0653] In addition, measuring tumor volumes of individual mice on Day 56, the results show the first control mouse reached terminal size, while the tumor size for QXL138AM, Meso-YNSM5.2, and Meso-YNS sustained inhibition. (See, Figure 61). Example 47: Methods of Evaluating Activity of Meso-YNSM5.2 Fusion Protein In vivo.

[0654] in another experiment, 1x106Capan-1 ceils were injected s.c into NCG mice in a 200ul volume 1:1 PBS Matrigel. Mice were treated twice a week with either 2mg / kg, 1 mg / kg or 0,5mg / kg of MESO-YNS5.2 (Q158) on day 11, 14, 17, 21, 24, and 28. Tumor volumes were measured over time, when mice reached terminal tumor volumes (>1000mm3) values were kept to calculate the mean until all mice in the group reached terminal size.

[0655] The results show that while there were no complete responses (CRs) at this dose level, there is shown an elevated level of efficacy in ail three (3) doses (2 mg / kg, 1 mg / kg, and 0.5 mg / kg) in Capan-1. (See, Figure 62).

[0656] In addition, measuring tumor volumes of individual mice on Day 51, the results show the first control mouse reached terminal size, while the tumor size for all three (3) doses (2 mg / kg, 1 mg / kg, and 0.5 mg / kg) sustained inhibition. (See, Figure 63).

[0657] Finally, during the entirety of the treatment period, the results show no significant body weight change for all three (3) doses (2 mg / kg, 1 mg / kg, and 0.5 mg / kg) as well as no signs of toxicity. (See, Figure 64).

[0658] Example 48: Human Clinical Trials for the Treatment of Human Carcinomas through the Use of Masked IFN Fusion Protein which bind specific TAAs.

[0659] Masked IFN fusion protein which binds specific TAAs are synthesized in accordance with the present invention which specifically accumulates in a tumor cell and are used in the treatment of certain tumors and other immunological disorders and / or other diseases. In connection with each of these indications, two clinical approaches are successfully pursued.

[0660] I.) Adjunctive therapy: In adjunctive therapy, patients are treated with masked IFN fusion protein which bind specific TAAs in combination with a chemotherapeutic or pharmaceutical or biopharmaceutical agent or a combination thereof. Protocol designs address effectiveness as assessed by the following examples, including but not limited to, reduction in tumor mass of primary or metastatic lesions, increased progression free survival, overall survival, improvement of patient’s health, disease stabilization, as well as the ability to reduce usual doses of standard chemotherapy and other biologic agents. These dosage reductions allow additional and / or prolonged therapy by reducing dose-related toxicity of the chemotherapeutic or biologic agent.

[0661] II.) Monotherapy: In connection with the use of the masked IFN fusion protein which bind specific TAAs in monotherapy of tumors, the masked IFN fusion protein which bind specific TAAs are administered to patients without a chemotherapeutic or pharmaceutical or biological agent In one embodiment, monotherapy is conducted clinically in end-stage cancer patients with extensive metastatic disease. Protocol designs address effectiveness as assessed by the following examples, including but not limited to, reduction in tumor mass of primary or metastatic lesions, increased progression free survival, overall survival, improvement of patient’s health, disease stabilization, as well as the ability to reduce usual doses of standard chemotherapy and other biologic agents.

[0662] Dosage

[0663] Dosage regimens may be adjusted to provide the optimum desired response. For example, a single masked IFN fusion protein which bind specific TAA injection may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. “Dosage Unit Form” as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention is dictated by and directly dependent on (a) the unique characteristics of the masked IFN fusion protein which bind a specific TAA. and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such a compound for the treatment of sensitivity in individuals.

[0664] Clinical Development Plan (GDP)

[0665] The CDP follows and develops treatments of cancer(s) and / or immunological disorders using masked IFN fusion protein which binds specific TAAs of the disclosure in connection with adjunctive therapy or monotherapy. Trials initially demonstrate safety and thereafter confirm efficacy in repeat doses. Trials are open label comparing standard chemotherapy with standard therapy plus masked IFN fusion protein which bind specific TAAs. As will be appreciated, one non-limiting criteria that can be utilized in connection with enrollment of patients is concentration of masked IFN fusion protein which bind specific TAAs in a tumor as determined by standard detection methods known in the art.

[0666] The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any that are functionally equivalent are within the scope of the invention. Various modifications to the models, methods, and life cycle methodology of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention. Table I. Select Tumor Associated Antigen(s).

[0667] CD138 HPV E6 E7 CEA MUC1 EGFRvlll STEAP LMP2 HER-2 / neu MARTI CD20 TMPRSS2 PSMA gptOO NA17 PLAC1 EGFR B7-H4 GM3 PR1 ALK BORIS FAP NECTIN4 Tn Tyrosinase Cyclin B1 GloboH Folate Receptor Alpha CSPG4 ETV6-AML PSA RCC NY-BR-1 SLC34A2 TRP-2 RGS5 EphA2 GD3 SART3 PAP Fucosyl GM1 STn B7-H3 Mesothelin PAX5 AFP PSCA OY-TES1 EpCAM MAGE A1 SLITRK6 AKAP-4 XAGE 1 TAA 5T4 CD38 GPR94 SSX2 AXL CD-37

[0668]

[0669] Table IL List of Interferon(s) and functional mutants. Approved Symbol Approved Name IFNA1 Interferon alpha 1 IFNA2 Interferon alpha 2 IFNA4 Interferon alpha 4 IFNA5 Interferon alpha 5 IFNA6 Interferon alpha 6 IFNA7 Interferon alpha 7 IFNA8 Interferon alpha 3 IFNA10 Interferon alpha 10 IFNA11P Interferon alpha 11 IFNA12P Interferon alpha 12 IFNA13 Interferon alpha 13 IFNA14 Interferon alpha 14 IFNA16 Interferon alpha 16 IFNA17 Interferon alpha 17 IFNA20P Interferon alpha 20, pseudogene IFNA21 Interferon alpha 21 IFNA22P Interferon alpha 22, pseudogene IFNB1 Interferon beta 1 YNS IFNA

[0670]

[0671] Table HI. Amino Acid Abbreviations.

[0672] SINGLE LETTER THREE LETTER^ FULL NAMEH F Phe j phenylalanine L Leu leucine

[0673] S Ser serine

[0674] Y Tyr i tyrosine

[0675] C Cys i cysteine W Trp tryptophan P Pro | proline

[0676] H His histidine

[0677] Q Gin | glutamine R Arg I arginine

[0678] 1 lie I isoleucine [M Met methionine Thr threonine [N Asn j asparagine K Lys lysine

[0679] V Vai | valine

[0680] A Ala alanine

[0681] D Asp aspartic acid E Glu | glutamic acid G Gly! glycine

[0682]

Claims

1. CLAIMS:2.1) A “Targeted Masked IFN” comprising:3.a. an antibody which specifically binds to a Mesotheiin antigen, wherein said antibody comprises a heavy chain variable region at least 90% identical to (SEQ ID NO: 53) and a heavy chain constant region at least 90% identical to (SEQ ID NO: 52), and a variable light chain at least 95% identical to (SEQ ID NO: 20); and4.b. an I FNa further comprising a YNS Mutation (H57Y, E58N, and Q61S) (SEQ ID NO: 21) whereby said I FNa is attached at the C-terminal of said heavy chain constant region (SEQ ID NO: 52).5.2) The Targeted Masked IFN of claim 1, wherein the N-terminal of said I FNa is fused to the C- terminal of said antibody heavy and / or light chain further comprising a flexible peptide linker set forth in (SEQ ID NO: 5).6.3) The Targeted Masked IFN of claim 1, comprising the sequence set forth in (SEQ ID NO.: 54).7.4) The Targeted Masked IFN of claim 1, comprising the sequence set forth in (SEQ ID NO.: 55).8.5) The Targeted Masked IFN of claim 1, wherein said antibody binds mesotheiin.9.6) The Targeted Masked IFN of claim 1, wherein the heavy chain comprises a CDR sequence set forth In (SEQ ID NO: 56), (SEQ ID NO: 57), or (SEQ ID NO: 58).10.7) The Targeted Masked IFN of claim 1, wherein the light chain comprises a CDR sequence set forth in (SEQ ID NO: 59), (SEQ ID NO: 60), or (SEQ ID NO: 61).11.8) A method of making a Targeted Masked IFN of any of claim(s) 1.12.9) A kit comprising a Targeted Masked IFN of clalm(s) 1.13.10) A method of treating cancer in a subject, comprising administering to said subject a therapeutically effective amount of the Targeted Masked IFN according to claim(s) 1. 11) The method of claim 11, wherein the subject is human.14.12) The method of claim 11, wherein the cancer is pancreatic cancer.15.13) The method of claim 11, wherein the cancer is pancreatic mesothelioma.16.14) The method of claim 11, wherein the cancer is gastric carcinoma.17.15) A composition comprising the sequence set forth in Figure 58 (SEQ ID NO: 54).18.16) A composition comprising the sequence set forth in Figure 58 (SEQ ID NO: 55).19.17) The composition of claim 13, further comprising the CDR sequences set forth in (SEQ ID NO:20.56), (SEQ ID NO: 57), and (SEQ ID NO: 58). 18) The composition of claim 14, further comprising the CDR sequences set forth in (SEQ ID NO: 59), (SEQ ID NO: 60), and (SEQ ID NO: 61).21.19) A kit comprising the composition of claim 13.22.20) A kit comprising the composition of claim 14.

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