Preinterferon proteins and uses thereof

Abstract

The present disclosure provides protease activated IFN proproteins. The IFN proprotein comprises an IFN moiety that is made so as not to bind to its receptor by attachment to the Fc moiety via a protease cleavable linker so as to be sterically hindered. The protease cleavable linker is cleaved under the action of a protease, e.g., at a tumor site, thereby releasing and activating the IFN moiety. The IFN proprotein optionally further comprises a targeting moiety, e.g., a targeting moiety that recognizes a tumor-associated antigen and directs the proprotein to a tumor site. The disclosure further provides pharmaceutical compositions comprising the IFN proproteins and methods of using the IFN proproteins in therapy, as well as nucleic acids encoding the IFN proproteins, recombinant cells expressing the IFN proproteins, and methods of producing the IFN proproteins.

Description

[0001] 1. Cross - reference to related applications

[0002] This application claims the priority benefit of U.S. Provisional Application No. 63 / 399,040, filed on August 18, 2022, U.S. Provisional Application No. 63 / 383,804, filed on November 15, 2022, and U.S. Provisional Application No. 63 / 481,303, filed on January 24, 2023. The content of each of these U.S. Provisional Applications is hereby incorporated by reference in its entirety.

[0003] 2. Sequence Listing

[0004] This application contains a Sequence Listing that has been electronically submitted and is hereby incorporated by reference in its entirety. The copy created on August 15, 2023, is named RGN-020WO_SL.xml and is 361,113 bytes in size. 3. Background of the Invention

[0005] Type I interferons (IFNs) are thought to directly inhibit tumor cell proliferation. Type I IFNs have utility in the treatment of several types of cancer, including hematological malignancies (chronic myeloid leukemia, hairy cell leukemia, multiple myeloma, and non-Hodgkin lymphoma) and solid tumors (melanoma, renal cell carcinoma, and Kaposi sarcoma). See, e.g., Zitvogel et al., 2015, Nat Rev Immunol 15:405-414 and Antonelli et al., 2015, Cytokine Growth Factor Rev 26:121-131.

[0006] A particular advantage of type I IFN therapy is its ability to intervene at multiple points in the generation of the anti-tumor immune response, including stimulation of innate and adaptive cytotoxic lymphocyte populations, negative regulation of inhibitory cell types, effects on tumor cells by inhibiting proliferation and regulating apoptosis, differentiation, migration, and cell surface antigen expression (Parker et al., 2016, Nature Reviews Cancer 16:131–144).

[0007] One of the greatest obstacles to the clinical use of type I IFN is the severe side effects associated with such treatments. The most common side effects are flu-like symptoms, hematotoxicity, elevated transaminases, nausea, fatigue, and psychiatric sequelae. These side effects prevent the attainment and maintenance of the doses required to achieve maximal therapeutic efficacy, and their occurrence may completely outweigh the clinical benefits of type I IFN treatment (Lotrich, 2009, Dialogues Clin Neurosci 11:417-425). Type I IFN signals through the IFNAR1 / IFNAR2 complex, which is expressed on most cells and tissues in the body. Thus, the ability to preferentially or specifically deliver active type I IFN to tumor-reactive immune cells (see, e.g., Diamond et al., 2011, J Exp Med. 208(10):1989–2003; Cauwels et al., 2018, Cancer Res. 78(2):463–474) or to the tumor microenvironment is crucial for the continued clinical use of type I IFN. Strategies are needed to modify type I IFN to obtain a drug that preferentially acts on tumor-reactive immune cells and / or exerts its activity at the tumor site and also reduces side effects on normal IFNAR-expressing cells.

[0008] Accordingly, there is a need in the art for new type I IFN therapies with improved therapeutic efficacy and safety profiles. 4. Summary of the Invention

[0009] The present disclosure relates to IFN proproteins that are activated by proteases (e.g., proteases expressed in the tumor environment).

[0010] The IFN proprotein comprises an IFN moiety that is sterically hindered from binding to its receptor and is constructed such that the IFN moiety is activated by protease cleavage of a linker in the IFN proprotein, resulting in alleviation of the steric hindrance of the IFN moiety. The IFN proprotein preferably further comprises a targeting moiety that directs the IFN proprotein to a particular tissue or cell type.

[0011] Generally, the IFN proproteins of the present disclosure comprise two polypeptide chains, each polypeptide chain comprising, from the N-terminus to the C-terminus, a first linker, an interferon (IFN) moiety, a second linker, and an Fc domain. In some embodiments, both the first linker and the second linker are protease-cleavable linkers (PCLs). In other embodiments, only one of the first linker and the second linker is a PCL and the other is a non-cleavable linker (NCL). Thus, in some embodiments, the first linker is a PCL and the second linker is an NCL. In other embodiments, the first linker is an NCL and the second linker is a PCL.

[0012] The IFN proprotein can further comprise a targeting moiety (or a component thereof, e.g., one chain of a Fab) at the N-terminus of, for example, one or two Fc domains. The targeting moiety comprises an antigen-binding domain ("ABD") that can bind, for example, to a target molecule present on the surface of a tumor (e.g., a tumor-associated antigen) or other components in the tumor microenvironment (e.g., the extracellular matrix) ("ECM") or tumor lymphocytes).

[0013] Exemplary IFN moieties for use in the IFN proproteins of the present disclosure are described in Section 6.3.

[0014] The protease-cleavable linkers for use in the IFN proproteins of the present disclosure are described in Section 6.4.

[0015] The non-cleavable linkers for use in the IFN proproteins of the present disclosure are described in Section 6.5.

[0016] The targeting moieties for use in the IFN proproteins of the present disclosure are described in Section 6.6, and the targeting moiety forms are disclosed in Section 6.7.

[0017] The Fc domains that can be incorporated into the IFN proproteins of the present disclosure are described in Section 6.8.

[0018] Exemplary IFN proproteins of the present disclosure are described in Section 6.2 and numbered Examples 1 to 146.

[0019] The present disclosure further provides nucleic acids encoding the IFN proproteins of the present disclosure. The nucleic acids encoding the IFN proproteins can be a single nucleic acid (e.g., a vector encoding all the polypeptide chains of the IFN proprotein) or multiple nucleic acids (e.g., two or more vectors encoding different polypeptide chains of the IFN proprotein). The present disclosure further provides host cells and cell lines engineered to express the nucleic acids and IFN proproteins of the present disclosure. The present disclosure further provides methods for producing the IFN proproteins of the present disclosure. Exemplary nucleic acids, host cells and cell lines, and methods for producing IFN proproteins are described in Section 6.9 and numbered Examples 147 to 149.

[0020] The present disclosure further provides pharmaceutical compositions comprising the IFN proproteins of the present disclosure. Exemplary pharmaceutical compositions are described in Section 6.10 and numbered Example 150.

[0021] The present disclosure further provides methods of using the IFN proproteins and pharmaceutical compositions of the present disclosure, for example, to treat cancer. Exemplary methods are described in Section 6.11 and numbered Examples 151 to 201. 5. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figures 1A - 1CIs a schematic diagram showing the configurations of three IFN proproteins of the present disclosure, where a protease-cleavable linker flanks the interferon portion. Figure 1A Represents an IFN proprotein having the overall configuration antibody-PCL-IFN-PCL-Fc; Figure 1B Represents a single-hinge IFN proprotein having the configuration Fab-PCL-IFN-PCL-hinge-Fc; and Figure 1C Represents a double-hinge IFN proprotein having the configuration Fab-hinge-PCL-IFN-PCL-hinge-Fc. Although the targeting portion is shown in the form of a Fab, the VH, VL, and CL domains of the Fab are optional for non-targeted IFN proproteins or can be replaced by other targeting portions (such as scFv).

[0023] Figures 2A - 2F Is a schematic diagram showing the configurations of six IFN proproteins of the present disclosure, where only the protease-cleavable linker (PCL) is located on one side of the interferon portion and the non-cleavable linker (NCL) is located on the other side of the interferon portion. Figure 2A Represents an IFN proprotein having the overall configuration antibody-NCL-IFN-PCL-Fc; Figure 2B Represents a single-hinge IFN proprotein having the configuration Fab-NCL-IFN-PCL-hinge-Fc; Figure 2C Represents a double-hinge IFN proprotein having the configuration Fab-hinge-NCL-IFN-PCL-hinge-Fc; Figure 2D Represents an IFN proprotein having the overall configuration antibody-PCL-IFN-NCL-Fc; Figure 2E Represents a single-hinge IFN proprotein having the configuration Fab-PCL-IFN-NCL-hinge-Fc; and Figure 2F Represents a double-hinge IFN proprotein having the configuration Fab-hinge-PCL-IFN-NCL-hinge-Fc. Although the targeting portion is shown in the form of a Fab, the VH, VL, and CL domains of the Fab are optional for non-targeted IFN proproteins or can be replaced by other targeting portions (such as scFv).

[0024] Figure 3Table of exemplary targeting IFN pro - proteins and their constituent polypeptide chains according to Figure 1. TM refers to the targeting moiety; HC refers to the heavy chain of the antibody; LC refers to the light chain of the antibody; IFN refers to the interferon (IFN) moiety, and ΔN and ΔC refer to the N - terminal and C - terminal truncations in the IFN sequence of the IFN moiety (e.g., as described in Section 6.3); PCL refers to the protease - cleavable linker (e.g., as described in Section 6.4), Fc refers to the Fc domain (e.g., as described in Section 6.8); hinge refers to the hinge sequence of the antibody, and long hinge and short hinge refer to the full - length or truncated versions of the immunoglobulin hinge sequence (e.g., as described in Section 6.8.3). Further, although shown with protease - cleavable linkers (PCL) flanking the IFN moiety, one PCL in each of Chain 1 and Chain 2 can be replaced with a non - cleavable linker (NCL), e.g., as described in Section 6.5.

[0025] Figures 4A - 4C Is the size - exclusion ultra - performance liquid chromatography (SE - UPLC) profile of exemplary IFN molecules that can be incorporated into the IFN pro - protein constructs of the present disclosure. Figure 4A Illustrates the SEC profile of the IFN molecule Fc - IFNα1 with an N - terminal Fc domain and a C - terminal IFN moiety; Figure 4B Illustrates the SEC profile of the IFN molecule Fc - IFNα2b with an N - terminal Fc and a C - terminal IFN moiety; and Figure 4C Illustrates the SEC profile of the IFN molecule IFNα2b - Fc with an N - terminal IFN moiety and a C - terminal Fc domain.

[0026] Figures 5A - 5C Depicts the in vitro activity of exemplary IFN molecules linked to the Fc molecule at the N - terminal or C - terminal. Figure 5A The sketch images in represent the N - terminal and C - terminal Fc fusions of IFN. Fc - IFN is the overall representation of an IFN molecule with an N - terminal Fc domain and a C - terminal IFN moiety, while IFN - Fc is the overall representation of an IFN molecule with an N - terminal IFN moiety and a C - terminal Fc domain. Figure 5B Is a graph showing the in vitro activity of exemplary IFN molecules, Fc - IFNα2b and IFNα2b - Fc compared to unlinked IFNα2b. Figure 5C Is a graph showing the activity of Fc - IFN molecules compared to different unlinked IFNs.

[0027] Figures 6A - 6D Is the SE - UPLC profile of exemplary mutant IFN molecules that can be incorporated into the IFN pro - protein constructs of the present disclosure. Figure 6AShows the SE-UPLC chromatogram of the mutant IFN molecule Fc-IFNα2bR33A. Figure 6B Shows the SE-UPLC chromatogram of the mutant IFN molecule Fc-IFNα2bR149A. Figure 6C Shows the SE-UPLC chromatogram of the mutant IFN molecule Fc-IFNα2bR120A. Figure 6D Shows the SE-UPLC chromatogram of the mutant IFN molecule Fc-IFNα2bS152A.

[0028] Figures 7A - 7B Depicts the in vitro activity of exemplary mutant IFN molecules that can be incorporated into the IFN preprotein constructs of the present disclosure. Figure 7A The schematic images in show the overall structures of wild-type (WT) or mutant (Mut) Fc-IFN molecules. Figure 7B Is a graph showing the in vitro activity of Fc-IFNα2b molecules with mutations affecting the IFNAR1 or IFNAR2 interface.

[0029] Figures 8A - 8C Is the SE-UPLC chromatogram of an exemplary IFN preprotein construct. Figure 8A Shows the SE-UPLC chromatogram of the single-hinge full-length IFN preprotein aPD1-single-hinge-FLIFN. Figure 8B Shows the SE-UPLC chromatogram of the double-hinge IFN preprotein aPD1-double-hinge-long-IFN. Figure 8C Shows the SEC chromatogram of an IFN preprotein with two Fc regions.

[0030] Figures 9A - 9B Shows the in vitro enzymatic cleavage of single and double-hinge IFN preproteins. Figure 9A Is a gel image showing the cleavage of three single-hinge preproteins, two double-hinge IFN preproteins, and a positive control by uPA. Similarly, Figure 9B Is a gel image showing the cleavage of the same single and double IFN preproteins by MMP2 and MMP9.

[0031] Figures 10A - 10B Depicts the in vitro activity of exemplary IFN preproteins compared to IFNα2b and Fc-IFNα2b. Figure 10A Is a graph showing the in vitro activity of two single-hinge IFN preproteins (full-length and truncated) and one double-hinge IFN preprotein. Figure 10B Is a graph showing Figure 10A The change in activity of the same IFN preprotein in by adding only MMP buffer (dashed line) or MMP buffer and enzyme mixture (dashed line) in. 6. Specific Embodiments

[0032] 6.1. Definitions

[0033] The following terms as used herein have the following meanings:

[0034] ABD chain, targeting partial chain : The targeting moiety and the antigen-binding site (ABD) therein can exist as a single polypeptide chain (e.g., in the case of scFv or scFab) or be formed by the association of more than one polypeptide chain (e.g., in the case of Fab or Fv). As used herein, the terms "ABD chain" and "targeting moiety chain" refer to all or part of the ABD or targeting moiety present on a single polypeptide chain. The use of the terms "ABD chain" or "targeting moiety chain" is for convenience and descriptive purposes only and does not imply a particular configuration or method of production. In addition, references to ABD or targeting moiety when describing an IFN proprotein encompass ABD chain or targeting moiety chain unless the context indicates otherwise. Thus, when describing an IFN proprotein in which an Fc domain is operably linked to a targeting moiety, the Fc domain can be directly or indirectly covalently linked via a peptide bond (e.g., via a linker) to, for example, (1) the first ABD or targeting moiety chain of a Fab or Fv (where the other components of the Fab or Fv are on a second relevant ABD or targeting moiety chain) or (2) an ABD or targeting moiety chain containing an scFv or scFab.

[0035] about : Throughout the specification, the use of terms such as "about", "approximately", etc. in front of a number indicates that the number is not necessarily exact (e.g., taking into account variations in fractions, measurement accuracy and / or precision, timing, etc.). It should be understood that the disclosure of "about X" or "approximately X" for a number X is also a disclosure of "X". Thus, for example, the disclosure of an embodiment in which one sequence has "about X% sequence identity" with another sequence is also a disclosure of an embodiment in which the sequence has "X% sequence identity" with the other sequence.

[0036] activate (activation) : Terms such as "activation" etc. when used in connection with the IFN proproteins of the present disclosure refer to protease-mediated enzymatic cleavage of a protease-cleavable linker, which enzymatic cleavage results in the release of the IFN moiety from the spatially blocking constant domain.

[0037] and / or : Unless otherwise indicated, the disjunctive "or" should be used in its proper sense as a Boolean logical operator, covering the selection of features in the alternatives (A or B, where the selection of A is mutually exclusive with B) and the selection of combined features (A or B, where both A and B are selected). In some places in this text, the term "and / or" is used for the same purpose, which should not be construed as implying that "or" is used to refer to mutually exclusive alternatives.

[0038] antibody:As used herein, the term "antibody" refers to a polypeptide (or group of polypeptides) of the immunoglobulin family that can bind an antigen non-covalently, reversibly, and specifically. For example, a naturally occurring "antibody" of the IgG type is a tetramer that contains at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region contains one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) that are interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, bispecific or multispecific antibodies, and anti-idiotypic (anti-Id) antibodies. Antibodies can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Both the light and heavy chains are divided into structurally and functionally homologous regions. The terms "constant" and "variable" are used functionally. In this regard, it should be understood that the variable domains of both the light chain (VL) and heavy chain (VH) portions determine antigen recognition and specificity. In contrast, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the numbering of the constant domain structures increases as they become more distal from the antigen-binding domain or amino terminus of the antibody. The N-terminus is the variable region, and the C-terminus is the constant region; the CH3 and CL domains represent the carboxyl termini of the heavy and light chains of a native antibody, respectively. For convenience, and unless the context dictates otherwise, a reference to an antibody also refers to antibody fragments and engineered antibodies that include non-naturally occurring antigen-binding domains and / or antigen-binding domains having non-natural structures.

[0039] antigen - binding domain:As used herein, the term "antigen-binding domain" or "ABD" refers to the portion of an antibody or antibody fragment (e.g., targeting moiety) that has the ability to non-covalently, reversibly, and specifically bind to an antigen. Examples of antibody fragments that may contain an ABD include, but are not limited to, single-chain Fv (scFv), Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments, which are divalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragments, which consist of VH and CH1 domains; Fv fragments, which consist of VL and VH domains of a single arm of an antibody; dAb fragments (Ward et al., 1989, Nature 341:544-546), which consist of VH domains; and isolated complementarity-determining regions (CDRs). Thus, the term "antibody fragment" encompasses proteolytic fragments of an antibody (e.g., Fab and F(ab) 2 fragments) and engineered proteins containing one or more portions of an antibody (e.g., scFv). Antibody fragments can also be incorporated into single-domain antibodies, gigantibodies, minibodies, intracellular antibodies, diabodies, triabodies, tetra-bodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology 23:1126-1136).

[0040] associate :In the context of an IFN precursor protein, the term "association" refers to the functional relationship between two or more polypeptide chains. Specifically, the term "association" means that two or more polypeptides associate with each other (e.g., non-covalently through molecular interactions or covalently through one or more disulfide bridges or chemical crosslinks) to produce a functional IFN precursor protein. Examples of associations that may be present in the IFN precursor proteins of the present disclosure include, but are not limited to, associations between Fc domains that are to form an Fc region (such as the homodimeric or heterodimeric types described in Section 6.8), associations between the VH and VL regions in a Fab or Fv, and associations between CH1 and CL in a Fab.

[0041] cancer: The term "cancer" refers to diseases characterized by the uncontrolled (and often rapid) growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein, and these examples include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, adrenal cancer, ganglionic cancer, biliary tract cancer, bone cancer, endometrial cancer, eye cancer, fallopian tube cancer, genital tract cancer, large bowel cancer, meningeal cancer, esophageal cancer, peritoneal cancer, pituitary cancer, penile cancer, placental cancer, pleural cancer, salivary gland cancer, small intestine cancer, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, upper respiratory and digestive system cancers, urinary tract cancer, vaginal cancer, vulvar cancer, lymphoma, leukemia, lung cancer, etc., for example, any TAA-positive cancer of any of the foregoing types.

[0042] complementary determining region: As used herein, the term "complementary determining region" or "CDR" refers to the sequences of amino acids within the variable region of an antibody that confer antigen specificity and binding affinity. For example, typically, there are three CDRs in each heavy chain variable region (e.g., CDR-H1, CDR-H2, and CDR-H3), and three CDRs in each light chain variable region (CDR-L1, CDR-L2, and CDR-L3). The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al., 1991, "Sequences of Proteins of Immunological Interest," 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., 1997, JMB 273:927-948 ("Chothia" numbering scheme), and the ImMunoGenTics (IMGT) numbering (Lefranc, 1999, The Immunologist 7:132-136; Lefranc et al., 2003, Dev. Comp. Immunol. 27:55-77 ("IMGT" numbering scheme). For example, for the classical form, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3). Under Chothia, the CDR amino acid numbering in VH is 26-32 (CDR-H1), 52-56 (CDR-H2), and 95-102 (CDR-H3); and the amino acid residues in VL are numbered 26-32 (CDR-L1), 50-52 (CDR-L2), and 91-96 (CDR-L3). By combining the CDR definitions of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) in human VH and amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3) in human VL.Under IMGT, the CDR amino acid residue numbers in VH are approximately 26 - 35 (CDR-H1), 51 - 57 (CDR-H2), and 93 - 102 (CDR-H3), and the CDR amino acid residue numbers in VL are approximately 27 - 32 (CDR-L1), 50 - 52 (CDR-L2), and 89 - 97 (CDR-L3) (according to "Kabat" numbering). Under IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0043] constant domain The term "constant domain" refers to the CH1, CH2, CH3, or CL domain of an immunoglobulin.

[0044] The term "CH1 domain" refers to the heavy chain constant region that connects the variable domain to the hinge in the heavy chain constant domain. In some embodiments, the term "CH1 domain" refers to the region of an immunoglobulin molecule spanning amino acids 118 to 215 (EU numbering). The term "CH1 domain" encompasses wild-type CH1 domains and their variants (e.g., non-naturally occurring CH1 domains or modified CH1 domains). For example, the term "CH1 domain" includes wild-type IgG1, IgG2, IgG3, and IgG4 CH1 domains and their variants having 1, 2, 3, 4, 5, 1 to 3, 1 to 5, 3 to 5, and / or up to 5, 4, 3, 2, or 1 mutations (e.g., substitutions, deletions, and / or additions). Exemplary CH1 domains include CH1 domains having mutations that alter the biological activity of the antibody, such as ADCC, CDC, or half-life.

[0045] The term "CH2 domain" refers to the heavy chain constant region that connects the hinge to the CH3 domain in the heavy chain constant domain. In some embodiments, the term "CH2 domain" refers to the region of an immunoglobulin molecule spanning amino acids 238 to 340 (EU numbering). The term "CH2 domain" encompasses wild-type CH2 domains and their variants (e.g., non-naturally occurring CH2 domains or modified CH2 domains). For example, the term "CH2 domain" includes wild-type IgG1, IgG2, IgG3, and IgG4 CH2 domains and their variants having 1, 2, 3, 4, 5, 1 to 3, 1 to 5, 3 to 5, and / or up to 5, 4, 3, 2, or 1 mutations (e.g., substitutions, deletions, and / or additions). Exemplary CH2 domains include CH2 domains having mutations that alter the biological activity of the antibody, such as ADCC, CDC, purification, dimerization, and half-life.

[0046] The term "CH3 domain" refers to the constant region of the heavy chain located at the C-terminus of the CH2 domain within the heavy chain constant domain. In some embodiments, the term "CH3 domain" refers to the region of an immunoglobulin molecule spanning amino acids 341 to 447 (EU numbering). The term "CH3 domain" encompasses wild-type CH3 domains and variants thereof (e.g., non-naturally occurring CH3 domains or modified CH3 domains). For example, the term "CH3 domain" includes wild-type IgG1, IgG2, IgG3, and IgG4 CH3 domains and variants thereof having 1, 2, 3, 4, 5, 1 to 3, 1 to 5, 3 to 5, and / or up to 5, 4, 3, 2, or 1 mutations (e.g., substitutions, deletions, and / or additions). Exemplary CH3 domains include CH3 domains having mutations that alter the biological activity of the antibody, such as ADCC, CDC, purification, dimerization, and half-life.

[0047] The term "CL domain" refers to the constant region of the immunoglobulin light chain. The term "CL domain" encompasses wild-type CL domains (e.g., κ or λ light chain constant regions) and variants thereof (e.g., non-naturally occurring CL domains or modified CL domains). For example, the term "CL domain" includes wild-type κ and λ constant domains and variants thereof having 1, 2, 3, 4, 5, 1 to 3, 1 to 5, 3 to 5, and / or up to 5, 4, 3, 2, or 1 mutations (e.g., substitutions, deletions, and / or additions).

[0048] effector function:The term "effector function" refers to the activity of an antibody molecule mediated by the binding of domains other than the antigen-binding domain of the antibody (usually mediated by the binding of effector molecules). Effector functions include complement-mediated effector functions, which are mediated, for example, by binding the C1 component of complement to the antibody. Activation of complement is important in the opsonization and lysis of cellular pathogens. Activation of complement also stimulates an inflammatory response and may also be involved in autoimmune hypersensitivity reactions. Effector functions also include Fc receptor (FcR)-mediated effector functions, which can be triggered upon binding of the constant domain of the antibody to an Fc receptor (FcR). Binding of an antibody to an Fc receptor on the cell surface initiates many important and diverse biological responses, which include phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (referred to as antibody-dependent cell-mediated cytotoxicity or ADCC), release of inflammatory mediators, control of placental transfer, and immunoglobulin production. The effector function of an antibody can be altered by changing (e.g., enhancing or decreasing) the affinity of the antibody for effector molecules such as Fc receptors or complement components. The binding affinity is usually altered by modifying the effector molecule binding site, and in such cases, it is appropriate to localize the site of interest and modify at least a portion of the site in a suitable manner. It is also contemplated that alterations in the binding site on the antibody for an effector molecule need not significantly alter the overall binding affinity but may alter the geometry of the interaction such that effector mechanisms are rendered ineffective as in non-productive binding. It is further contemplated that effector functions can also be altered by modifying sites that do not directly participate in effector molecule binding but otherwise participate in the execution of effector functions.

[0049] epitope :An epitope or antigenic determinant is a portion of an antigen recognized by an antibody or other antigen-binding moiety as described herein. Epitopes can be linear or conformational.

[0050] Fab: The term "Fab" refers to a pair of polypeptide chains, where the first polypeptide chain contains the variable heavy chain (VH) domain of an antibody, which is operably linked (usually at the N-terminus) to a first constant domain (referred to herein as C1), and the second polypeptide chain contains the variable light chain (VL) domain at the N-terminus of the antibody, which is operably linked (usually at the N-terminus) to a second constant domain (referred to herein as C2) that is capable of pairing with the first constant domain. In a native antibody, VH is located at the N-terminus of the first constant domain (CH1) of the heavy chain, and VL is located at the N-terminus of the constant domain of the light chain (CL). The Fabs of the present disclosure can be arranged according to the native orientation or include domain substitutions or exchanges that promote correct VH and VL pairing. For example, the CH1 and CL domain pair in a Fab can be replaced with a CH3 domain pair to promote correct modified Fab chain pairing in a heterodimeric molecule. It is also possible to reverse CH1 and CL such that CH1 is attached to VL and CL is attached to VH, and this configuration is generally referred to as Crossmab. The term "Fab" encompasses single-chain Fabs.

[0051] Fc domain and Fc region : The term "Fc domain" refers to a portion of the heavy chain that pairs with the corresponding portion of another heavy chain. In some embodiments, the Fc domain contains a CH2 domain followed by a CH3 domain, which may or may not have a hinge region at the N-terminus of the CH2 domain. The term "Fc region" refers to the region formed by the association of two heavy chain Fc domains. The two Fc domains within the Fc region can be the same as or different from each other. In a native antibody, the Fc domains are typically the same, but one or both Fc domains may be modified to allow heterodimerization, for example, via a knob-into-hole interaction.

[0052] Fv : The term "Fv" refers to the smallest antibody fragment that can be derived from an immunoglobulin containing the complete target recognition and binding site. This region consists of a dimer (VH-VL dimer) formed by a non-covalent tight association of a variable heavy chain domain and a variable light chain domain. It is in this configuration that the three CDRs of each variable domain interact to define the target binding site on the surface of the VH-VL dimer. Generally, six CDRs confer the target binding specificity of an antibody. However, in some cases, even a single variable domain (or half of the Fv containing only the three CDRs with target specificity) can have the ability to recognize and bind a target. The reference to the VH-VL dimer herein does not imply any particular configuration. When present on a single polypeptide chain (e.g., scFv), VH is located at the N-terminus or C-terminus of VL.

[0053] half - antibody:The term "half-antibody" refers to a molecule that comprises at least one Fc domain and can associate with another Fc-containing molecule through, for example, disulfide bridges or molecular interactions. A half-antibody can consist of one polypeptide chain or more than one polypeptide chain (e.g., the two polypeptide chains of a Fab). Examples of half-antibodies are molecules that comprise the heavy and light chains of an antibody (e.g., an IgG antibody). Another example of a half-antibody is a molecule that comprises a first polypeptide containing a VL domain and a CL domain and a second polypeptide containing a VH domain, a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, wherein the VL and VH domains form an ABD. Yet another example of a half-antibody is a polypeptide that comprises an scFv domain, a CH2 domain, and a CH3 domain.

[0054] The IFN proprotein of the present disclosure typically comprises two half-antibodies, each half-antibody comprising an IFN moiety flanked by protease-cleavable linkers, with its Fc domain at the C-terminus of the C-terminal protease-cleavable linker and the constant domain at the N-terminus of the N-terminal protease-cleavable linker. One or both of the half-antibodies in the IFN proprotein can further comprise a targeting moiety.

[0055] The term "half-antibody" is used for descriptive purposes only and does not imply a particular configuration or method of production. Describing a half-antibody as a "first" half-antibody, "second" half-antibody, "left" half-antibody, "right" half-antibody, etc. is for convenience and descriptive purposes only.

[0056] host cell or recombinant host cell:The term "host cell" or "recombinant host cell" refers to, for example, a cell that has been genetically engineered by the introduction of heterologous nucleic acid. It should be understood that such terms refer not only to a particular subject cell but also to progeny of such a cell. Since certain modifications may occur in progeny due to mutation or environmental influences, such progeny may in fact differ from the parental cell but are still included within the scope of the term "host cell" as used herein. A host cell may transiently carry heterologous nucleic acid on an episomal heterologous expression vector, or may stably carry heterologous nucleic acid, for example, by integration of the heterologous nucleic acid into the host cell genome. For the purpose of expressing the IFN precursor protein of the present disclosure, the host cell is preferably a cell line of mammalian origin or mammalian-like characteristics, such as monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293), baby hamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2 / 0, HeLa, Madin-Darby bovine kidney (MDBK), myeloma and lymphoma cells or derivatives and / or engineered variants thereof. Engineered variants include, for example, derivatives that grow at a higher density than the original cell line and / or derivatives with modified glycan profiles and / or derivatives with site-specific integration sites.

[0057] interferon :The term "interferon" as used herein refers to full-length interferon or modified interferon, such as truncated and / or mutant interferon. In some embodiments, the modified interferon is attenuated compared to the corresponding wild-type interferon (e.g., retains less than 50%, less than 40%, less than 30%, less than 20% or less than 10%, less than 1%, less than 0.1% or less than 0.05% activity in an in vitro luciferase reporter assay as described in Section 8.2.4). In some embodiments, the modified interferon attenuates within a range defined by any two of the foregoing values, e.g., 0.05% to 50%, 0.1% to 20%, 0.1% to 10%, 0.05% to 5%, 1% to 20%, and so on. In other embodiments, the modified interferon substantially retains the biological activity of the corresponding wild-type interferon (e.g., retains at least 50% activity in an in vitro luciferase reporter assay as described in Section 8.2.4). Interferons include type I interferons (e.g., interferon-α and interferon-β) and type II interferons (e.g., interferon-γ).

[0058] linker :As used herein, the term "linker" refers to a protease-cleavable linker or a non-cleavable linker.

[0059] non - cleavable linker:As used herein, an uncleavable linker refers to a peptide whose amino acid sequence lacks the substrate sequence of a protease (e.g., the protease described in Section 6.4.1, which recognizes and cleaves a specific sequence motif, e.g., the substrate described in Section 6.4.2).

[0060] operably linked :The term "operably linked" refers to a functional relationship between two or more peptide or polypeptide domains or nucleic acid (e.g., DNA) segments. In the context of a fusion protein or other polypeptide, the term "operably linked" means that two or more amino acid fragments are joined to produce a functional polypeptide. For example, in the context of the IFN proprotein of the present disclosure, the individual components (e.g., the Fc domain and the IFN portion) can be operably linked directly or via a peptide linker sequence. In the context of a nucleic acid encoding a fusion protein (such as a half-antibody of the IFN proprotein of the present disclosure), "operably linked" means that two nucleic acids are joined such that the amino acid sequences encoded by the two nucleic acids remain in-frame. In the context of transcriptional regulation, the term refers to the functional relationship between a transcriptional regulatory sequence and a transcriptional sequence. For example, if a promoter or enhancer sequence stimulates or regulates the transcription of a coding sequence in a suitable host cell or other expression system, then the promoter or enhancer sequence is operably linked to the coding sequence.

[0061] polypeptide, peptide and protein :The terms "polypeptide", "peptide" and "protein" are used interchangeably herein and refer to polymers of amino acid residues.

[0062] pre - protein :A "proprotein" is an inactive protein precursor and can be activated by proteolytic cleavage by a protease. Thus, a proprotein is "protease activatable".

[0063] protease :As used herein, the term "protease" refers to any enzyme that catalyzes the hydrolysis of peptide bonds. Generally, proteases useful in the present disclosure, e.g., the proteases described in Section 6.4.1, recognize and cleave specific sequence motifs, e.g., the substrates described in Section 6.4.2. Preferably, proteases are expressed at higher levels in cancerous tissues compared to normal tissues.

[0064] protease - cleavable linker :As used herein, the term "protease-cleavable linker" or "PCL" refers to a peptide whose amino acid sequence contains one or more (e.g., two, three or more) substrate sequences of one or more proteases. Exemplary protease-cleavable linkers are described in Section 6.4, and exemplary protease-cleavable linker sequences are disclosed in Section 6.4.4.

[0065] recognize: As used herein, the term "recognize" refers to an antibody or antibody fragment (e.g., targeting moiety) that finds its epitope and interacts (e.g., binds) with that epitope.

[0066] single - chain Fab or scFab : As used herein, the term "single-chain Fab" or "scFab" refers to an ABD comprising a VH domain, a CH1 domain, a VL domain, a CL domain, and a linker. In some embodiments, the foregoing domains and linker are arranged in one of the following orders in an N-terminus to C-terminus orientation: (a) VH-CH1-linker-VL-CL, (b) VL-CL-linker-VH-CH1, (c) VH-CL-linker-VL-CH1, or (d) VL-CH1-linker-VH-CL. The linker is preferably a non-cleavable linker of at least 30 amino acids, more preferably between 32 and 50 amino acids. The single-chain Fab fragment is typically stabilized by the native disulfide bond between the CL domain and the CH1 domain. In addition, these single-chain Fab molecules can be further stabilized by generating interchain disulfide bonds via the insertion of cysteine residues (e.g., at position 44 in the VH domain and position 100 in the VL domain according to Kabat numbering).

[0067] single - chain Fv or scFv : As used herein, the term "single-chain Fv" or "scFv" refers to an ABD comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the required structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenberg and Moore eds. (1994), Springer-Verlag, New York, pp. 269-315. The VH and VL can be arranged in an N-terminus to C-terminus (i.e., VH-VL or VL-VH) order, typically separated by a linker, e.g., as listed in Table E.

[0068] spacer : As used herein, the term "spacer" refers to a peptide incorporated into a linker containing a substrate, the amino acid sequence of which is not a substrate for a protease. The spacer can be used to separate the substrate from other domains (e.g., ABD) in a molecule. In some aspects, the residues in the spacer minimize aminopeptidase and / or exopeptidase action to prevent cleavage of the N-terminal amino acid.

[0069] specific (or selective) binding: The term "specifically (or selectively) binds" to an antigen or epitope refers to a binding reaction that determines the presence of a homologous antigen or epitope in a heterogeneous population of proteins and other molecules. The binding reaction can, but need not, be mediated by an antibody or antibody fragment. The term "specific binding" does not exclude cross-species reactivity. For example, an antigen-binding domain (e.g., an antigen-binding fragment of an antibody) that "specifically binds" to an antigen from one species may also "specifically bind" to that antigen in one or more other species. Thus, such cross-species reactivity per se does not change the classification of the antigen-binding domain as a "specific" binder. In certain embodiments, an antigen-binding domain of the present disclosure that specifically binds to a human antigen has cross-species reactivity with one or more non-human mammalian species (e.g., primate species (including but not limited to one or more of cynomolgus monkeys, rhesus monkeys, and pig-tailed monkeys)) or rodents (e.g., Mus musculus).

[0070] subject : The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. In a preferred embodiment, the subject is a human.

[0071] substrate : The term "substrate" refers to a peptide sequence on which a protease will act and in which the protease will cleave a peptide bond.

[0072] target molecule : As used herein, the term "target molecule" refers to any biomolecule (e.g., protein, carbohydrate, lipid, or a combination thereof) expressed on the cell surface or in the extracellular matrix that can be specifically bound by a targeting moiety in the IFN precursor protein of the present disclosure.

[0073] targeting moiety : As used herein, the term "targeting moiety" refers to any molecule or binding moiety (e.g., an immunoglobulin or antigen-binding fragment) that can bind to a cell surface or extracellular matrix molecule at a site (e.g., on a tumor cell or on a lymphocyte in the tumor microenvironment) to which the IFN precursor protein of the present disclosure is to be targeted. In some embodiments, the targeting moiety binds to a TAA. In other embodiments, the targeting moiety binds to a TCA. In addition to localizing the IFN precursor protein to a specific site, the targeting moiety may also have functional activity. For example, a targeting moiety that binds to a checkpoint inhibitor such as PD1 may also exhibit anti-tumor activity or enhance the anti-tumor activity of IFN, e.g., by inhibiting PD1 signaling.

[0074] T - cell antigen, TCA:The term "T cell antigen" or "TCA" refers to a molecule (usually a protein, carbohydrate, lipid, or some combination thereof) expressed on the surface of T lymphocytes and can be used to preferentially target a medicament to a specific site. In some embodiments, the site is cancer tissue and / or the T cell antigen is a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or virus lymphocyte, or a checkpoint inhibitor expressed on T lymphocytes.

[0075] tumor :The term "tumor" is used interchangeably herein with the term "cancer", e.g., both terms encompass solids and liquids, e.g., diffuse or circulating tumors. As used herein, the terms "cancer" or "tumor" include pre-cancerous as well as malignant cancers and tumors.

[0076] tumor - associated antigen, TAA :The term "tumor-associated antigen" or "TAA" refers to a molecule (usually a protein, carbohydrate, lipid, or some combination thereof) expressed on the surface of cancer cells either fully or as a fragment (e.g., MHC / peptide), and which can be used to preferentially target a medicament to cancer cells. In some embodiments, the TAA is a marker expressed by normal and cancer cells (e.g., a lineage marker). In some embodiments, the TAA is a cell surface molecule overexpressed in cancer cells compared to normal cells, e.g., 1-fold overexpressed, 2-fold overexpressed, 3-fold overexpressed, or more compared to normal cells. In some embodiments, the TAA is a cell surface molecule synthesized inappropriately in cancer cells, e.g., a molecule containing deletions, additions, or mutations compared to the molecule expressed on normal cells. In some embodiments, the TAA is expressed either fully or as a fragment (e.g., MHC / peptide) specifically on the cell surface of cancer cells and is not synthesized or expressed on the surface of normal cells. Thus, the term "TAA" encompasses cancer cell-specific antigens, sometimes referred to in the art as tumor-specific antigens ("TSA").

[0077] treat (treatment / treating): As used herein, the terms "treat", "treatment" and "treating" refer to a decrease or improvement in the progression, severity and / or duration of a proliferative disorder, or an improvement in one or more symptoms (preferably, one or more distinguishable symptoms) of a proliferative disorder caused by administration of one or more IFN receptor agonists of the present disclosure (e.g., an IFN proprotein capable of activating an IFN receptor agonist, for example, upon activation). In some embodiments, the disorder is a proliferative disorder, and the terms "treat", "treatment" and "treating" refer to an improvement in at least one measurable physical parameter of a proliferative disease (such as the growth of a tumor) that may not necessarily be distinguishable by the patient. In other embodiments, the terms "treat", "treatment" and "treating" refer to inhibiting the progression of a proliferative disease physically, for example, by stabilizing distinguishable symptoms, physiologically, for example, by stabilizing physical parameters, or both. In other embodiments, the terms "treat", "treatment" and "treating" refer to a decrease or stabilization of tumor size or cancer cell count.

[0078] universal light chain, UCL : As used herein, the term "universal light chain" or "ULC" refers to a variable light chain (VL) that can pair with more than one variable heavy chain (VH). In the context of a targeting moiety, the term "universal light chain" or "ULC" refers to a light chain polypeptide that is capable of pairing with the heavy chain region of a targeting moiety and is also capable of pairing with other heavy chain regions. The ULC may also include a constant domain, such as the CL domain of an antibody. The universal light chain is also referred to as the "common light chain".

[0079] VH : The term "VH" refers to the variable region of the immunoglobulin heavy chain of an antibody (including the heavy chain of an Fv, scFv, dsFv or Fab).

[0080] VL : The term "VL" refers to the variable region of the immunoglobulin light chain (including the light chain of an Fv, scFv, dsFv or Fab).

[0081] 6.2. IFN proprotein

[0082] The present disclosure relates to IFN proproteins that comprise an IFN moiety attenuated by steric hindrance from flanking constant domains. The IFN proproteins are constructed such that upon encounter with a protease, such as a protease overexpressed in the tumor environment, the protease-cleavable linker is cleaved and IFN is released. This is achieved by incorporating the IFN moiety between the constant / Fc domains of an antibody, which IFN moiety is flanked by a protease-cleavable linker (PCL). Thus, these proproteins are sometimes referred to as “internal” IFN constructs. IFN proproteins that are capable of, for example, agonizing the IFN receptor upon activation are sometimes referred to herein as “IFN receptor agonists”.

[0083] Typically, the IFN proproteins consist of two half-antibodies that comprise a pair of Fc domains that associate to form an Fc region (usually comprising a hinge sequence), at its N-terminus is a linker that may or may not be cleavable, an IFN moiety, and an additional linker that may or may not be cleavable. In some embodiments, each half-antibody comprises two protease-cleavable linkers flanking the IFN moiety. Exemplary IFN proproteins having two protease-cleavable linkers in each half-antibody are depicted in Figures 1A - 1C . In other embodiments, each half-antibody comprises a single protease-cleavable linker on one side of the IFN moiety and a non-cleavable linker on the other side of the IFN moiety. Exemplary IFN proproteins having one protease-cleavable linker in each half-antibody are depicted in Figures 2A - 2F . Further N-terminal to the protease-cleavable linker is an antibody constant domain, or the entire constant domain comprising the Fc domain, which associates to form another Fc region, or just a part of the constant domain (e.g., the CH1 domain).

[0084] Exemplary configurations of the IFN proproteins of the present disclosure are depicted in Figures 1A - 1C and Figures 2A - 2F . As shown, the IFN moiety and the surrounding linker may comprise a hinge domain only at its C-terminus (“single hinge”, e.g., as shown in Figure 1A , Figure 1B , 2A , 2B, 2D, and 2E) or at both its N-terminus and C-terminus (“double hinge”, e.g., as shown in Figure 1C , 2C , and 2F).

[0085] Typically, the IFN proprotein comprises:

[0086] a) A first polypeptide chain that comprises:

[0087] i) A first immunoglobulin constant domain;

[0088] ii) A first linker;

[0089] iii) The first type I interferon (IFN) moiety;

[0090] iv) A second linker and

[0091] v) A first Fc domain; and

[0092] b) A second polypeptide chain comprising:

[0093] i) A second immunoglobulin constant domain;

[0094] ii) A third linker;

[0095] iii) A second type I interferon (IFN) moiety;

[0096] iv) A fourth linker; and

[0097] v) A second Fc domain that associates with the first Fc domain to form an Fc region;

[0098] In some embodiments, the first linker, the second linker, the third linker, and the fourth linker are all protease-cleavable linkers. In other embodiments, only two of the first linker, the second linker, the third linker, and the fourth linker are protease-cleavable linkers. For example, in certain embodiments, the first linker and the third linker are protease-cleavable linkers, and the second linker and the fourth linker are non-cleavable linkers. In other embodiments, the first linker and the third linker are non-cleavable linkers, and the second linker and the fourth linker are protease-cleavable linkers.

[0099] Preferably, the IFN moiety in the IFN proprotein is sterically hindered by the Fc domain and / or the constant domain from binding to the IFN receptor.

[0100] Exemplary IFN proproteins are depicted in Figure 1A where the targeting moiety (depicted as a Fab domain represented by VH-CH1 paired with VL-CL) is optional. Figure 1A The IFN proprotein of

[0101] a) The first polypeptide chain comprises:

[0102] i) An optional first VH1 domain;

[0103] ii) A first CH1 domain;

[0104] iii) A first Fc domain comprising a hinge domain, a CH2 domain, and a CH3 domain, wherein the CH3 domain is the first immunoglobulin constant domain;

[0105] iv) a first protease-cleavable linker (PCL);

[0106] v) a first type I interferon (IFN) moiety;

[0107] vi) a second protease-cleavable linker (PCL); and

[0108] vii) a second Fc domain;

[0109] b) The second polypeptide chain comprises:

[0110] i) an optional second VH1 domain;

[0111] ii) a second CH1 domain;

[0112] iii) a third Fc domain comprising a hinge domain, a CH2 domain, and a CH3 domain, wherein the CH3 domain is the second immunoglobulin constant domain;

[0113] iv) a third protease-cleavable linker (PCL);

[0114] v) a second type I interferon (IFN) moiety;

[0115] vi) a fourth protease-cleavable linker (PCL); and

[0116] vii) a fourth Fc domain;

[0117] c) The optional third polypeptide chain comprises:

[0118] i) a first VL domain;

[0119] ii) a first CL domain; and

[0120] d) The fourth polypeptide chain comprises:

[0121] i) a second VL domain; and

[0122] ii) a second CL domain;

[0123] wherein the first polypeptide chain associates with the second polypeptide chain such that the first Fc domain and the third Fc domain associate to form an Fc region, and the second Fc domain and the fourth Fc domain form another Fc region.

[0124] When present, the third polypeptide chain associates with the first polypeptide chain such that the first VH, CH1, VL, and CL form a first targeting moiety, and the fourth polypeptide chain associates with the second polypeptide chain such that the second VH, CH1, VL, and CL form a second targeting moiety. Alternatively, the first polypeptide chain and the second polypeptide chain may comprise an scFv at their N-terminus.

[0125] Thus, the first polypeptide chain (along with the third polypeptide chain, if present) represents the first half-antibody, and the second polypeptide chain (along with the fourth polypeptide chain, if present) represents the second half-antibody.

[0126] Another exemplary IFN precursor protein is depicted in Figure 1B , wherein the targeting moiety (depicted as the Fab domain represented by VH-CH1 paired with VL-CL) is optional. Figure 1B The IFN precursor protein of

[0127] a) The first polypeptide chain comprises:

[0128] i) An optional first VH1 domain;

[0129] ii) A first CH1 domain;

[0130] iii) A first protease-cleavable linker (PCL);

[0131] iv) A first type I interferon (IFN) moiety;

[0132] v) A second protease-cleavable linker (PCL); and

[0133] vi) A first Fc domain;

[0134] b) The second polypeptide chain comprises:

[0135] i) An optional second VH1 domain;

[0136] ii) A second CH1 domain;

[0137] iii) A third protease-cleavable linker (PCL);

[0138] iv) A second type I interferon (IFN) moiety;

[0139] v) A fourth protease-cleavable linker (PCL); and

[0140] vi) A second Fc domain;

[0141] c) The optional third polypeptide chain comprises:

[0142] i) A first VL domain;

[0143] ii) A first CL domain; and

[0144] d) The fourth polypeptide chain comprises:

[0145] i) A second VL domain; and

[0146] ii) The second CL domain;

[0147] Wherein the first polypeptide chain associates with the second polypeptide chain such that the first Fc domain and the second Fc domain form an Fc region.

[0148] When present, a third polypeptide chain associates with the first polypeptide chain such that the first VH, CH1, VL, and CL form a first targeting moiety, and a fourth polypeptide chain associates with the second polypeptide chain such that the second VH, CH1, VL, and CL form a second targeting moiety. Alternatively, the first polypeptide chain and the second polypeptide chain may comprise an scFv at their N-terminus.

[0149] Thus, the first polypeptide chain (along with the third polypeptide chain, if present) represents a first half-antibody, and the second polypeptide chain (along with the fourth polypeptide chain, if present) represents a second half-antibody.

[0150] Figure 1B Variants of the IFN preprotein of Figure 1C are depicted in Figure 1C The IFN preprotein of

[0151] Another exemplary IFN preprotein is depicted in Figure 2A wherein the targeting moiety (depicted as a Fab domain represented by VH-CH1 paired with VL-CL) is optional. Figure 2A The IFN preprotein of

[0152] a) The first polypeptide chain comprises:

[0153] i) An optional first VH1 domain;

[0154] ii) A first CH1 domain;

[0155] iii) A first Fc domain, which comprises a hinge domain, a CH2 domain, and a CH3 domain, wherein the CH3 domain is the first immunoglobulin constant domain;

[0156] iv) A first non-cleavable linker (NCL);

[0157] v) A first type I interferon (IFN) moiety;

[0158] vi) A first protease-cleavable linker (PCL); and

[0159] vii) A second Fc domain;

[0160] b) The second polypeptide chain comprises:

[0161] i) Optionally, a second VH1 domain;

[0162] ii) A second CH1 domain;

[0163] iii) A third Fc domain, which comprises a hinge domain, a CH2 domain and a CH3 domain, wherein the CH3 domain is the second immunoglobulin constant domain;

[0164] iv) A second non-cleavable linker (NCL);

[0165] v) A second type I interferon (IFN) moiety;

[0166] vi) A second protease-cleavable linker (PCL); and

[0167] vii) A fourth Fc domain;

[0168] c) The optional third polypeptide chain comprises:

[0169] i) A first VL domain;

[0170] ii) A first CL domain; and

[0171] d) The fourth polypeptide chain comprises:

[0172] i) A second VL domain; and

[0173] ii) A second CL domain;

[0174] wherein the first polypeptide chain associates with the second polypeptide chain such that the first Fc domain and the third Fc domain associate to form an Fc region, and the second Fc domain and the fourth Fc domain form another Fc region.

[0175] When present, the third polypeptide chain associates with the first polypeptide chain such that the first VH, CH1, VL and CL form a first targeting moiety, and the fourth polypeptide chain associates with the second polypeptide chain such that the second VH, CH1, VL and CL form a second targeting moiety. Alternatively, the first polypeptide chain and the second polypeptide chain may comprise an scFv at their N-terminus.

[0176] Thus, the first polypeptide chain (along with the third polypeptide chain, if present) represents a first half-antibody, and the second polypeptide chain (along with the fourth polypeptide chain, if present) represents a second half-antibody.

[0177] Another exemplary IFN proprotein is depicted in Figure 2Bin which the targeting moiety (depicted as the Fab domain represented by VH-CH1 paired with VL-CL) is optional. Figure 2B The IFN precursor protein of Figure 2B comprises a first polypeptide chain, a second polypeptide chain, an optional third polypeptide chain, and an optional fourth polypeptide chain, wherein:

[0178] a) The first polypeptide chain comprises:

[0179] i) an optional first VH1 domain;

[0180] ii) a first CH1 domain;

[0181] iii) a first non-cleavable linker (NCL);

[0182] iv) a first type I interferon (IFN) moiety;

[0183] v) a first protease-cleavable linker (PCL); and

[0184] vi) a first Fc domain;

[0185] b) The second polypeptide chain comprises:

[0186] i) an optional second VH1 domain;

[0187] ii) a second CH1 domain;

[0188] iii) a second non-cleavable linker (NCL);

[0189] iv) a second type I interferon (IFN) moiety;

[0190] v) a second protease-cleavable linker (PCL); and

[0191] vi) a second Fc domain;

[0192] c) The optional third polypeptide chain comprises:

[0193] i) a first VL domain;

[0194] ii) a first CL domain; and

[0195] d) The fourth polypeptide chain comprises:

[0196] i) a second VL domain; and

[0197] ii) a second CL domain;

[0198] wherein the first polypeptide chain associates with the second polypeptide chain such that the first Fc domain and the second Fc domain form an Fc region.

[0199] When present, the third polypeptide chain associates with the first polypeptide chain such that the first VH, CH1, VL, and CL form a first targeting moiety, and the fourth polypeptide chain associates with the second polypeptide chain such that the second VH, CH1, VL, and CL form a second targeting moiety. Alternatively, the first polypeptide chain and the second polypeptide chain may comprise an scFv at their N-terminus.

[0200] Thus, the first polypeptide chain (along with the third polypeptide chain, if present) represents a first half-antibody, and the second polypeptide chain (along with the fourth polypeptide chain, if present) represents a second half-antibody.

[0201] Figure 2B Variants of the IFN preprotein of Figure 2C are depicted in Figure 2C The IFN preprotein of

[0202] further comprises a first hinge domain between the first CH1 domain and the first non-cleavable linker and a second hinge domain between the second CH1 domain and the second non-cleavable linker. Figure 2D Another exemplary IFN preprotein is depicted in Figure 2D wherein the targeting moiety (depicted as a Fab domain represented by VH-CH1 paired with VL-CL) is optional.

[0203] a) The first polypeptide chain comprises:

[0204] i) an optional first VH1 domain;

[0205] ii) a first CH1 domain;

[0206] iii) a first Fc domain, which comprises a hinge domain, a CH2 domain, and a CH3 domain, wherein the CH3 domain is the first immunoglobulin constant domain;

[0207] iv) a first protease-cleavable linker (PCL);

[0208] v) a first type I interferon (IFN) moiety;

[0209] vi) a first non-cleavable linker (NCL); and

[0210] vii) a second Fc domain;

[0211] b) The second polypeptide chain comprises:

[0212] i) an optional second VH1 domain;

[0213] ii) a second CH1 domain;

[0214] iii) A third Fc domain, which comprises a hinge domain, a CH2 domain and a CH3 domain, wherein the CH3 domain is the second immunoglobulin constant domain;

[0215] iv) A second protease-cleavable linker (PCL);

[0216] v) A second type I interferon (IFN) moiety;

[0217] vi) A second non-cleavable linker (NCL); and

[0218] vii) A fourth Fc domain;

[0219] c) The optional third polypeptide chain comprises:

[0220] i) A first VL domain;

[0221] ii) A first CL domain; and

[0222] d) The fourth polypeptide chain comprises:

[0223] i) A second VL domain; and

[0224] ii) A second CL domain;

[0225] wherein the first polypeptide chain associates with the second polypeptide chain such that the first Fc domain and the third Fc domain associate to form an Fc region, and the second Fc domain and the fourth Fc domain form another Fc region.

[0226] When present, the third polypeptide chain associates with the first polypeptide chain such that the first VH, CH1, VL and CL form a first targeting moiety, and the fourth polypeptide chain associates with the second polypeptide chain such that the second VH, CH1, VL and CL form a second targeting moiety. Alternatively, the first polypeptide chain and the second polypeptide chain may comprise an scFv at their N-terminus.

[0227] Thus, the first polypeptide chain (along with the third polypeptide chain, if present) represents a first half-antibody, and the second polypeptide chain (along with the fourth polypeptide chain, if present) represents a second half-antibody.

[0228] Another exemplary IFN proprotein is depicted in Figure 2E wherein the targeting moiety (depicted as a Fab domain represented by VH-CH1 paired with VL-CL) is optional. Figure 2E The IFN proprotein of

[0229] a) The first polypeptide chain comprises:

[0230] i) An optional first VH1 domain;

[0231] ii) A first CH1 domain;

[0232] iii) A first protease-cleavable linker (PCL);

[0233] iv) A first type I interferon (IFN) moiety;

[0234] v) A first non-cleavable linker (NCL); and

[0235] vi) A first Fc domain;

[0236] b) The second polypeptide chain comprises:

[0237] i) An optional second VH1 domain;

[0238] ii) A second CH1 domain;

[0239] iii) A second protease-cleavable linker (PCL);

[0240] iv) A second type I interferon (IFN) moiety;

[0241] v) A second non-cleavable linker (NCL); and

[0242] vi) A second Fc domain;

[0243] c) The optional third polypeptide chain comprises:

[0244] i) A first VL domain;

[0245] ii) A first CL domain; and

[0246] d) The fourth polypeptide chain comprises:

[0247] i) A second VL domain; and

[0248] ii) A second CL domain;

[0249] Wherein the first polypeptide chain associates with the second polypeptide chain such that the first Fc domain and the second Fc domain form an Fc region.

[0250] When present, the third polypeptide chain associates with the first polypeptide chain such that the first VH, CH1, VL, and CL form a first targeting moiety, and the fourth polypeptide chain associates with the second polypeptide chain such that the second VH, CH1, VL, and CL form a second targeting moiety. Alternatively, the first polypeptide chain and the second polypeptide chain may comprise an scFv at their N-terminus.

[0251] Thus, the first polypeptide chain (along with the third polypeptide chain, if present) represents the first half-antibody, and the second polypeptide chain (along with the fourth polypeptide chain, if present) represents the second half-antibody.

[0252] Figure 2E Variants of the IFN preprotein of Figure 2F are depicted in Figure 2E The IFN preprotein of

[0253] further comprises a first hinge domain between the first CH1 domain and the first protease-cleavable linker, and a second hinge domain between the second CH1 domain and the second protease-cleavable linker. Figures 1A - 1C and Figures 2A - 2F The Fc domain in the IFN preprotein depicted in

[0254] usually includes a hinge domain.

[0255] Thus, the IFN preprotein usually contains two to four protease-cleavable linkers. Cleavage of all protease-cleavable linkers in the IFN preprotein with four protease-cleavable linkers results in the release of an activated IFN protein that contains the IFN portion and lacks the C-terminal Fc portion, the N-terminal constant domain, and the targeting portion (if present). Cleavage of all protease-cleavable linkers in the IFN preprotein with two protease-cleavable linkers results in the removal of one of the steric blocking portions and produces an IFN molecule that contains the targeting portion (antibody-IFN) or the Fc domain (IFN-Fc).

[0256] The order and length of the hinge and linker sequences can vary, and the order of the IFN portion (containing the full-length or N-terminal and / or C-terminal truncated IFN sequences) can also vary. Exemplary IFN portions are described in Section 6.3 and include IFNα- and IFNβ-based portions as described in Sections 6.3.1 and 6.3.2 below. Exemplary protease-cleavable linker sequences are disclosed in Section 6.4. Exemplary non-cleavable linker and hinge sequences are disclosed in Sections 6.5 and 6.8.3, respectively. Exemplary targeting portions are disclosed in Section 6.6.

[0257] 6.3. IFN Portion

[0258] There are two major classes of IFN: type I (IFN-α subtypes, IFN-β, etc.) and type II (IFN-γ). Additional IFNs (IFN-like cytokines; IFN-λ subtypes) have also been identified.

[0259] The IFN moiety of the present disclosure can include any wild-type or modified (e.g., truncated and / or mutant) IFN or IFN-like cytokine sequence, but preferably a type I IFN moiety. Type I IFN binds to the heterodimeric plasma membrane receptor IFNAR composed of IFNAR1 and IFNAR2, which is widely expressed in all nucleated cells. Ligand binding is initiated by the high-affinity receptor subunit IFNAR2 (Piehler et al., 2012, Immunological Reviews, doi.org / 10.1111 / imr.12001). Thus, type I IFN is capable of acting on almost all cells of the body. Sixteen type I interferon subtypes have been identified, which vary in their intrinsic variability of affinity and activity for IFNAR2.

[0260] In some embodiments, the type I IFN moiety is an interferon-α (IFNα) moiety. In other embodiments, the type I IFN moiety is an interferon-β (IFNβ) moiety.

[0261] In other embodiments, the type I IFN moiety is an interferon-ω (IFNω), interferon-ε (IFNε), or interferon-κ (IFNκ) moiety.

[0262] The type I IFN moiety can include sequences that differ from the wild-type IFN sequence due to one or more mutations (e.g., substitutions, deletions, or insertions). Substitutions that weaken IFN activity by reducing receptor binding can be appropriately used. Amino acids with N-terminal or C-terminal deletions (or truncations) can also be used, e.g., truncations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids from the N-terminus and / or C-terminus of mature type I IFN. Without being bound by theory, the inventors believe that terminal truncation imposes additional steric constraints on the IFN moiety and reduces IFN activity until the protease-cleavable linker in the IFN precursor protein is cleaved.

[0263] Further details of exemplary type I IFN moieties are provided below.

[0264] 6.3.1. Interferon-α moiety

[0265] The IFNα gene is a member of the interferon-alpha gene cluster on chromosome 9. The encoded cytokine is a member of the type I interferon family, which is produced in response to viral infection and is a key part of the innate immune response, having potent antiviral, antiproliferative, and immunomodulatory properties. IFNα is a protein family that has at least 15 known human IFNα subtypes. The major subtypes that have been identified are IFNα1, IFNα2, IFNα8, IFNα10, IFNα14, and IFNα21.

[0266] The IFNα1 gene has two allelic variants: IFNα1a and IFNα1b. The amino acid sequence of human IFNα1a has been assigned the UniProtKB accession number P01562, reproduced below, with the signal peptide underlined:

[0267] MASPFALLMVLVVLSCKSSCSLG CDLPETH SLDNRRTLML LAQMSRISPS

[0268] SCLMDRHDFG FPQEEFDGNQ FQKAPAISVL HELIQQIFNL FTTKDSSAAW

[0269] DEDLLDKFCT ELYQQLNDLE ACVMQEERVG ETPLMNADSI LAVKKYFRRI

[0270] TLYLTEKKYS PCAWEVVRAE IMRSLSLSTN LQERLRRKE(SEQ ID NO:1)

[0271] The human IFNα1b gene differs from the IFNα1a allelic variant by a single base change in the coding region, resulting in a single change in the amino acid sequence (Val114 instead of Ala114 in the mature protein, corresponding to Val137 instead of Ala137 in the full-length polypeptide).

[0272] The IFNα2 allele has 3 allelic variants: IFNα2a, IFNα2b, and IFNα2c. The allele IFNα2b is the dominant allele, while the allele IFNα2a is subdominant, and IFNα2c is only a minor allelic variant. The amino acid sequence of human IFNα2 has been assigned the UniProtKB accession number P01563. The sequence of the IFNα2b allele is reproduced below, with the signal peptide underlined:

[0273] MALTFALLVALLVLSCKSSCSVG CDLPQTH SLGSRRTLML LAQMRRISLF

[0274] SCLKDRHDFG FPQEEFGNQF QKAETIPVLH EMIQQIFNLF STKDSSAAWD

[0275] ETLLDKFYTE LYQQLNDLEA CVIQGVGVTE TPLMKEDSIL AVRKYFQRIT LYLKEKKYSPCAWEVVRAEI MRSFSLSTNL QESLRSKE(SEQ ID NO:2)

[0276] IFNα2b has arginine (R) at position 23 of the mature protein, while IFNα2a has lysine (K). Thus, in some embodiments, the IFNα2 moiety has arginine at the position corresponding to position 23 of the mature protein. In other embodiments, the IFNα2 moiety has lysine at the position corresponding to position 23 of the mature protein.

[0277] In various aspects, the IFNα moiety comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to a fragment of the amino acid sequence of mature IFNα1a, IFNα1b, and / or IFNα2b, IFNα2a, or IFNα2c or a truncation thereof having at most 15 amino acids at its N-terminus and / or C-terminus (e.g., a truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids from the N-terminus and / or C-terminus of mature IFNα1a, IFNα1b, and / or IFNα2b, IFNα2a, or IFNα2c).

[0278] In some embodiments, the IFNα moiety has one or more amino acid substitutions, e.g., substitutions that alter IFNAR binding and / or agonistic activity. Exemplary substitutions are found in WO 2013 / 107791, U.S. Patent No. 8,258,263, WO2007 / 000769A2, WO2008 / 124086, WO2010 / 030671, WO2018 / 144999A1, and WO2015 / 007520, WO 2013 / 059885, WO2020156467A1, WO2021 / 126929A1. In some embodiments, the IFNα moiety comprises:

[0279] a) one or more substitutions selected from: L15A, A19W, R22A, R23A, L26A, F27A,

[0280] L30A, L30V, K31A, D32A, R33K or R33A or R33Q, H34A, D35A, Q40A,

[0281] H57Y, E58N, Q61S, F64A, N65A, T69A, L80A, D82E, Y85A, T86I, Y89A,

[0282] D114R or D114A, L117A, R120A or R120E or R120K, K121E, R125A, K133A,

[0283] K134A, R144A, A145G or A145M, M148A, R149A, R149K, S152A, L153A,

[0284] N156A; and / or

[0285] b) one or more substitutions at amino acids 57 to 89 and 159 to 165 as described in WO2007000769A2; and / or

[0286] c) one or more amino acid substitutions of alanine, glycine or threonine at positions 9, 17, 47, 65, 66,

[0287] 117, 123, 128, 147 and 157 as described in WO2021126929A1.

[0288] The amino acid positions of the foregoing substitutions are given with reference to mature IFNα2b.

[0289] In a further embodiment, the IFNα moiety comprises one or more amino acid substitutions listed in Table 1. Table 1 lists IFNα substitutions identified by amino acid positions within the IFNα2 sequence.

[0290]

[0291]

[0292]

[0293] In some embodiments, the IFNα moiety comprises an amino acid sequence that includes the amino acid substitutions R33A or R33K, Q90A, E96A, R120A, A145M, R149A or R149K, S152A, or any combination of two or more of the foregoing, e.g., Q90A + R120A or A145M + R149K.

[0294] Sequences of exemplary IFNα moieties that can be used in the IFN proproteins of the present disclosure are listed in Table 2 below:

[0295]

[0296]

[0297] 6.3.2. Interferon 1-β moiety

[0298] Interferon 1-β (IFN1β or IFN 1-β) is a cytokine naturally produced by the immune system in response to biological and chemical stimuli. IFN1β is a glycosylated secreted monomer with a molecular weight of approximately 22 kDa, which is produced abundantly by fibroblasts, and thus it is also known as fibroblast interferon. IFN1β binds to the IFNAR receptor composed of the IFNAR1 and IFNAR2 dimers to induce signal transduction via the JAK / STAT pathway and other pathways. IFN1β can also act by binding to IFNAR1 alone and signaling independently of the Jak-STAT pathway (see, for example, Stanifer et al., 2019, Int. J Mol. Sci. 20(6):1445).

[0299] IFN1β contains 5 α-helices, which are named A (YNLLGFLQRSSNFQCQKLL (SEQ ID NO:18)), B (KEDAALTIYEMLQNIFAIF (SEQ ID NO:19)), C (ETIVENLLANVYHQINHLKTVLEEKL (SEQ ID NO:20)), D (SSLHLKRYYGRILHYLKA (SEQ ID NO:21)) and E (HCAWTIVRVEILRNFYFINRLT (SEQ ID NO:22)). The five α-helices are interconnected by loops of 2 to 28 residues named AB, BC, CD, and DE loops. It has been reported that the A helix in the AB loop and the E helix in the DE loop are involved in the binding of IFN1β to the IFNAR receptor.

[0300] Two types of IFN1β have been described: interferon 1-β1 (IFN1β1) and interferon 1-β3 (IFN1β3).

[0301] The amino acid sequence of the human IFNβ precursor is listed under GenBank: accession number AAA36040.1, and is reproduced below (where the signal peptide is underlined):

[0302] MTNKCLLQIALLLCFSTTALSMSYNLLGFL QRSSNFQCQK LLWQLNGRLE YCLKDRMNFDIPEEIKQLQQ FQKEDAALTI YEMLQNIFAI FRQDSSSTGW NETIVENLLA NVYHQINHLK TVLEEKLEKEDFTRGKLMSS LHLKRYYGRI LHYLKAKEYS HCAWTIVRVE ILRNFYFINR LTGYLRN(SEQ ID NO:23)

[0303] In various aspects, the IFNβ moiety comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity with the amino acid sequence of a fragment of mature IFN1β1 or a truncation thereof having at most 15 amino acids at its N-terminus and / or C-terminus (e.g., a truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids from the N-terminus and / or C-terminus of IFN1β1).

[0304] In various embodiments, the IFNβ moiety comprises one or more amino acid substitutions and / or deletions compared to IFN1β1. In some embodiments, the substitution is C17S (referring to mature IFN1β1), and the deletion is one of the C-terminal truncations described in US2009 / 0025106A1, such as IFN-ΔΙ, IFNΑ2, IFNΑ3, IFNΑ4, IFNΑ5, IFNΑ6, IFN-Δ7, IFN-Δδ, IFNΑ9 and IFN-ΔΙΟ.

[0305] 6.3.3. Other type I interferons

[0306] In certain aspects, the type I IFN moiety is different from the IFNα or IFNβ moiety, e.g., the interferon-ω (IFNω), interferon-ε (IFNε) or interferon-κ (IFNκ) moiety.

[0307] Human IFNω is identified by the UniProt accession number P05000, and the IFNω1 allele has the amino acid sequence listed below, with the signal sequence underlined:

[0308] MALLFPLLAALVMTSYSPVGSLGCD LPQNHGLLSRNTLVLLHQMRRISPFLCLKD

[0309] RRDFRFPQEMVKGSQLQKAHVMSVLHEMLQQIFSLFHTERSSAAWNMTLLDQL

[0310] HTGLHQQLQHLETCLLQVVGEGESAGAISSPALTLRRYFQGIRVYLKEKKYSDCAWEVVRMEIMKSLFLSTNMQERLRSKDRDLGSS(SEQ ID NO:24)

[0311] In various aspects, the IFNω moiety comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence of a fragment of mature IFN1ω1 or a truncation thereof having at most 15 amino acids at its N-terminus and / or C-terminus (e.g., a truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids from the N-terminus and / or C-terminus of IFN1ω1).

[0312] Human IFNε is identified by UniProt accession number Q86WN2 and has the amino acid sequence listed below, with the signal sequence underlined:

[0313] MIIKHFFGTVLVLLASTTIFS LDLKLIIFQQRQVNQESLKLLNKLQTLSIQQCLPHR

[0314] KNFLLPQKSLSPQQYQKGHTLAILHEMLQQIFSLFRANISLDGWEENHTEKFLIQL

[0315] HQQLEYLEALMGLEAEKLSGTLGSDNLRLQVKMYFRRIHDYLENQDYSTCAWAI VQVEISRCLFFVFSLTEKLSKQGRPLNDMKQELTTEFRSPR(SEQ ID NO:25)

[0316] In various aspects, the IFNε moiety comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence of mature IFN1ε or a truncated fragment thereof having up to 15 amino acids at its N-terminus and / or C-terminus (e.g., truncations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids from the N-terminus and / or C-terminus of IFN1ε).

[0317] Human IFNκ is identified by UniProt accession number Q9P0W0 and has the amino acid sequence set forth below, with the signal sequence underlined:

[0318] MSTKPDMIQKCLWLEILMGIFIAGTLS LDCNLLNVHLRRVTWQNLRHLSSMSNSF

[0319] PVECLRENIAFELPQEFLQYTQPMKRDIKKAFYEMSLQAFNIFSQHTFKYWKERH

[0320] LKQIQIGLDQQAEYLNQCLEEDKNENEDMKEMKENEMKPSEARVPQLSSLELRR

[0321] YFHRIDNFLKEKKYSDCAWEIVRVEIRRCLYYFYKFTALFRRK(SEQ ID NO:26)

[0322] In various aspects, the IFNκ moiety comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence of mature IFN1κ or a truncated fragment thereof having up to 15 amino acids at its N-terminus and / or C-terminus (e.g., truncations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids from the N-terminus and / or C-terminus of IFN1κ).

[0323] 6.4. Protease-Cleavable Linkers

[0324] The IFN proprotein of the present disclosure generally comprises four linkers, referred to as the first linker, the second linker, the third linker, and the fourth linker in the numbered examples below, wherein the first linker and the second linker are on one polypeptide chain and the third linker and the fourth linker are on another polypeptide chain. Two to four of the linkers are protease-cleavable linkers. In some embodiments, the first linker, the second linker, the third linker, and the fourth linker are all protease-cleavable linkers. In other embodiments, only two of the first linker, the second linker, the third linker, and the fourth linker are protease-cleavable linkers. For example, in certain embodiments, the first linker and the third linker are protease-cleavable linkers, and the second linker and the fourth linker are non-cleavable linkers. In other embodiments, the first linker and the third linker are non-cleavable linkers, and the second linker and the fourth linker are protease-cleavable linkers.

[0325] The protease-cleavable linker can range from 8 amino acids to 100 or more amino acids. In various embodiments, the protease-cleavable linker can range from 8 amino acids to 15 amino acids, 10 amino acids to 20 amino acids, 20 amino acids to 80 amino acids, and in some aspects, the non-cleavable peptide linker can range in length from 20 amino acids to 60 amino acids, 20 amino acids to 40 amino acids, 30 amino acids to 50 amino acids, 20 amino acids to 80 amino acids, or 30 amino acids to 70 amino acids.

[0326] The protease-cleavable linker comprises one or more substrate sequences for one or more proteases (e.g., one or more of the proteases listed in Section 6.4.1). The one or more substrate sequences (e.g., one or more of the substrate sequences listed in Section 6.4.2) are typically (but not necessarily) flanked by one or more spacer sequences (e.g., spacer sequences as described in Section 6.4.3). Each protease-cleavable linker can comprise one, two, three, or more substrate sequences. The spacer sequences can be adjacent, overlapping, or separated by spacer sequences. Preferably, the C-terminus and N-terminus of the protease-cleavable linker contain spacer sequences.

[0327] In various aspects of the IFN proprotein comprising four protease-cleavable linkers, the first protease-cleavable linker and the third protease-cleavable linker can be cleaved by the same protease and / or the second protease-cleavable linker and the fourth protease-cleavable linker can be cleaved by the same protease. In some embodiments, the protease is a protease listed in Table A.

[0328] In a further aspect of the IFN proprotein comprising four protease-cleavable linkers, the first protease-cleavable linker and the third protease-cleavable linker comprise the same substrate sequence and / or the second protease-cleavable linker and the fourth protease-cleavable linker comprise the same substrate sequence. In some embodiments, the substrate sequences are listed in Table B. In a further embodiment, the first protease-cleavable linker and the third protease-cleavable linker also comprise the same spacer sequence and / or the second protease-cleavable linker and the fourth protease-cleavable linker also comprise the same spacer sequence. In some embodiments, the spacer sequences are listed in Table C.

[0329] In a further aspect of the IFN proprotein comprising four protease-cleavable linkers, the first linker and the third linker comprise the same linker sequence and / or the second linker and the fourth linker comprise the same linker sequence. In some embodiments, the linker sequences are listed in Table D.

[0330] In some embodiments of the IFN proprotein comprising four protease-cleavable linkers, the first protease-cleavable linker and the third protease-cleavable linker are the same as the second protease-cleavable linker and the fourth protease-cleavable linker.

[0331] In other embodiments, the first protease-cleavable linker and the third protease-cleavable linker are different from the second protease-cleavable linker and the fourth protease-cleavable linker.

[0332] In the foregoing aspects and embodiments, different linkers may be cleaved by the same protease, different proteases, or when the linker comprises multiple substrate sequences, different linkers may be cleaved by multiple proteases, where one or more proteases are common and one or more proteases are different.

[0333] Exemplary protease-cleavable linker sequences are listed in Section 6.4.4

[0334] 6.4.1. Proteases

[0335] Exemplary proteases whose substrate sequences can be incorporated into protease-cleavable linkers are listed in Table A below.

[0336]

[0337] In certain embodiments, the protease is matrix metalloproteinase (MMP)-2, MMP-9, asparaginyl endopeptidase, thrombin, fibroblast activation protease (FAP), MMP-1, MMP-3, MMP-7, MMP-8, MMP-12, MMP-13, MMP-14, membrane type 1 matrix metalloproteinase (MT1-MMP), plasmin, transmembrane protease, serine (TMPRSS-3 / 4), cathepsin A, cathepsin B, cathepsin D, cathepsin E, cathepsin F, cathepsin H, cathepsin K, cathepsin L, cathepsin L2, cathepsin O, cathepsin S, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, human neutrophil elastase, urokinase / urokinase-type plasminogen activator (uPA), a disintegrin and metalloproteinase (ADAM) 10, ADAM12, ADAM17, ADAM with thrombospondin motifs (ADAMTS), ADAMTS5, beta-secretase (BACE), granzyme A, granzyme B, guanidobenzoate hydrolase, serine protease, proteolytic enzyme, proteolytic enzyme 2, transmembrane peptidase, neutral lysosome, prostate-specific membrane antigen (PSMA), tumor necrosis factor converting enzyme (TACE), kallikrein-related peptidase (KLK) 3, KLK5, KLK7, KLK11, NS3 / 4 protease of hepatitis C virus (HCV-NS3 / 4), tissue plasminogen activator (tPA), calpain, calpain 2, glutamate carboxypeptidase II, plasma kallikrein, AMSH-like protease, AMSH, gamma-secretase component, antiplasmin-lytic enzyme (APCE), des-cysteine 1, apoptosis-related cysteine peptidase or N-acetylated alpha-linked acidic dipeptidase-like 1.

[0338] 6.4.2. Substrate

[0339] Exemplary substrate sequences that can be cleaved by tumor proteases and can be incorporated into protease-cleavable linkers are listed in Table B below.

[0340]

[0341]

[0342]

[0343]

[0344]

[0345] 6.4.3. Spacer

[0346] Exemplary spacer sequences that can be incorporated into the protease-cleavable linker are listed in Table C below. In addition to the spacer sequences listed in Table C, any non-cleavable linker sequences described in Section 6.5 (e.g., the non-cleavable linker sequences listed in Table E) or portions thereof can be used as spacer sequences. In some embodiments, no spacer sequence is present in the protease-cleavable linker at all.

[0347]

[0348]

[0349] In some embodiments, as used in Table C above, n is an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0350] 6.4.4. Exemplary Protease-Cleavable Linkers

[0351] Exemplary protease-cleavable linkers containing one or more substrate sequences and spacer sequences are listed in Table D below.

[0352]

[0353]

[0354]

[0355]

[0356] In certain aspects, the protease-cleavable linker comprises an amino acid sequence having up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitutions compared to the sequences listed in Table D. Thus, in some embodiments, the protease-cleavable linker comprises any amino acid sequence in Table D or consists of such, where these amino acid sequences have 1 - 5 amino acid substitutions compared to the sequences listed in Table D.

[0357] 6.5. Non-Cleavable Linkers

[0358] In certain aspects, the present disclosure provides an IFN proprotein, wherein two or more components of the IFN proprotein are linked to each other by a peptide linker. By way of example and not limitation, the linker can be used to link the Fc domain and the targeting moiety or different domains within the targeting moiety (e.g., the VH and VL domains in a scFv).

[0359] Preferably, all linkers in the IFN precursor protein are non-cleavable linkers (NCLs), except for the protease-cleavable linker whose cleavage results in IFN activation.

[0360] The range of the non-cleavable linker can be from 2 amino acids to 60 or more amino acids, and in some aspects, the non-cleavable peptide linker can range in length from 3 amino acids to 50 amino acids, 4 to 30 amino acids, 5 to 25 amino acids, 10 to 25 amino acids, 10 amino acids to 60 amino acids, 12 amino acids to 20 amino acids, 20 amino acids to 50 amino acids, or 25 amino acids to 35 amino acids.

[0361] In certain aspects, the non-cleavable linker is at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids in length, and optionally at most 30 amino acids, at most 40 amino acids, at most 50 amino acids, or at most 60 amino acids in length.

[0362] In some of the foregoing embodiments, the non-cleavable linker ranges in length from 5 amino acids to 50 amino acids, for example, ranges in length from 5 to 50 amino acids, 5 to 45 amino acids, 5 to 40 amino acids, 5 to 35 amino acids, 5 to 30 amino acids, 5 to 25 amino acids, or 5 to 20 amino acids. In other of the foregoing embodiments, the non-cleavable linker ranges in length from 6 amino acids to 50 amino acids, for example, ranges in length from 6 to 50 amino acids, 6 to 45 amino acids, 6 to 40 amino acids, 6 to 35 amino acids, 6 to 30 amino acids, 6 to 25 amino acids, or 6 to 20 amino acids. In still other embodiments, the non-cleavable linker ranges in length from 7 amino acids to 50 amino acids, for example, ranges in length from 7 to 50 amino acids, 7 to 45 amino acids, 7 to 40 amino acids, 7 to 35 amino acids, 7 to 30 amino acids, 7 to 25 amino acids, or 7 to 20 amino acids.

[0363] Charged (e.g., charged hydrophilic linkers) and / or flexible non-cleavable linkers are particularly preferred.

[0364] Examples of flexible non-cleavable linkers that can be used in the IFN precursor proteins of the present disclosure include those disclosed by Chen et al., 2013, Adv Drug Deliv Rev. 65(10):1357-1369 and Klein et al., 2014, Protein Engineering, Design & Selection 27(10):325-330. Particularly useful flexible non-cleavable linkers are or include repeats of glycine and serine, for example, G n S (SEQ ID NO:299) or SGn A monomer or polymer of (SEQ ID NO:300), where n is an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the non-cleavable linker is or comprises G 4 A monomer or polymer of S(SEQ ID NO:301) repeat sequences, for example, (GGGGS) n (SEQ ID NO:301).

[0365] The polyglycine non-cleavable linker can be suitably used in the IFN proprotein of the present disclosure. In some embodiments, the peptide non-cleavable linker comprises two consecutive glycines (2Gly), three consecutive glycines (3Gly), four consecutive glycines (4Gly(SEQ ID NO:302)), five consecutive glycines (5Gly(SEQ ID NO:303)), six consecutive glycines (6Gly(SEQID NO:304)), seven consecutive glycines (7Gly(SEQ ID NO:305)), eight consecutive glycines (8Gly(SEQ IDNO:306)), or nine consecutive glycines (9Gly(SEQ ID NO:307)).

[0366] Exemplary non-cleavable linker sequences are listed in Table E below.

[0367]

[0368]

[0369]

[0370] In certain aspects, the IFN proprotein of the present disclosure can comprise a polypeptide chain (e.g., as depicted in Figure 1A ) that, in an N-terminus to C-terminus orientation, comprises a targeting moiety (or targeting moiety chain), a hinge domain, a CH2 domain, and a CH3 domain, or a polypeptide chain that comprises a targeting moiety (or targeting moiety chain), a hinge domain, followed by a protease-cleavable linker (e.g., as depicted in Figure 1C ). Thus, the hinge domain can be said to constitute a type of linker. Exemplary hinge domains are listed in Section 6.8.3.

[0371] 6.6. Targeting Moiety

[0372] Incorporating a targeting moiety into the IFN proprotein of the present disclosure allows for delivery of a high concentration of IFN to the tumor microenvironment while reducing systemic exposure, resulting in fewer side effects than those obtained with untargeted IFN molecules.

[0373] Any type of target molecule that is expected to be present at or capable of driving the IFN proprotein at a particular lesion or tissue can be targeted by the IFN proproteins of the present disclosure. In some embodiments, the IFN proprotein is intended to treat cancer, for example, by inducing a local immune response against tumor tissue. Thus, the target molecule can be any local tumor and associated target molecule. Target molecules recognized by the targeting portion of the IFN proproteins of the present disclosure are typically present on, for example, the surface of activated T cells, the surface of tumor cells, the surface of dendritic cells or other antigen-presenting cells, the surface of natural killer (NK) cells, the surface of virus-infected cells, the surface of other diseased cells, free in serum, present in the extracellular matrix (ECM), or in immune cells present at the target site, such as tumor-reactive lymphocytes.

[0374] In various embodiments, the target molecule is an extracellular matrix (“ECM”) antigen, a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or virus lymphocyte, a T cell antigen (“TCA”), a checkpoint inhibitor, a tumor-associated antigen (“TAA), a dendritic cell (DC) or other antigen-presenting cell (APC) antigen, or a natural killer (NK) cell antigen. One of ordinary skill in the art will recognize that the foregoing categories of target molecules are not mutually exclusive, and thus a given target molecule can fall into more than one of the foregoing categories of target molecules. For example, some molecules can be considered both TAA and ECM proteins, while other molecules can be considered both TCA and checkpoint inhibitors.

[0375] Exemplary cancer types that can be targeted include acute lymphoblastic leukemia, acute myeloid leukemia, cholangiocarcinoma, B cell leukemia, B cell lymphoma, cholangiocarcinoma, bone cancer, brain cancer, breast cancer, triple negative breast cancer, cervical cancer, Burkitt lymphoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastric cancer, gastrointestinal cancer, glioma, hairy cell leukemia, head and neck cancer, Hodgkin lymphoma, liver cancer, lung cancer, medullary thyroid cancer, melanoma, multiple myeloma, ovarian cancer, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, lung cancer, kidney cancer, sarcoma, skin cancer, testicular cancer, urothelial cancer, and other bladder cancers. However, one of ordinary skill in the art will recognize that TAAs and other target molecules associated with the tumor microenvironment are known for almost any type of cancer.

[0376] Non-limiting examples of ECM antigens include syndecan, heparanase, integrin, osteopontin, connexin, cadherin, laminin, laminin-type EGF, lectin, fibronectin, notch protein, nectin (e.g., nectin-4), tenascin, collagen (e.g., type X collagen), and matrix protein.

[0377] Other target molecules are cell surface molecules of tumors or virus lymphocytes, such as T cell co-stimulatory proteins such as CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0378] In certain embodiments, the target molecule is a checkpoint inhibitor, such as CTLA-4, PD1, PDL1, PDL2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2. In a specific embodiment, the target molecule is PD1. In other embodiments, the target molecule is LAG3. In still other embodiments, the target molecule is PDL1.

[0379] In some embodiments, the target molecule is located on the surface of dendritic cells or other antigen-presenting cells, such as XCR1, Clec9a, CD1c, CD11c, CD14, PDL1, macrophage mannose receptor (CD206), and DEC-205.

[0380] In further embodiments, the target molecule is located on the surface of natural killer (NK) cells, such as CD335, CD38, CD2, NKG2D, NKp44, NKp30, CD16, LFA-1, CD27, KIR, NKH1A, and NKp46.

[0381] Antibodies and antigen-binding portions generally bind to specific antigenic determinants and are capable of directing the IFN proprotein to a target site, such as a specific type of tumor cell or tumor stroma carrying the antigenic determinant. In a specific embodiment, the targeting portion recognizes a tumor-associated antigen (TAA). Preferably, the TAA is a human TAA. The antigen may or may not be present on normal cells. In certain embodiments, the TAA is preferentially expressed or upregulated on tumor cells compared to normal cells. In other embodiments, the TAA is a lineage marker. Exemplary TAAs include fibroblast activation protein (FAP), the A1 domain of tenascin-C (TNC A1), the A2 domain of tenascin-C (TNC A2), extra domain B of fibronectin (EDB), melanoma-associated chondroitin sulfate proteoglycan (MCSP), MART-1 / Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cathepsin b, colorectal-associated antigen (CRC)-C017-1A / GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T cell receptor / CD3-zeta chain, MAGE family tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE family tumor antigens (e.g.,GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, alpha-fetoprotein, E-cadherin, alpha-catenin, beta-catenin and gamma-catenin, p120ctn, gp100 Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products (such as human papillomavirus proteins), Smad tumor antigen family, Imp-1, P1A, Epstein-Barr virus-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, c-erbB-2, Her2, EGFR, IGF-1R, CD2 (T cell surface antigen), CD3 (heteromultimer associated with TCR), CD22 (B cell receptor), CD23 (low affinity IgE receptor), CD30 (cytokine receptor), CD33 (myeloid cell surface antigen), CD40 (tumor necrosis factor receptor), IL-6R- (IL6 receptor), CD20, MCSP, PDGFβR (beta-platelet-derived growth factor receptor), ErbB2 epithelial cell adhesion molecule (EpCAM), EGFR variant III (EGFRvIII), CD19, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glioma-associated antigen, beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostate enzyme, prostate-specific antigen (PSA), PAP, LAGA-1a, p53, prostate, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, ephrin B2, insulin-like growth factor (IGF1)-I, IGF-II, IGFI receptor, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin and A1 domain of tenascin-C (TnC A1).

[0382] Suitable forms of the targeting moiety are described in Section 6.7. The targeting moiety is preferably an antigen-binding moiety, such as an antibody or an antigen-binding portion of an antibody, e.g., an scFv as described in Section 6.7.2 or a Fab as described in Section 6.7.1.

[0383] In some embodiments, the targeting moiety targets an exemplary target molecule listed in Table F below, with reference to the exemplary antibody or antibody sequence on which the targeting moiety may be based.

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400] In some aspects, the targeting moiety competes with the antibodies listed in Table F for binding to the target molecule. In a further aspect, the targeting moiety comprises CDRs having the CDR sequences of the antibodies listed in Table F. In some embodiments, the targeting moiety comprises all 6 CDR sequences of the antibodies listed in Table F. In other embodiments, the targeting moiety comprises at least the heavy chain CDR sequences (CDR-H1, CDR-H2, CDR-H3) of the antibodies listed in Table F and the light chain CDR sequences of a common light chain. In a further aspect, the targeting moiety comprises a VH comprising the amino acid sequence of the VH of the antibodies listed in Table F. In some embodiments, the targeting moiety further comprises a VL comprising the amino acid sequence of the VL of the antibodies listed in Table F. In other embodiments, the targeting moiety further comprises a common light chain VL sequence.

[0401] In some embodiments, the target molecule is PDL1. Table F-1 below provides exemplary anti-PDL1 antibodies and / or antibody sequences on which the targeting moiety can be based, e.g., which can be incorporated into the targeting moiety for the interferon precursor proteins of the present disclosure.

[0402]

[0403]

[0404]

[0405] In some aspects, the targeting moiety competes with the anti-PDL1 antibodies listed in Table F-1 for binding to PDL1. In a further aspect, the targeting moiety comprises CDRs having the CDR sequences of the anti-PDL1 antibodies listed in Table F-1. In some embodiments, the targeting moiety comprises all 6 CDR sequences of the anti-PDL1 antibodies listed in Table F-1. In other embodiments, the targeting moiety comprises at least the heavy chain CDR sequences (CDR-H1, CDR-H2, CDR-H3) of the anti-PDL1 antibodies listed in Table F-1 and the light chain CDR sequences of a common light chain. In a further aspect, the targeting moiety comprises a VH comprising the amino acid sequence of the VH of the anti-PDL1 antibodies listed in Table F-1. In some embodiments, the targeting moiety further comprises a VL comprising the amino acid sequence of the VL of the anti-PDL1 antibodies listed in Table F-1. In other embodiments, the targeting moiety further comprises a common light chain VL sequence.

[0406] In some embodiments, the target molecule is PD1. Table F-2 below provides exemplary anti-PD1 antibodies and / or antibody sequences on which the targeting moiety can be based, e.g., which can be incorporated into the targeting moiety for the interferon precursor proteins of the present disclosure.

[0407]

[0408]

[0409]

[0410]

[0411] In some aspects, the targeting moiety competes with the anti-PD1 antibodies listed in Table F-2 for binding to PD1. In further aspects, the targeting moiety comprises CDRs having the CDR sequences of the anti-PD1 antibodies listed in Table F-2. In some embodiments, the targeting moiety comprises all 6 CDR sequences of the anti-PD1 antibodies listed in Table F-2. In other embodiments, the targeting moiety comprises at least the heavy chain CDR sequences (CDR-H1, CDR-H2, CDR-H3) of the anti-PD1 antibodies listed in Table F-2 and the light chain CDR sequences of a common light chain. In further aspects, the targeting moiety comprises a VH comprising the amino acid sequence of the VH of the anti-PD1 antibodies listed in Table F-2. In some embodiments, the targeting moiety further comprises a VL comprising the amino acid sequence of the VL of the anti-PD1 antibodies listed in Table F-2. In other embodiments, the targeting moiety further comprises a common light chain VL sequence.

[0412] In cases where the target molecule is a checkpoint inhibitor, in some embodiments, the checkpoint inhibitor targeting moiety is non-blocking or weakly blocking to ligand-receptor binding. Examples of non-blocking or weakly blocking anti-PD1 antibodies include those having SEQ ID No: 2 / 10 of PCT Publication No. WO2015 / 112800A1; SEQ ID No: 16 / 17 of U.S. Patent No. 11,034,765B2; and VH / VL amino acid sequences of SEQ ID No. 164 / 178, 165 / 179, 166 / 180, 167 / 181, 168 / 182, 169 / 183, 170 / 184, 171 / 185, 172 / 186, 173 / 187, 174 / 188, 175 / 189, 176 / 190, and 177 / 190 of U.S. Patent No. 10,294,299B2. Examples of non-blocking or weakly blocking anti-LAG3 antibodies include those having VH / VL amino acid sequences of SEQ ID No 23 / 24, 3 / 4, and 11 / 12 of U.S. Publication US2022 / 0056126A1.

[0413] Other target molecules that can be targeted by the IFN preprotein are disclosed in Table I and, for example, in Hafeez et al., 2020, Molecules 25:4764, doi:10.3390 / molecules25204764 (particularly in Table 1). The entire content of Table 1 of Hafeez et al. is incorporated herein by reference.

[0414] 6.7. Targeting moiety formats

[0415] In some aspects, the targeting moiety of the IFN preprotein of the present disclosure can be any type of antibody or fragment thereof that retains specific binding to an epitope. In one embodiment, the targeting moiety is an immunoglobulin molecule or a fragment thereof, particularly an immunoglobulin molecule of the IgG class, more particularly IgG 1 or IgG 4 immunoglobulin molecule. Antibody fragments include, but are not limited to, VH (or V H ) fragments, VL (or V L ) fragments, Fab fragments, F(ab') 2 fragments, scFv fragments, Fv fragments, minibodies, diabodies, triabodies, and tetra-bodies.

[0416] 6.7.1. Fab

[0417] Fab domains are traditionally generated by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. Fab domains can contain constant domain and variable region sequences from any suitable species and can thus be murine, chimeric, human, or humanized.

[0418] The Fab domain typically contains a CH1 domain attached to the VH domain, which pairs with a CL domain attached to the VL domain. In wild-type immunoglobulins, the VH domain pairs with the VL domain to form the Fv region, and the CH1 domain pairs with the CL domain to further stabilize the binding site. A disulfide bond between the two constant domains can further stabilize the Fab domain. When the targeting moiety is a Fab, the CH1 of the Fab can represent the constant domain located at the N-terminus of the IFN moiety, for example, as Figure 1B and 1C shown.

[0419] For the IFN preprotein of the present disclosure, particularly when the light chain of the targeting moiety is not a common or universal light chain, it is advantageous to use a Fab heterodimerization strategy to allow correct association of Fab domains belonging to the same targeting moiety and to minimize aberrant pairing of Fab domains belonging to different targeting moieties. For example, the Fab heterodimerization strategy shown in Table G below can be used:

[0420]

[0421]

[0422] Thus, in certain embodiments, correct association between the two polypeptides of a Fab is facilitated by exchanging the VL and VH domains of the Fab with each other or by exchanging the CH1 and CL domains with each other, e.g., as described in WO 2009 / 080251.

[0423] Correct Fab pairing can also be facilitated by introducing one or more amino acid modifications in the CH1 domain of the Fab, one or more amino acid modifications in the CL domain of the Fab, and / or one or more amino acid modifications in the VH domain, and one or more amino acid modifications in the VL domain. The modified amino acids are typically part of the VH:VL and CH1:CL interfaces such that the Fab components preferentially pair with each other rather than with components of other Fabs.

[0424] In one embodiment, the one or more amino acid modifications are limited to the conserved framework residues of the variable domains (VH, VL) and constant domains (CH1, CL), as indicated by the Kabat numbering of the residues. Almagro, 2008, Frontiers In Bioscience 13:1619-1633 provides definitions of framework residues based on the Kabat, Chothia, and IMGT numbering schemes.

[0425] In one embodiment, the modifications introduced in the VH and CH1 and / or VL and CL domains are complementary to each other. Complementarity at the heavy and light chain interface can be achieved based on steric and hydrophobic contacts, electrostatic / charge interactions, or a combination of various interactions. Complementarity between protein surfaces has been widely described in the literature as lock and key fit, peg in hole, protrusion and cavity, donor and acceptor, etc., all of which imply the nature of a structural and chemical match between two interacting surfaces.

[0426] In one embodiment, one or more of the introduced modifications introduce new hydrogen bonds at the interface of the Fab components. In one embodiment, one or more of the introduced modifications introduce new salt bridges across the interface of the Fab components. Exemplary substitutions are described in WO 2014 / 150973 and WO 2014 / 082179, the contents of which are incorporated herein by reference.

[0427] In some embodiments, the Fab domain comprises a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain, which introduce a salt bridge between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0428] In some embodiments, the Fab domain comprises 143Q and 188V substitutions in the CH1 domain and 113T and 176V substitutions in the CL domain, which serve to exchange the hydrophobic and polar regions of contact between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0429] In some embodiments, the Fab domain may contain modifications in some or all of the VH, CH1, VL, and CL domains to introduce orthogonal Fab interfaces that facilitate correct assembly of the Fab domain (Lewis et al., 2014, Nature Biotechnology 32:191-198). In one embodiment, 39K and 62E modifications are introduced in the VH domain, H172A and F174G modifications are introduced in the CH1 domain, 1R, 38D, (36F) modifications are introduced in the VL domain, and L135Y and S176W modifications are introduced in the CL domain. In another embodiment, a 39Y modification is introduced in the VH domain and a 38R modification is introduced in the VL domain.

[0430] The Fab domain can also be modified to replace the native CH1:CL disulfide bond with an engineered disulfide bond, thereby enhancing the efficiency of Fab assembly pairing. For example, an engineered disulfide bond can be introduced by introducing 126C in the CH1 domain and 121C in the CL domain (see, e.g., Mazor et al., 2015, Mabs 7:377-89).

[0431] The Fab domain can also be modified by replacing the CH1 and CL domains with alternative domains that promote correct assembly. For example, Wu et al., 2015, Mabs 7:364-76, describe replacing the CH1 domain with the constant domain of a T cell receptor and the CL domain with the b domain of a T cell receptor, and pairing these domain replacements with additional charge-charge interactions between the VL and VH domains by introducing a 38D modification in the VL domain and a 39K modification in the VH domain.

[0432] Instead of or in addition to using Fab heterodimerization strategies to promote correct VH-VL pairing, the VL of a common light chain (also referred to as a universal light chain) can be used for each unique ABD in the IFN proprotein of the present disclosure. In various embodiments, use of the common light chain described herein reduces the number of inappropriate species in the IFN proprotein compared to using the original cognate VL. In various embodiments, the VL domain of the ABD is identified from a monospecific antibody that includes the common light chain. In various embodiments, the VH region of the ABD in the IFN proprotein comprises human heavy chain variable gene segments that are rearranged in vivo within murine B cells that have previously been engineered to express a limited human light chain repertoire or a single human light chain homologous to the human heavy chain, and in response to exposure to an antigen of interest, an antibody repertoire is generated that contains multiple human VHs that are homologous to one of a possible human VL or two possible human VLs, wherein the antibody repertoire is specific for the antigen of interest. The common light chain is derived from a rearranged human Vκ1-39Jκ5 sequence or a rearranged human Vκ3-20Jκ1 sequence and includes somatically mutated (e.g., affinity matured) versions. See, e.g., U.S. Patent No. 10,412,940.

[0433] 6.7.2.scFv

[0434] A single-chain Fv or "scFv" antibody fragment contains the VH and VL domains of an antibody in a single polypeptide chain, is capable of being expressed as a single polypeptide chain, and retains the specificity of the intact antibody from which it is derived. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for target binding. Examples of linkers suitable for linking the VH and VL chains of an scFv are the non-cleavable linkers identified in Section 6.5.

[0435] Unless otherwise specified, as used herein, an scFv can have the VL and VH variable regions in either order. For example, relative to the N-terminus and C-terminus of the polypeptide, an scFv can comprise VL-linker-VH or can comprise VH-linker-VL.

[0436] An scFv can comprise VH and VL sequences from any suitable species, such as murine, human, or humanized VH and VL sequences.

[0437] To generate a nucleic acid encoding an scFv, DNA fragments encoding VH and VL are operably linked to another fragment encoding a linker, e.g., any of the linkers described in Section 6.5 (usually a repeat sequence containing the amino acids glycine and serine, such as the amino acid sequence (Gly4-Ser)3 (SEQ ID NO:180)), such that the VH and VL sequences can be expressed as a continuous single-chain protein, where the VL and VH regions are linked by a flexible linker (see, e.g., Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature 348:552-554).

[0438] 6.8. Fc Region

[0439] The IFN proproteins of the present disclosure generally include a pair of Fc domains that associate to form an Fc region. In native antibodies, the Fc region contains a hinge region at its N-terminus to form the constant domains. Throughout the present disclosure, unless otherwise specified, references to Fc domains encompass Fc domains having a hinge domain at their N-terminus.

[0440] The Fc domain can be derived from any suitable species operably linked to an ABD or a component thereof. In one embodiment, the Fc domain is derived from a human Fc domain. In a preferred embodiment, the targeting moiety or a component thereof is fused to an IgG Fc molecule. The targeting moiety or a component thereof can be fused to the N-terminus or C-terminus or both of the IgG Fc domain.

[0441] The Fc domain can be derived from any suitable class of antibodies, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In one embodiment, the Fc domain is derived from IgG1, IgG2, IgG3, or IgG4. In one embodiment, the Fc domain is derived from IgG1. In one embodiment, the Fc domain is derived from IgG4. Exemplary sequences of Fc domains from IgG1, IgG2, IgG3, and IgG4 are provided in Table Y below.

[0442]

[0443]

[0444] In some aspects, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 410. Where the Fc domain has at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 410 (e.g., has a sequence identity between 90% and 99% to SEQ ID NO: 410), the Fc domain may further comprise one or more amino acid substitutions as described herein, such as one or more substitutions that reduce effector function (e.g., as described in Section 6.8.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.8.2).

[0445] In some aspects, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 411. Where the Fc domain has at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 411 (e.g., has a sequence identity between 90% and 99% to SEQ ID NO: 411), the Fc domain may further comprise one or more amino acid substitutions as described herein, such as one or more substitutions that reduce effector function (e.g., as described in Section 6.8.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.8.2).

[0446] In some aspects, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 412. Where the Fc domain has at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 412 (e.g., has a sequence identity between 90% and 99% to SEQ ID NO: 412), the Fc domain may further comprise one or more amino acid substitutions as described herein, such as one or more substitutions that reduce effector function (e.g., as described in Section 6.8.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.8.2).

[0447] In some aspects, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 413. In cases where the Fc domain has at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 413 (e.g., has a sequence identity between 90% and 99% to SEQ ID NO: 413), the Fc domain may further comprise one or more of the amino acid substitutions described herein, such as one or more substitutions that reduce effector function (e.g., as described in Section 6.8.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.8.2).

[0448] The two Fc domains within the Fc region can be the same as or different from each other. In native antibodies, the Fc domains are typically identical, but for the purpose of generating multispecific binding molecules, such as the IFN proproteins of the present disclosure and the MBMs generated through their activation, the Fc domains may advantageously be different to allow heterodimerization, as described in Section 6.8.2 below.

[0449] In native antibodies, the heavy chain Fc domains of IgA, IgD, and IgG are composed of two heavy chain constant domains (CH2 and CH3), while the heavy chain Fc domains of IgE and IgM are composed of three heavy chain constant domains (CH2, CH3, and CH4). These dimers form the Fc region.

[0450] In the IFN proproteins of the present disclosure, the Fc region and / or the Fc domains therein may comprise heavy chain constant domains from one or more different classes (e.g., one, two, or three different classes) of antibodies.

[0451] In one embodiment, the Fc region comprises the CH2 and CH3 domains derived from IgG1.

[0452] In one embodiment, the Fc region comprises the CH2 and CH3 domains derived from IgG2.

[0453] In one embodiment, the Fc region comprises the CH2 and CH3 domains derived from IgG3.

[0454] In one embodiment, the Fc region comprises the CH2 and CH3 domains derived from IgG4.

[0455] In one embodiment, the Fc region comprises the CH4 domain from IgM. The IgM CH4 domain is typically located at the C-terminus of the CH3 domain.

[0456] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG and a CH4 domain derived from IgM.

[0457] It should be understood that the heavy chain constant domains of the Fc region used to generate the IFN proprotein of the present disclosure may include variants of the above-described naturally occurring constant domains. Such variants may contain one or more amino acid variations compared to the wild-type constant domain. In one example, the Fc region of the present disclosure comprises at least one constant domain that is different in sequence from the wild-type constant domain. It should be understood that the variant constant domain may be longer or shorter than the wild-type constant domain. Preferably, the variant constant domain is at least 60% identical or similar to the wild-type constant domain. In another example, the variant constant domain is at least 70% identical or similar. In another example, the variant constant domain is at least 80% identical or similar. In another example, the variant constant domain is at least 90% identical or similar. In another example, the variant constant domain is at least 95% identical or similar.

[0458] IgM and IgA naturally exist in the human body as covalent multimers of common H2L2 antibody units. When IgM incorporates the J chain, it appears as a pentamer; or when IgM lacks the J chain, it appears as a hexamer. IgA appears as a monomer and a dimer. The heavy chains of IgM and IgA have 18 amino acids extending to the C-terminal constant domain, called the tailpiece. The tailpiece contains a cysteine residue that forms a disulfide bond between the heavy chains of the polymer and is thought to play an important role in polymerization. The tailpiece also contains glycosylation sites. In certain embodiments, the IFN proprotein of the present disclosure does not contain a tailpiece.

[0459] The Fc domain incorporated into the IFN proprotein of the present disclosure may include one or more modifications that alter the function of the protein, such as binding to Fc receptors such as FcRn or leukocyte receptors, binding to complement, modified disulfide bond structures, or altered glycosylation patterns. Exemplary Fc modifications that alter effector function are described in Section 6.8.1.

[0460] The Fc domain may also be altered to include modifications that improve the manufacturability of the asymmetric IFN proprotein, such as by allowing heterodimerization, which is the preferential pairing of non-identical Fc domains relative to identical Fc domains. Heterodimerization allows the production of IFN proproteins in which different polypeptide components are linked to each other through Fc regions containing Fc domains with different sequences. Examples of heterodimerization strategies are illustrated in Section 6.8.2.

[0461] It should be understood that any of the above modifications may be combined in any suitable manner to achieve the desired functional properties and / or combined with other modifications to alter the properties of the IFN proprotein.

[0462] 6.8.1 Fc domains with altered effector function

[0463] In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and / or effector function.

[0464] In certain embodiments, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In specific embodiments, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, and most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group consisting of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and cytokine secretion. In specific embodiments, the effector function is ADCC.

[0465] In one embodiment, the Fc domain (e.g., the Fc domain of an IFN proprotein half-antibody) or Fc region (e.g., one or two Fc domains of an IFN proprotein that can associate to form an Fc region) contains an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331, and P329 (numbering according to the Kabat EU index). In a more specific embodiment, the Fc domain or Fc region contains an amino acid substitution at a position selected from L234, L235, and P329 (numbering according to the Kabat EU index). In some embodiments, the Fc domain or Fc region contains the amino acid substitutions L234A and L235A (numbering according to the Kabat EU index). In one such embodiment, the Fc domain or region is an Igd Fc domain or region, particularly a human Igd Fc domain or region. In one embodiment, the Fc domain or Fc region contains an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numbering according to the Kabat EU index). In one embodiment, the Fc domain or Fc region contains an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297, and P331 (numbering according to the Kabat EU index). In a more specific embodiment, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a specific embodiment, the Fc domain or Fc region contains amino acid substitutions at positions P329, L234, and L235 (numbering according to the Kabat EU index). In a more specific embodiment, the Fc domain contains the amino acid mutations L234A, L235A, and P329G (“P329GLALA”, “PGLALA”, or “LALAPG”).

[0466] Generally, the same one or more amino acid substitutions are present in each Fc domain of the two Fc domains of the Fc region. Thus, in a specific embodiment, each Fc domain of the Fc region contains the amino acid substitutions L234A, L235A, and P329G (Kabat EU index number), i.e., in each of the first and second Fc domains of the Fc region, the leucine residue at position 234 is replaced by an alanine residue (L234A), the leucine residue at position 235 is replaced by an alanine residue (L235A), and the proline residue at position 329 is replaced by a glycine residue (P329G) (numbering according to the Kabat EU index).

[0467] In one embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In some embodiments, the IgG1 Fc domain is a variant IgG1 comprising D265A, N297A mutations (EU numbering) to reduce effector function.

[0468] In another embodiment, the Fc domain is an IgG4 Fc domain that reduces binding to Fc receptors. Exemplary IgG4 Fc domains that reduce binding to Fc receptors can comprise an amino acid sequence selected from Table H below. In some embodiments, the Fc domain only includes the bolded portion of the sequence shown below:

[0469]

[0470]

[0471]

[0472]

[0473]

[0474] In certain embodiments, the IgG4 with reduced effector function includes the bolded portion of the amino acid sequence of SEQ ID NO:31 of WO2014 / 121087, sometimes referred to herein as IgG4 or hIgG4, which has the following amino acid sequence: ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:396).

[0475] For heterodimeric Fc regions, combinations of the above-described variant IgG4 Fc sequences can be incorporated, such as an Fc region comprising an Fc domain containing the amino acid sequence of SEQ ID NO:30 of WO2014 / 121087 (or the bolded portion thereof) and an Fc domain containing the amino acid sequence of SEQ ID NO:37 of WO2014 / 121087 (or the bolded portion thereof), or an Fc region comprising an Fc domain containing the amino acid sequence of SEQ ID NO:31 of WO2014 / 121087 (or the bolded portion thereof) and an Fc domain containing the amino acid sequence of SEQ ID NO:38 of WO2014 / 121087 (or the bolded portion thereof).

[0476] 6.8.2. Fc heterodimerization variants

[0477] Certain IFN precursors require dimerization between two Fc domains, which, unlike native immunoglobulins, are operably linked to different N-terminal or C-terminal regions. Insufficient heterodimerization of the two Fc domains to form the Fc region can be an obstacle to increasing the yield of the desired heterodimeric molecule and poses challenges for purification. A variety of methods available in the art can be used to enhance the dimerization of Fc domains that may be present in the IFN precursors of the present disclosure, such as those disclosed in EP 1870459A1; U.S. Patent No. 5,582,996; U.S. Patent No. 5,731,168; U.S. Patent No. 5,910,573; U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,441; U.S. Patent No. 7,183,076; U.S. Patent Application Publication No. 2006204493A1; and PCT Publication No. WO 2009 / 089004A1.

[0478] In some embodiments, the present disclosure provides an IFN precursor comprising an Fc heterodimer, i.e., an Fc region comprising heterologous, different Fc domains. Generally, each Fc domain in the Fc heterodimer comprises the CH3 domain of an antibody. The CH3 domain is derived from the constant region of an antibody of any isotype, class, or subclass, and preferably from the constant region of an antibody of the IgG (IgG1, IgG2, IgG3, and IgG4) class, as described in the previous section.

[0479] Heterodimerization of two different heavy chains at the CH3 domain results in the desired IFN precursor, while homodimerization of identical heavy chains will reduce the yield of the desired IFN precursor. Thus, in a preferred embodiment, the polypeptides that associate to form the IFN precursors of the present disclosure will contain CH3 domains having modifications that favor heterodimeric association relative to unmodified Fc domains.

[0480] In a specific embodiment, the modification that promotes Fc heterodimer formation is the so-called "knob-into-hole" or "knob-hole" modification, which includes a "knob" modification in one Fc domain of the Fc domain and a "hole" modification in the other Fc domain. The knob-into-hole technology is described in, for example, U.S. Patent No. 5,731,168; US 7,695,936; Ridgway et al., 1996, Prot Eng 9:617-621, and Carter, 2001, Immunol Meth 248:7-15. Generally, the method includes introducing a protrusion ("knob") at the interface of the first polypeptide and introducing a corresponding cavity ("hole") in the interface of the second polypeptide, such that the protrusion can be positioned in the cavity, thereby promoting heterodimer formation and hindering homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensatory cavity of the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0481] Thus, in some embodiments, the amino acid residues in the CH3 domain of the first subunit of the Fc domain are replaced with amino acid residues having a larger side chain volume, thereby creating a protrusion within the CH3 domain of the first subunit that can be positioned in a cavity within the CH3 domain of the second subunit, and the amino acid residues in the CH3 domain of the second subunit of the Fc domain are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit in which the protrusion within the CH3 domain of the first subunit can be positioned. Preferably, the amino acid residues having a larger side chain volume are selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residues having a smaller side chain volume are selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protrusion and the cavity can be prepared by altering the nucleic acid encoding the polypeptide, e.g., by site-specific mutagenesis or by peptide synthesis. An exemplary substitution is Y470T.

[0482] In specific such embodiments, in the first Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V), and optionally, the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (according to Kabat EU index numbering). In a further embodiment, in the first Fc domain, the serine residue at position 354 is additionally replaced with a cysteine residue (S354C), or the glutamate residue at position 356 is replaced with a cysteine residue (E356C) (especially the serine residue at position 354 is replaced with a cysteine residue), and in the second Fc domain, the tyrosine residue at position 349 is additionally replaced with a cysteine residue (Y349C) (according to Kabat EU index numbering). In a specific embodiment, the first Fc domain comprises the amino acid substitutions S354C and T366W, and the second Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (according to Kabat EU index numbering).

[0483] In some embodiments, electrostatic steering (e.g., as described in Gunasekaran et al., 2010, J Biol Chem 285(25):19637-46) can be used to facilitate the association of the first and second Fc domains of the Fc region.

[0484] As an alternative or addition to using Fc domains modified to promote heterodimerization, the Fc domains can be modified to allow for a purification strategy that enables selection of Fc heterodimers. In one such embodiment, a polypeptide comprises a modified Fc domain that eliminates its binding to Protein A, thus enabling a purification method for producing heterodimeric proteins. See, e.g., U.S. Patent No. 8,586,713. Thus, the IFN proprotein comprises a first CH3 domain and a second Ig CH3 domain, wherein the first Ig CH3 domain and the second Ig CH3 domain differ by at least one amino acid from each other, and wherein the at least one amino acid difference reduces the binding of the IFN proprotein to Protein A compared to a corresponding IFN proprotein lacking the amino acid difference. In one embodiment, the first CH3 domain binds Protein A and the second CH3 domain contains a mutation / modification that reduces or eliminates Protein A binding, such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second CH3 may further comprise a Y96F modification (by IMGT; Y436F by EU). Such modifications are referred to herein as "star" mutations.

[0485] In some embodiments, the Fc may contain one or more mutations (e.g., knobs-into-holes mutations) to facilitate heterodimerization and star mutations to facilitate purification.

[0486] 6.8.3. Hinge Domain

[0487] The IFN proprotein of the present disclosure may comprise an Fc domain that includes a hinge domain at its N-terminus. The hinge region may be a native or modified hinge region. The hinge region is typically located at the N-terminus of the Fc region. Unless the context otherwise dictates, the term "hinge domain" refers to a native or non-naturally occurring hinge sequence that is a monomeric hinge domain in the context of a single or monomeric polypeptide chain and may comprise two associated hinge sequences located on separate polypeptide chains in the context of a dimeric polypeptide (e.g., a homodimeric or heterodimeric IFN proprotein formed by the association of two Fc domains). Sometimes, the two related hinge sequences are referred to as the "hinge region". In certain embodiments of the IFN proprotein, additional iterations of the hinge region may be incorporated into the polypeptide sequence.

[0488] The native hinge region is the hinge region typically found between the Fab and Fc domains of a naturally occurring antibody. A modified hinge region is any hinge that differs in length and / or composition from the native hinge region. Such hinges may include hinge regions from other species, such as human, mouse, rat, rabbit, shark, pig, hamster, camel, llama, or goat hinge regions. Other modified hinge regions may comprise the complete hinge region from an antibody of a different class or subclass from the heavy chain Fc domain or Fc region. Alternatively, a modified hinge region may comprise a native hinge or a portion of a repeating unit, where each unit in the repeat is derived from the native hinge region. In additional alternatives, the native hinge region may be altered by converting one or more cysteine or other residues to neutral residues, such as serine or alanine, or by converting appropriately placed residues to cysteine residues. In this way, the number of cysteine residues in the hinge region may be increased or decreased. Other modified hinge regions may be fully synthetic and may be designed to have desired properties, such as length, cysteine composition, and flexibility.

[0489] Many modified hinge regions have been described, for example, in U.S. Patent No. 5,677,425, WO 99 / 15549, WO2005 / 003170, WO 2005 / 003169, WO 2005 / 003170, WO 98 / 25971, and WO 2005 / 003171 and these are incorporated herein by reference.

[0490] In one embodiment, the IFN proprotein of the present disclosure comprises an Fc region, wherein one or both Fc domains have a complete hinge domain at their N-terminus.

[0491] In various embodiments, positions 233 - 236 within the hinge region can be G, G, G, and unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied, unoccupied, and unoccupied; or all unoccupied, where the positions are numbered by EU numbering.

[0492] In some embodiments, the IFN pro - protein of the present disclosure comprises a modified hinge region that reduces the binding affinity for Fcγ receptors relative to the wild - type hinge region of the same isotype (e.g., human IgG1 or human IgG4).

[0493] In one embodiment, the IFN pro - protein of the present disclosure comprises an Fc region, wherein each Fc domain has a complete hinge domain at its N - terminus, wherein each Fc domain and hinge domain are derived from IgG4 and each hinge domain comprises the modified sequence CPPC (SEQ ID NO:375). The core hinge region of human IgG4 contains the sequence CPSC (SEQ ID NO:376) compared to IgG1 containing the sequence CPPC (SEQ ID NO:375). The presence of the serine residue in the IgG4 sequence results in increased flexibility in this region, such that a portion of the molecule forms disulfide bonds within the same protein chain (intra - chain disulfide bonds) rather than bridging to other heavy chains in the IgG molecule to form inter - chain disulfide bonds. (Angel et al., 1993, Mol Immunol 30(1):105 - 108). Changing the serine residue to a proline to obtain the same core sequence as IgG1 can fully form inter - chain disulfide bonds in the IgG4 hinge region, thereby reducing heterogeneity in the purified product. This altered isotype is referred to as IgG4P.

[0494] The hinge sequence incorporated into the IFN pro - protein of the present disclosure can be full - length (“long”) or truncated (“short”). An example of a full - length hinge sequence is ESKYGPPCPPCPAPPVA (SEQ ID NO:377). An example of a truncated hinge sequence is ESKYGPPCPPC (SEQ ID NO:378). ESKYGPPCPPC (SEQ ID NO:378) is truncated by 6 amino acids compared to the full - length hinge sequence ESKYGPPCPPCPAPPCA (SEQ ID NO:379). In various aspects, compared to a full - length hinge sequence, e.g., any of the full - length hinge sequences disclosed herein, the truncated hinge can have a C - terminal deletion of 1, 2, 3, 4, 5, or 6 amino acids. Without being bound by theory, it is believed that the truncated hinge sequence may confer improved spatial constraints on the IFN moiety. An IFN pro - protein that contains two hinge domains in each half - antibody (e.g., having Figure 1C 、 2CIn the IFN proprotein of the configuration depicted in FIGS. 1 and 2F, the two hinge domains can both be full-length, truncated, or a combination thereof. Thus, the N-terminal hinge domain can be truncated, the C-terminal hinge domain can be truncated, or both the N-terminal hinge domain and the C-terminal hinge domain can be truncated.

[0495] 6.8.3.1. Chimeric hinge sequences

[0496] The hinge domain can be a chimeric hinge domain.

[0497] For example, the chimeric hinge can comprise an "upper hinge" sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4, which is combined with a "lower hinge" sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4.

[0498] In certain embodiments, the chimeric hinge region comprises the amino acid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 380) (previously disclosed as SEQ ID NO: 8 in WO2014 / 121087, which is incorporated herein by reference in its entirety) or ESKYGPPCPPCPAPPVA (SEQ ID NO: 377) (previously disclosed as SEQ ID NO: 9 in WO2014 / 121087). Such chimeric hinge sequences can be suitably linked to the IgG4 CH2 region (e.g., by incorporating an IgG4 Fc domain, such as a human or murine Fc domain, which can be further modified in the CH2 and / or CH3 domains to reduce effector function, as described, for example, in Section 6.8.1).

[0499] 6.8.3.2. Hinge sequences with reduced effector function

[0500] In further embodiments, the hinge region can be modified to reduce effector function, as described, for example, in WO2016161010A2, which is incorporated herein by reference in its entirety. In various embodiments, positions 233-236 of the modified hinge region are G, G, G and unoccupied; G, G, unoccupied and unoccupied; G, unoccupied, unoccupied and unoccupied; or all unoccupied, where the positions are numbered by EU numbering (as shown in FIG. 1 of WO2016161010A2). These segments can be represented as GGG-, GG--, G---, or ----, where "-" represents an unoccupied position.

[0501] Position 236 is not occupied in canonical human IgG2 but is occupied in other canonical human IgG isotypes. In all four human isotypes, positions 233 - 235 are occupied by residues other than G (as shown in Figure 1 of WO2016161010A2).

[0502] Hinge modifications within positions 233 - 236 can be combined with position 228 occupied by P. Position 228 is naturally occupied by P in human IgG1 and IgG2, but by S in human IgG4 and by R in human IgG3. The S228P mutation in IgG4 antibodies favors the stabilization of IgG4 antibodies and reduces the exchange of heavy - chain light - chain pairs between exogenous and endogenous antibodies. Preferably, positions 226 - 229 are occupied by C, P, P, and C, respectively.

[0503] Exemplary hinge regions have residues 226 - 236, sometimes referred to as the middle (or core) and lower hinge, occupied by modified hinge sequences designated GGG-(233 - 236), GG--(233 - 236), G---(233 - 236), and no G (233 - 236). Optionally, the hinge domain amino acid sequence includes CPPCPAPGGG - GPSVF (SEQ ID NO:381) (previously disclosed as SEQ ID NO:1 of WO2016161010A2), CPPCPAPGG--GPSVF (SEQ ID NO:382) (previously disclosed as SEQ ID NO:2 of WO2016161010A2), CPPCPAPG---GPSVF (SEQ ID NO:383) (previously disclosed as SEQ ID NO:3 of WO2016161010A2), or CPPCPAP----GPSVF (SEQ ID NO:384) (previously disclosed as SEQ ID NO:4 of WO2016161010A2).

[0504] The above - described modified hinge regions can be incorporated into the heavy - chain constant region, which typically includes CH2 and CH3 domains and can have additional hinge segments (e.g., upper hinge) flanking the designated region. Such additional constant - region fragments that are present generally have the same isotype, preferably a human isotype, although they can be heterozygotes of different isotypes. The isotype of such additional human constant - region fragments is preferably human IgG4, but can also be human IgG1, IgG2, or IgG3 or a heterozygote with domains of different isotypes. Exemplary sequences of human IgG1, IgG2, and IgG4 are shown in Figures 2 to 4 of WO2016161010A2.

[0505] In a specific embodiment, the modified hinge sequence can be linked to the IgG4 CH2 region (e.g., by incorporating an IgG4 Fc domain, such as a human or murine Fc domain, which can be further modified in the CH2 and / or CH3 domains to reduce effector function, e.g., as described in Section 6.8.1).

[0506] 6.9. Nucleic Acids and Host Cells

[0507] In another aspect, the present disclosure provides nucleic acids encoding the IFN preproteins of the present disclosure. In some embodiments, the IFN preprotein is encoded by a single nucleic acid. In other embodiments, the IFN preprotein can be encoded by multiple (e.g., two, three, four, or more) nucleic acids.

[0508] A single nucleic acid can encode an IFN preprotein comprising a single polypeptide chain, an IFN preprotein comprising two or more polypeptide chains, or a portion of an IFN preprotein comprising more than two polypeptide chains (e.g., a single nucleic acid can encode two polypeptide chains of an IFN preprotein comprising three, four, or more polypeptide chains, or three polypeptide chains of an IFN preprotein comprising four or more polypeptide chains). For separate control of expression, the open reading frames encoding two or more polypeptide chains can be under the control of separate transcriptional regulatory elements (e.g., promoters and / or enhancers). The open reading frames encoding two or more polypeptides can also be under the control of the same transcriptional regulatory element and separated by an internal ribosome entry site (IRES) sequence, thereby allowing translation into separate polypeptides.

[0509] In some embodiments, the IFN preprotein comprising two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding the IFN preprotein can be equal to or less than the number of polypeptide chains in the IFN preprotein (e.g., when more than one polypeptide chain is encoded by a single nucleic acid).

[0510] The nucleic acids of the present disclosure can be DNA or RNA (e.g., mRNA).

[0511] In another aspect, the present disclosure provides host cells and vectors containing the nucleic acids of the present disclosure. The nucleic acids can be present in a single vector or in separate vectors in the same host cell or separate host cells, as described in more detail below.

[0512] 6.9.1. Vectors

[0513] The present disclosure provides vectors comprising nucleotide sequences encoding the IFN preproteins or components thereof described herein, such as one or two polypeptide chains of a half-antibody of an IFN preprotein. Vectors include, but are not limited to, viruses, plasmids, cosmids, λ phages, or yeast artificial chromosomes (YACs).

[0514] A variety of vector systems can be employed. For example, one class of vectors utilizes DNA elements derived from animal viruses, such as, for example, bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retroviruses (Rous sarcoma virus, MMTV or MoMLV) or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses, such as Semliki Forest virus, Eastern equine encephalitis virus and flavivirus.

[0515] Additionally, cells in which DNA has been stably integrated into their chromosomes can be selected by introducing one or more markers that permit selection of the transfected host cells. The markers can provide, for example, prototrophy for auxotrophic hosts, biocide resistance (e.g., antibiotics) or resistance to heavy metals (e.g., copper). The selectable marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Optimal synthesis of mRNA may also require additional elements. These elements can include splice signals as well as transcriptional promoters, enhancers and termination signals.

[0516] Once the expression vector or the DNA sequence containing the construct has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be employed to achieve this, such as, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques. Methods and conditions for culturing the resulting transfected cells and for recovering the expressed polypeptide are known to those skilled in the art and can vary or be optimized based on this specification according to the particular expression vector and mammalian host cell used.

[0517] 6.9.2. Cells

[0518] The present disclosure also provides host cells comprising the nucleic acids of the present disclosure.

[0519] In one embodiment, the host cell is genetically engineered to comprise one or more of the nucleic acids described herein.

[0520] In one embodiment, the host cell is genetically engineered using an expression cassette. The phrase "expression cassette" refers to a nucleotide sequence capable of affecting the expression of a gene in a host compatible with such sequences. Such cassettes can include a promoter, an open reading frame with or without introns and a termination signal. Additional factors necessary or helpful for affecting expression can also be used, such as, for example, an inducible promoter.

[0521] The present disclosure also provides host cells comprising the vectors described herein.

[0522] The cells can be, but are not limited to, eukaryotic cells, bacterial cells, insect cells, or human cells. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells.

[0523] 6.10. Pharmaceutical Compositions

[0524] The IFN proprotein of the present disclosure can be in the form of a composition comprising the IFN proprotein and one or more carriers, excipients, and / or diluents. The composition can be formulated for a specific use, such as for veterinary use or human pharmaceutical use. The form of the composition used (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents, and / or carriers will depend on the intended use of the IFN proprotein and the mode of administration for therapeutic use.

[0525] For therapeutic use, the composition can be provided as part of a sterile pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition can be in any suitable form (depending on the desired method of administering it to a patient). The pharmaceutical composition can be administered to a patient by a variety of routes, such as orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intratumorally, intrathecally, topically, or locally. The most suitable route of administration in any given case will depend on the specific IFN proprotein, the subject, and the nature and severity of the disease, as well as the physical condition of the subject. Generally, the pharmaceutical composition will be administered intravenously or subcutaneously.

[0526] The pharmaceutical composition can conveniently be in unit dosage form containing a predetermined amount of the IFN proprotein of the present disclosure. The amount of IFN proprotein contained in a unit dose will depend on the disease being treated and other factors well known in the art. Such unit doses can be in the form of a lyophilized dry powder containing an amount of IFN proprotein suitable for single administration, or in liquid form. The dry powder unit dosage form can be packaged in a kit together with a syringe, an appropriate amount of diluent, and / or other components for administration. The unit dose in liquid form can conveniently be provided in the form of a syringe prefilled with an amount of IFN proprotein suitable for single administration.

[0527] The pharmaceutical composition can also be supplied in bulk containing an amount of IFN proprotein suitable for multiple administrations.

[0528] A pharmaceutical composition can be prepared by mixing IFN proprotein having a desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (all of which are referred to herein as "carriers") commonly used in the art (i.e., buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, antioxidants, and various other additives) for storage as a lyophilized preparation or an aqueous solution. See Remington’s Pharmaceutical Sciences, 16th Edition (Osol, Ed. 1980). Such additives should be non-toxic to the recipient at the doses and concentrations employed.

[0529] Buffers help maintain the pH value within a range close to physiological conditions. They can be present in various concentrations, but are typically present at concentrations in the range of about 2 mM to about 50 mM. Buffers suitable for the present disclosure include organic and inorganic acids and their salts, such as citrate buffers (e.g., sodium citrate - disodium citrate mixture, citric acid - trisodium citrate mixture, citric acid - sodium citrate mixture, etc.), succinate buffers (e.g., succinic acid - sodium succinate mixture, succinic acid - sodium hydroxide mixture, succinic acid - disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid - sodium tartrate mixture, tartaric acid - potassium tartrate mixture, tartaric acid - sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid - sodium fumarate mixture, fumaric acid - disodium fumarate mixture, sodium fumarate - disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconic acid - sodium gluconate mixture, gluconic acid - sodium hydroxide mixture, gluconic acid - potassium gluconate mixture, etc.), oxalate buffers (e.g., oxalic acid - sodium oxalate mixture, oxalic acid - sodium hydroxide mixture, oxalic acid - potassium oxalate mixture, etc.), lactate buffers (e.g., lactic acid - sodium lactate mixture, lactic acid - sodium hydroxide mixture, lactic acid - potassium lactate mixture, etc.), and acetate buffers (e.g., acetic acid - sodium acetate mixture, acetic acid - sodium hydroxide mixture, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts (such as Tris) can also be used.

[0530] Preservatives can be added to retard microbial growth and can be added in an amount in the range of about 0.2% to 1% (w / v). Preservatives suitable for the present disclosure include phenol, benzyl alcohol, m-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, octadecyl dimethyl benzyl ammonium chloride, benzalkonium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkyl p-hydroxybenzoates (such as methyl or propyl p-hydroxybenzoate), catechol, resorcinol, cyclohexanol, and 3-pentanol. An isotonic agent (sometimes referred to as a "stabilizer") can be added to ensure the isotonicity of the liquid compositions of the present disclosure, and isotonic agents include polyhydric alcohols, such as trihydric or higher polyhydric alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a large class of excipients whose functional range includes from fillers to additives, which can dissolve therapeutic agents or help prevent denaturation or adhesion to the container walls. Typical stabilizers can be polyhydric alcohols (listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, inositol, galactitol, glycerol, etc., including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, lipoic acid, sodium thioacetate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides of 10 residues or less); proteins, such as human serum albumin, bovine serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose, fructose, glucose; disaccharides, such as lactose, maltose, sucrose, and trehalose; and trisaccharides, such as raffinose; and polysaccharides, such as dextran. The stabilizer can be present in an amount in the range of 0.5 to 10% by weight based on the weight of the IFN proprotein.

[0531] Nonionic surfactants or detergents (also referred to as "wetting agents") can be added to assist in dissolving the glycoprotein and to protect the glycoprotein from aggregation caused by agitation, which also allows the formulation to be exposed to shear surface stress without causing denaturation of the protein. Suitable nonionic surfactants include polysorbates (20, 80, etc.), poloxamers (184, 188, etc.), and Pluronic polyols. The nonionic surfactant can be present in a range of about 0.05 mg / mL to about 1.0 mg / mL (e.g., about 0.07 mg / mL to about 0.2 mg / mL).

[0532] Other miscellaneous excipients include fillers (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and co-solvents.

[0533] The IFN proprotein of the present disclosure can be formulated into a pharmaceutical composition comprising the IFN proprotein, for example, containing one or more pharmaceutically acceptable excipients or carriers. To prepare a pharmaceutical or sterile composition comprising the IFN proprotein of the present disclosure, the IFN proprotein preparation can be combined with one or more pharmaceutically acceptable excipients or carriers.

[0534] For example, a preparation of the IFN proprotein can be prepared by mixing the IFN proprotein with a physiologically acceptable carrier, excipient, or stabilizer in the form of, for example, a lyophilized powder, slurry, aqueous solution, lotion, or suspension (see, e.g., Hardman et al., 2001, Goodman and Gilman’s The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro, 2000, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis et al. (eds.), 1993, Pharmaceutical Dosage Forms: General Medications, Marcel Dekker, NY; Lieberman et al. (eds.), 1990, Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al. (eds.), 1990, Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie, 2000, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.).

[0535] The effective amount for a particular subject can vary depending on a variety of factors, such as the disorder being treated, the overall health of the subject, the method of administration, route and dosage, and the severity of side effects (see, e.g., Maynard et al. (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).

[0536] The compositions of the present disclosure can also be administered by one or more routes of administration using one or more methods known in the art. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired result. Selected routes of administration of the IFN proprotein include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, intraspinal, or other general routes of administration, such as by injection or infusion. General administration can represent a mode of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Alternatively, the compositions of the present disclosure can be administered by non-general routes, such as topical, epidermal, or mucosal routes of administration, e.g., intranasal, oral, vaginal, rectal, sublingual, or topical. In one embodiment, the IFN proprotein is administered by infusion. In another embodiment, the IFN proprotein of the present disclosure is administered subcutaneously.

[0537] 6.10.1. Pharmaceutical Compositions for Delivery of IFN Proproteins Encoding Nucleic Acids

[0538] The IFN proproteins of the present disclosure (e.g., IFN receptor agonists) can be delivered by any method useful for gene therapy, e.g., as mRNA or by a viral vector encoding the IFN proprotein (e.g., IFN receptor agonist) under the control of a suitable promoter.

[0539] Exemplary viral vectors include recombinant adenoviruses and adeno-associated virus vectors (rAAV). rAAV vectors are based on the defective and non-pathogenic parvovirus adeno-associated virus type 2. Most such vectors are derived from plasmids that retain only the AAV inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery achieved by integration into the genome of transduced cells are key features of this vector system. AAV serotypes can be used to deliver the IL27 transgene AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV 8.2, AAV9, and AAV rh10, as well as pseudotyped AAVs such as AAV2 / 8, AAV2 / 5, and AAV2 / 6.

[0540] AAV can be produced at the clinical scale by many different processes. Examples of systems that can be used include (1) plasmid DNA transfection in mammalian cells, (2) Ad infection of stable mammalian cell lines, (3) infection of mammalian cells with recombinant herpes simplex virus (rHSV), and (4) infection of insect cells (Sf9 cells) with recombinant baculovirus (reviewed by Penaud-Budloo et al., 2018, Mol Ther Methods Clin Dev. 8:166-180).

[0541] Replication-deficient recombinant adenovirus vectors (Ad) can be produced at high titers and easily infect many different cell types. Most adenovirus vectors are engineered to replace the Ad Ela, Elb, and / or E3 genes with a transgene; subsequently, the replication-deficient vectors are propagated in human 293 cells, which provide the missing transgene functions. Ad vectors can transduce multiple types of tissues in vivo, including non-dividing differentiated cells such as those found in the liver, kidney, and muscle. Conventional Ad vectors have a large carrying capacity.

[0542] Packaging cells are used to form virus particles capable of infecting host cells. Such cells include 293 cells for packaging adenovirus, and w2 cells or PA317 cells for packaging retrovirus. Viral vectors used in gene therapy are usually produced by producer cell lines that package nucleic acid vectors into virus particles. The vector usually contains the minimal viral sequences required for packaging and subsequent integration into the host (if applicable), and other viral sequences are replaced by an expression cassette encoding the protein to be expressed. The missing viral functions are provided in trans by the packaging cell line. For example, AAV vectors used in gene therapy usually only have the inverted terminal repeats (ITRs) from the AAV genome, which are necessary for packaging and integration into the host genome. The viral DNA is packaged in a cell line that contains a helper plasmid encoding other AAV genes, namely rep and cap, but lacks the ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus promotes the replication of the AAV vector and the expression of the AAV genes from the helper plasmid. Due to the lack of ITR sequences, the helper plasmid is not packaged in large amounts. Adenovirus contamination can be reduced, for example, by heat treatment, as adenovirus is more sensitive to heat treatment than AAV.

[0543] A nucleic acid molecule (e.g., mRNA) or virus can be formulated as the sole pharmaceutically active ingredient in a pharmaceutical composition, or can be combined with other active agents for a particular disorder being treated. Optionally, other medicaments, pharmaceutical agents, carriers, adjuvants, diluents can be included in the compositions provided herein. For example, wetting agents, emulsifying agents, and lubricants (such as sodium lauryl sulfate and magnesium stearate), as well as coloring agents, demolding agents, coating agents, sweetening agents, flavoring agents, and fragrances, preservatives, antioxidants, chelating agents, and inert gases, any one or more of which can also be present in the composition. Exemplary other reagents and excipients that can be included in the composition include, for example, water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0544] 6.11. Therapeutic Indications and Methods of Use

[0545] The present disclosure provides methods of use and applications for the IFN proprotein of the present disclosure.

[0546] The IFN proprotein of the present disclosure (e.g., IFN receptor agonist) can be used to stimulate an immune response in a variety of applications.

[0547] In some aspects, the present disclosure provides a method of treating cancer, which includes administering to a subject in need thereof the IFN proprotein or pharmaceutical composition described herein. In some embodiments, the activated IFN protein comprising the IFN moiety is produced by cleavage of one or more protease-cleavable linkers in the IFN proprotein by one or more proteases expressed by cancer tissue. Thus, the IFN proprotein is selectively activated in cancer tissue.

[0548] In some embodiments, the present disclosure provides a method of treating cancer with an IFN protein selectively activated in cancer tissue, which includes administering to a subject in need thereof the IFN proprotein or pharmaceutical composition as described herein, wherein the IFN proprotein has one or more protease-cleavable linkers, and each protease-cleavable linker comprises one or more substrates for one or more proteases expressed by the cancer tissue expected for the IFN protein. Thus, the activated IFN protein comprising the IFN moiety is produced by cleavage of one or more protease-cleavable linkers in the IFN proprotein by one or more proteases in the cancer tissue.

[0549] The present disclosure further provides a method of local delivery of an IFN protein, which includes administering to a subject the IFN proprotein or pharmaceutical composition as described herein, wherein the IFN proprotein has one or more protease-cleavable linkers, and each protease-cleavable linker comprises one or more substrates for one or more proteases expressed by the tissue to which local delivery of the IFN protein is to be made. As used herein, the term "local delivery" does not require local administration, but means that the active component of the IFN proprotein refers to activation of the protein at the lesion of interest by proteases active at the intended site, optionally in combination with targeting to the lesion of interest with a targeting moiety that recognizes a target molecule expressed by the tissue.

[0550] The present disclosure further provides a method of administering an IFN therapy with reduced systemic exposure and / or reduced systemic toxicity to a subject, which includes administering to the subject an IFN therapy in the form of the IFN proprotein or pharmaceutical composition as described herein, wherein the IFN proprotein has one or more protease-cleavable linkers, and each protease-cleavable linker comprises one or more substrates for one or more proteases expressed by the tissue for which IFN therapy is desired and / or expected.

[0551] Thus, due to the preferential activation of the IFN proprotein at the lesion where IFN treatment is expected, the foregoing methods allow for a reduction in off-target side effects of the IFN therapy.

[0552] In some embodiments of the foregoing methods, the IFN proprotein is also targeted and comprises one or more targeting moieties that recognize a target molecule expressed (e.g., by the tissue) at the site where treatment is expected.

[0553] Accordingly, the present disclosure provides a method of targeting an activated IFN protein to a site of intended treatment (e.g., cancer tissue), which comprises administering to a subject an IFN proprotein or pharmaceutical composition as described herein, wherein the IFN comprises one or more targeting moieties that recognize a target molecule expressed in a lesion or by the tissue of intended treatment (e.g., cancer tissue), and the IFN has one or more protease-cleavable linkers, each protease-cleavable linker comprising one or more substrates for one or more proteases expressed by the tissue for which IFN therapy is desired and / or intended.

[0554] The present disclosure further provides a method of locally inducing an immune response in a target tissue, which comprises administering to a subject an IFN proprotein or pharmaceutical composition as described herein, the IFN proprotein or pharmaceutical composition having one or more targeting moieties capable of binding to a target molecule expressed in the target tissue, and one or more protease-cleavable linkers, each protease-cleavable linker comprising one or more substrates for one or more proteases expressed in the target tissue. The activated IFN protein comprising the IFN moiety can subsequently be generated by cleavage of one or more protease-cleavable linkers in the IFN proprotein by one or more proteases in the target tissue. The resulting activated IFN protein can then induce an immune response against at least one cell type in the target tissue.

[0555] In some embodiments, the administration is not local to the tissue. For example, when the target tissue is cancer tissue, the administration can be systemic or subcutaneous.

[0556] The IFN proprotein of the present disclosure can be used to treat any proliferative disease (e.g., cancer) that expresses a target molecule (on tumor cells or in the tumor microenvironment, e.g., extracellular matrix or tumor lymphocytes). In a specific embodiment, the cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, brain tumor, cholangiocarcinoma, bladder cancer, bone cancer, breast cancer, bronchial tumor, Burkitt lymphoma, carcinoma of unknown primary, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasm, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hairy cell leukemia, hepatocellular carcinoma, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, midline tract carcinoma involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid carcinoma, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sézary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, or Wilms tumor.

[0557] Table I below shows exemplary indications for which IFN proproteins targeting specific target molecules can be used.

[0558]

[0559]

[0560]

[0561] Other target molecules and corresponding indications are disclosed, for example, in Hafeez et al., 2020, Molecules 25:4764, doi:10.3390 / molecules25204764, particularly in Table 1. Table 1 is hereby incorporated by reference in its entirety.

[0562] In further embodiments, the IFN proprotein (e.g., an IFN receptor agonist) can be used to enhance an immune response elicited by another agent. Thus, in some embodiments, the IFN proprotein (e.g., an IFN receptor agonist) of the present disclosure is administered as an adjuvant therapy to an immunogenic agent. In some embodiments, the immunogenic agent is an adjuvanted or non-adjuvanted vaccine. Thus, the IFN proprotein (e.g., an IFN receptor agonist) can enhance the antigen-specific immune response elicited by the vaccine. In various embodiments, the vaccine is a prophylactic or therapeutic cancer vaccine or a prophylactic or therapeutic vaccine against an infectious agent (e.g., a virus, bacterium, or parasite).

[0563] 7. Numbered embodiments

[0564] Although various specific embodiments have been shown and described, it should be understood that various changes can be made without departing from the spirit and scope of the present disclosure. The present disclosure is illustrated by the numbered embodiments set forth below.

[0565] In the following numbered embodiments, the targeting moiety is preferably bound to a mammalian target molecule, the IFN moiety is preferably derived from a mammalian IFN, the Fc domain is preferably derived from a mammalian antibody, and the subject is preferably a mammalian. More preferably, the mammalian is a human.

[0566] 1. A type I interferon (IFN) proprotein comprising:

[0567] (a) A first polypeptide chain comprising:

[0568] (i) A first immunoglobulin constant domain;

[0569] (ii) A first linker;

[0570] (iii) A first type I interferon (IFN) moiety;

[0571] (iv) A second linker; and

[0572] (v) A first Fc domain;

[0573] (b) A second polypeptide chain comprising:

[0574] (i) A second immunoglobulin constant domain;

[0575] (ii) The third linker;

[0576] (iii) The second type I interferon (IFN) moiety;

[0577] (iv) The fourth linker; and

[0578] (v) The second Fc domain, which associates with the first Fc domain to form an Fc region;

[0579] wherein at least two of the first linker, the second linker, the third linker, and the fourth linker are protease-cleavable linkers (PCLs), optionally wherein the IFN moiety in the IFN proprotein is sterically hindered by the Fc domain from binding to an IFN receptor.

[0580] 2. The IFN proprotein according to embodiment 1, wherein each of the first IFN moiety and the second IFN moiety comprises an amino acid sequence having at least about 90% sequence identity to: (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ, or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ having at most one 15-amino acid truncation at the N-terminus and / or C-terminus.

[0581] 3. The IFN proprotein according to embodiment 1, wherein each of the first IFN moiety and the second IFN moiety comprises an amino acid sequence having at least about 95% sequence identity to: (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ, or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ having at most one 15-amino acid truncation at the N-terminus and / or C-terminus.

[0582] 4. The IFN proprotein according to embodiment 1, wherein each of the first IFN moiety and the second IFN moiety comprises an amino acid sequence having at least about 98% sequence identity to: (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ, or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ having at most one 15-amino acid truncation at the N-terminus and / or C-terminus.

[0583] 5. The IFN proprotein according to any one of embodiments 1 to 4, wherein each of the first IFN moiety and the second IFN moiety comprises an amino acid sequence having one or more attenuation mutations compared to mature human IFNα1 or IFNα2b.

[0584] 6. The IFN proprotein according to any one of embodiments 1 to 5, which has one or more mutations selected from the following: L26A, F27A, R33A, R33K, L30A, D35E, H57Y, E58N, Q61S, H57S, E58S, H57A, E58A, Q61A, Q90A, E96A, R120A, L135A, R144A, R144S, R144T, R144Y, R144I, R144L, A145D, A145H, A145K, A145M, A145V, A145Y, R149A, R149K, S152A, R162A, and E165D.

[0585] 7. The IFN proprotein according to any one of embodiments 1 to 6, which comprises the amino acid substitution R33A.

[0586] 8. The IFN proprotein according to any one of embodiments 1 to 6, which comprises the amino acid substitution R33K.

[0587] 9. The IFN proprotein according to any one of embodiments 1 to 6, which comprises the amino acid substitution Q90A.

[0588] 10. The IFN proprotein according to any one of embodiments 1 to 6, which comprises the amino acid substitution E96A.

[0589] 11. The IFN proprotein according to any one of embodiments 1 to 6, which comprises the amino acid substitution R120A.

[0590] 12. The IFN proprotein according to any one of embodiments 1 to 6, which comprises the amino acid substitution A145M.

[0591] 13. The IFN proprotein according to any one of embodiments 1 to 6, which comprises the amino acid substitution R149A.

[0592] 14. The IFN proprotein according to any one of embodiments 1 to 6, which comprises the amino acid substitution R149K.

[0593] 15. The IFN proprotein according to any one of embodiments 1 to 6, which comprises the amino acid substitution S152A.

[0594] 16. The IFN proprotein according to any one of embodiments 1 to 6, which comprises the amino acid substitutions R33A, H57Y, E58N, and Q61S.

[0595] 17. The IFN proprotein according to any one of embodiments 1 to 6, which comprises the amino acid substitutions H57Y, E58N, Q61S, and R144A.

[0596] 18. The IFN proprotein according to any one of Embodiments 1 to 6, which comprises the amino acid substitutions Q90A and R120A.

[0597] 19. The IFN proprotein according to any one of Embodiments 1 to 6, which comprises the amino acid substitutions A145M and R149K.

[0598] 20. The IFN proprotein according to any one of Embodiments 1 to 19, wherein the first linker, the second linker, the third linker, and the fourth linker are protease-cleavable linkers (PCLs).

[0599] 21. The IFN proprotein according to any one of Embodiments 1 to 19, wherein the first linker and the third linker are protease-cleavable linkers (PCLs), optionally wherein the second linker and the fourth linker are non-cleavable linkers (NCLs).

[0600] 22. The IFN proprotein according to any one of Embodiments 1 to 19, wherein the second linker and the fourth linker are protease-cleavable linkers (PCLs), optionally wherein the first linker and the third linker are non-cleavable linkers (NCLs).

[0601] 23. The IFN proprotein according to any one of Embodiments 1 to 22, wherein each of the PCLs comprises a substrate sequence cleavable by any protease listed in Table A.

[0602] 24. The IFN proprotein according to any one of Embodiments 1 to 23, wherein each of the PCLs comprises one or more substrate sequences selected from the substrate sequences listed in Table B.

[0603] 25. The IFN proprotein according to any one of Embodiments 1 to 24, wherein each of the PCLs comprises one or more spacer sequences selected from the substrate sequences listed in Table C.

[0604] 26. The IFN proprotein according to any one of Embodiments 1 to 25, wherein each of the PCLs comprises the amino acid sequence of any one of the PCL sequences listed in Table D or a variant thereof having at most 5 amino acid substitutions, for example, a variant having 1 amino acid substitution, 2 amino acid substitutions, 3 amino acid substitutions, 4 amino acid substitutions, or 5 amino acid substitutions.

[0605] 27. The IFN proprotein according to any one of Embodiments 1 to 26, which comprises two or four PCLs, and the PCLs comprise or consist of the amino acid sequence ISSGLLSGRSDNH.

[0606] 28. The IFN proprotein according to any one of Embodiments 1 to 26, which comprises two or four PCLs, and the PCL comprises the amino acid sequence GGGISSGLLSGRSDNHGGGISSGLLSGRSDNHGGS or consists of the same.

[0607] 29. The IFN proprotein according to any one of Embodiments 1 to 26, which comprises two or four PCLs, and the PCL comprises the amino acid sequence GGSGGSIPVSLRSGGGISSGLLSGRSDNHGGSGGS or consists of the same.

[0608] 30. The IFN proprotein according to any one of Embodiments 1 to 26, which comprises two or four PCLs, and the PCL comprises the amino acid sequence GGSGGSVPLSLYSGGGISSGLLSGRSDNHGGSGGS or consists of the same.

[0609] 31. The IFN proprotein according to any one of Embodiments 1 to 26, which comprises two or four PCLs, and the PCL comprises the amino acid sequence GGSHPVGLLARGGGHPVGLLARGGGHPVGLLARGS or consists of the same.

[0610] 32. The IFN proprotein according to any one of Embodiments 1 to 26, which comprises two or four PCLs, and the PCL comprises the amino acid sequence GGSHPVGLLARGGGHPVGLLARGGSGRSAGGSGRSA or consists of the same.

[0611] 33. The IFN proprotein according to any one of Embodiments 1 to 32, wherein the first linker and the third linker are the same and / or the third and fourth linkers are the same.

[0612] 34. The IFN proprotein according to Embodiment 33, wherein the first linker, the second linker, the third linker and the fourth linker are the same.

[0613] 35. The IFN proprotein according to any one of Embodiments 1 to 33, wherein (i) the first linker and the third linker are non-cleavable linkers, or (ii) the second linker and the fourth linker are non-cleavable linkers.

[0614] 36. The IFN proprotein according to Embodiment 35, wherein the non-cleavable linker comprises any one of the NCL sequences listed in Table E or consists of the same.

[0615] 37. The IFN proprotein according to any one of Embodiments 1 to 33, wherein the first Fc domain and / or the second Fc domain comprises a hinge domain.

[0616] 38. An IFN proprotein according to any one of embodiments 1 to 37, further comprising one or more targeting moieties that bind to one or more target molecules.

[0617] 39. The IFN proprotein according to embodiment 38, comprising a first targeting moiety and a second targeting moiety.

[0618] 40. The IFN proprotein according to embodiment 39, wherein the first targeting moiety and the second targeting moiety are antibodies or antigen-binding fragments thereof.

[0619] 41. The IFN proprotein according to embodiment 40, wherein the first targeting moiety and the second targeting moiety are Fab.

[0620] 42. The IFN proprotein according to embodiment 40 or embodiment 41, wherein the first targeting moiety and the second targeting moiety comprise Fab domains.

[0621] 43. The IFN proprotein according to embodiment 42, wherein the first targeting moiety and the second targeting moiety further comprise a hinge sequence.

[0622] 44. The IFN proprotein according to embodiment 43, wherein the first targeting moiety and the second targeting moiety further comprise an Fc domain, each Fc domain comprising a CH2 domain and a CH3 domain.

[0623] 45. The IFN proprotein according to embodiment 44, wherein the Fc domain of the first targeting moiety and the Fc domain of the second targeting moiety associate with each other.

[0624] 46. The IFN proprotein according to any one of embodiments 39 to 45, wherein the first targeting moiety and the second targeting moiety are respectively located at the N-terminus of the first linker and the third linker.

[0625] 47. The IFN proprotein according to any one of embodiments 38 to 46, wherein a first immunoglobulin constant domain is part of the first targeting moiety and a second immunoglobulin constant domain is part of the second targeting moiety.

[0626] 48. The IFN proprotein according to embodiment 40, wherein the first immunoglobulin constant domain is a CH3 domain.

[0627] 49. The IFN proprotein according to embodiment 40, wherein the first immunoglobulin constant domain is a CH1 domain.

[0628] 50. The IFN proprotein according to any one of embodiments 1 to 49, which is constructed asFigure 1A , Figure 2A or Figure 2D as shown

[0629] 51. The IFN proprotein according to Example 50, which comprises the first polypeptide chain, the second polypeptide chain, the third polypeptide chain, and the fourth polypeptide chain, wherein:

[0630] (a) The first polypeptide chain comprises:

[0631] (i) A first VH1 domain;

[0632] (ii) A first CH1 domain;

[0633] (iii) A third Fc domain, which comprises a hinge domain, a CH2 domain, and a CH3 domain, wherein the CH3 domain is the first immunoglobulin constant domain;

[0634] (iv) The first linker;

[0635] (v) The first type I interferon (IFN) moiety;

[0636] (vi) The second linker; and

[0637] (vii) The first Fc domain;

[0638] (b) The second polypeptide chain comprises:

[0639] (i) A second VH1 domain;

[0640] (ii) A second CH1 domain;

[0641] (iii) A fourth Fc domain, which comprises a hinge domain, a CH2 domain, and a CH3 domain, wherein the CH3 domain is the second immunoglobulin constant domain;

[0642] (iv) The third linker;

[0643] (v) The second type I interferon (IFN) moiety;

[0644] (vi) The fourth linker; and

[0645] (vii) The third Fc domain;

[0646] (c) The third polypeptide chain comprises:

[0647] (i) A first VL domain;

[0648] (ii) A first CL domain; and

[0649] (d) The fourth polypeptide chain comprises:

[0650] (i) A second VL domain; and

[0651] (ii) A second CL domain;

[0652] Wherein the first polypeptide chain associates with the third polypeptide chain such that the first VH, CH1, VL, and CL form the first targeting moiety, and wherein the second polypeptide chain associates with the fourth polypeptide chain such that the second VH, CH1, VL, and CL form the second targeting moiety.

[0653] 52. The IFN proprotein according to embodiment 51, wherein the first linker, the second linker, the third linker, and the fourth linker are protease-cleavable linkers (PCL).

[0654] 53. The IFN proprotein according to embodiment 51, wherein the first linker and the third linker are non-cleavable linkers (NCL), and the second linker and the fourth linker are protease-cleavable linkers (PCL).

[0655] 54. The IFN proprotein according to embodiment 51, wherein the first linker and the third linker are protease-cleavable linkers (PCL), and the second linker and the fourth linker are non-cleavable linkers (NCL).

[0656] 55. The IFN proprotein according to any one of embodiments 1 to 49, which is constructed as Figure 1B , Figure 2B or Figure 2E shown.

[0657] 56. The IFN proprotein according to embodiment 55, wherein:

[0658] (a) The first polypeptide chain comprises:

[0659] (i) A first VH1 domain;

[0660] (ii) A first CH1 domain;

[0661] (iii) The first linker;

[0662] (iv) The first type I interferon (IFN) moiety;

[0663] (v) The second linker; and

[0664] (vi) The first Fc domain;

[0665] (b) The second polypeptide chain comprises:

[0666] (i) The second VH1 domain;

[0667] (ii) The second CH1 domain;

[0668] (iii) The third linker;

[0669] (iv) The second type I interferon (IFN) moiety;

[0670] (v) The fourth linker; and

[0671] (vi) The second Fc domain;

[0672] (c) The third polypeptide chain comprises:

[0673] (i) A first VL domain;

[0674] (ii) A second CL domain; and

[0675] (d) The fourth polypeptide chain comprises:

[0676] (i) A second VL domain;

[0677] (ii) A second CL domain;

[0678] wherein the first polypeptide chain associates with the third polypeptide chain such that the first VH, CH1, VL, and CL form the first targeting moiety, and wherein the second polypeptide chain associates with the fourth polypeptide chain such that the second VH, CH1, VL, and CL form the second targeting moiety.

[0679] 57. The IFN proprotein according to embodiment 56, wherein the first linker, the second linker, the third linker, and the fourth linker are protease-cleavable linkers (PCLs).

[0680] 58. The IFN proprotein according to embodiment 56, wherein the first linker and the third linker are non-cleavable linkers (NCLs), and the second linker and the fourth linker are protease-cleavable linkers (PCLs).

[0681] 59. The IFN proprotein according to embodiment 56, wherein the first linker and the third linker are protease-cleavable linkers (PCLs), and the second linker and the fourth linker are non-cleavable linkers (NCLs).

[0682] 60. The IFN proprotein according to any one of embodiments 1 to 49, which is constructed as Figure 1C , Figure 2C or Figure 2F shown.

[0683] 61. The IFN proprotein according to Example 60, which comprises the first polypeptide chain, the second polypeptide chain, the third polypeptide chain, and the fourth polypeptide chain, wherein:

[0684] (a) The first polypeptide chain comprises:

[0685] (i) A first VH1 domain;

[0686] (ii) A first CH1 domain;

[0687] (iii) A first hinge domain;

[0688] (iv) The first linker;

[0689] (v) The first type I interferon (IFN) moiety;

[0690] (vi) The second linker; and

[0691] (vii) The first Fc domain;

[0692] (b) The second polypeptide chain comprises:

[0693] (i) A second VH1 domain;

[0694] (ii) A second CH1 domain;

[0695] (iii) A second hinge domain;

[0696] (iv) The third linker;

[0697] (v) The second type I interferon (IFN) moiety;

[0698] (vi) The fourth linker; and

[0699] (vii) The second Fc domain;

[0700] (c) The third polypeptide chain comprises:

[0701] (i) A first VL domain;

[0702] (ii) A first CL domain; and

[0703] (d) The fourth polypeptide chain comprises:

[0704] (i) A second VL domain; and

[0705] (ii) A second CL domain;

[0706] The first polypeptide chain associates with the third polypeptide chain such that the first VH, CH1, VL, and CL form the first targeting moiety, and wherein the second polypeptide chain associates with the fourth polypeptide chain such that the second VH, CH1, VL, and CL form the second targeting moiety.

[0707] 62. The IFN proprotein according to embodiment 61, wherein the first linker, the second linker, the third linker, and the fourth linker are protease-cleavable linkers (PCLs).

[0708] 63. The IFN proprotein according to embodiment 61, wherein the first linker and the third linker are non-cleavable linkers (NCLs), and the second linker and the fourth linker are protease-cleavable linkers (PCLs).

[0709] 64. The IFN proprotein according to embodiment 61, wherein the first linker and the third linker are protease-cleavable linkers (PCLs), and the second linker and the fourth linker are non-cleavable linkers (NCLs).

[0710] 65. The IFN proprotein according to any one of embodiments 38 to 61, wherein the first targeting moiety and / or the second targeting moiety herein is capable of binding to an extracellular matrix (ECM) antigen, a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or virus lymphocyte, a T cell antigen (TCA), a checkpoint inhibitor, a tumor-associated antigen (TAA), a dendritic cell (DC) or other antigen-presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0711] 66. The IFN proprotein according to any one of embodiments 38 to 65, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to any target molecule identified in section 6.7.

[0712] 67. The IFN proprotein according to any one of embodiments 38 to 66, wherein the first targeting moiety and / or the second targeting moiety (a) comprises (i) a CDR or (ii) a VH sequence and a VL sequence of an antibody listed in Table F or (b) competes with the antibody listed in Table F for binding to the target molecule.

[0713] 68. The IFN proprotein according to any one of embodiments 38 to 66, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to an ECM antigen, which is optionally selected from syndecan, heparanase, integrin, osteopontin, tenascin, cadherin, laminin, laminin-type EGF, lectin, fibronectin, notch protein, nectin (e.g., nectin-4), tenascin, collagen (e.g., type X collagen), and matrix protein.

[0714] 69. The IFN proprotein according to embodiment 68, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to fibronectin, for example, fibronectin 4.

[0715] 70. The IFN proprotein according to embodiment 68, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to collagen, for example, collagen X.

[0716] 71. The IFN proprotein according to any one of embodiments 38 to 66, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to a cell surface molecule of a tumor or a virus lymphocyte.

[0717] 72. The IFN proprotein according to embodiment 71, wherein the antigen is a T cell costimulatory protein.

[0718] 73. The IFN proprotein according to embodiment 72, wherein the T cell costimulatory protein is CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C or B7-H3.

[0719] 74. The IFN proprotein according to embodiment 73, wherein the T cell costimulatory protein is B7-H3.

[0720] 75. The IFN proprotein according to any one of embodiments 38 to 66, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to a checkpoint inhibitor.

[0721] 76. The IFN proprotein according to embodiment 75, wherein the checkpoint inhibitor is CTLA-4, PD1, PDL1, PDL2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1 or CHK2.

[0722] 77. The IFN proprotein according to embodiment 76, wherein the checkpoint inhibitor is PDL1.

[0723] 78. The IFN proprotein according to embodiment 76, wherein the checkpoint inhibitor is PD1.

[0724] 79. The IFN proprotein according to embodiment 76, wherein the checkpoint inhibitor is LAG3.

[0725] 80. The IFN proprotein according to any one of embodiments 38 to 66, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to a tumor-associated antigen (TAA).

[0726] 81. The IFN proprotein according to embodiment 80, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to AFP, ALK, BAGE protein, BIRC5 (survivin), BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CEACAM5 (also known as carcinoembryonic antigen or CEA), CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE protein (e.g., GAGE-1 or -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, LMP2, MAGE protein (e.g., MAGE-1, -2, -3, -4, -6 and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase or uroplakin-3.

[0727] 82. The IFN proprotein according to embodiment 81, wherein the TAA is EGFR.

[0728] 83. The IFN proprotein according to embodiment 81, wherein the TAA is HER2.

[0729] 84. The IFN proprotein according to embodiment 81, wherein the TAA is EPCAM.

[0730] 85. The IFN proprotein according to embodiment 81, wherein the TAA is CEACAM5.

[0731] 86. The IFN proprotein according to embodiment 81, wherein the TAA is CD20.

[0732] 87. The IFN proprotein according to any one of embodiments 38 to 66, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to a dendritic cell (DC) antigen, which DC antigen is optionally selected from XCR1, Clec9a, CD1c, CD11c, CD14, PDL1, macrophage mannose receptor (CD206), and DEC-205.

[0733] 88. The IFN proprotein according to embodiment 87, wherein the dendritic cell antigen is XCR1.

[0734] 89. The IFN proprotein according to embodiment 87, wherein the dendritic cell antigen is Clec9a.

[0735] 90. The IFN proprotein according to embodiment 87, wherein the dendritic cell antigen is DEC-205.

[0736] 91. The IFN proprotein according to any one of embodiments 38 to 66, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to a natural killer (NK) cell antigen.

[0737] 92. The IFN proprotein according to any one of embodiments 1 to 37, which further comprises one or more targeting moieties, each targeting moiety comprising means for binding to one or more target molecules.

[0738] 93. The IFN proprotein according to embodiment 92, which comprises a first targeting moiety and a second targeting moiety, the first targeting moiety and the second targeting moiety each comprising means for binding to a target molecule.

[0739] 94. The IFN proprotein according to embodiment 93, wherein the first targeting moiety and the second targeting moiety are antibodies or antigen-binding fragments thereof.

[0740] 95. The IFN proprotein according to embodiment 94, wherein the first targeting moiety and the second targeting moiety are Fab.

[0741] 96. The IFN proprotein according to embodiment 94, wherein the first targeting moiety and the second targeting moiety comprise Fab domains.

[0742] 97. The IFN proprotein according to any one of embodiments 92 to 96, wherein the first targeting moiety and the second targeting moiety further comprise a hinge sequence.

[0743] 98. The IFN proprotein according to embodiment 97, wherein the first targeting moiety and the second targeting moiety further comprise an Fc domain, and each Fc domain comprises a CH2 domain and a CH3 domain.

[0744] 99. The IFN proprotein according to embodiment 98, wherein the Fc domain of the first targeting moiety and the Fc domain of the second targeting moiety associate with each other.

[0745] 100. The IFN proprotein according to any one of embodiments 92 to 99, wherein the first targeting moiety and the second targeting moiety are respectively located at the N-terminus of the first linker and the third linker.

[0746] 101. The IFN proprotein according to any one of embodiments 92 to 100, wherein the first immunoglobulin constant domain is part of the first targeting moiety, and the second immunoglobulin constant domain is part of the second targeting moiety.

[0747] 102. The IFN proprotein according to embodiment 101, wherein the first immunoglobulin constant domain is a CH3 domain.

[0748] 103. The IFN proprotein according to embodiment 101, wherein the first immunoglobulin constant domain is a CH1 domain.

[0749] 104. The IFN proprotein according to any one of embodiments 92 to 103, which is constructed as shown in Figure 1A 、 Figure 2A or Figure 2D shown.

[0750] 105. The IFN proprotein according to embodiment 104, which comprises the first polypeptide chain, the second polypeptide chain, the third polypeptide chain and the fourth polypeptide chain, wherein:

[0751] (a) The first polypeptide chain comprises:

[0752] (i) A first VH1 domain;

[0753] (ii) A first CH1 domain;

[0754] (iii) A third Fc domain, which comprises a hinge domain, a CH2 domain and a CH3 domain, wherein the CH3 domain is the first immunoglobulin constant domain;

[0755] (iv) the first linker;

[0756] (v) the first type I interferon (IFN) moiety;

[0757] (vi) the second linker; and

[0758] (vii) the first Fc domain;

[0759] (b) The second polypeptide chain comprises:

[0760] (i) a second VH1 domain;

[0761] (ii) a second CH1 domain;

[0762] (iii) a fourth Fc domain, which comprises a hinge domain, a CH2 domain and a CH3 domain, wherein the CH3 domain is the second immunoglobulin constant domain;

[0763] (iv) the third linker;

[0764] (v) the second type I interferon (IFN) moiety;

[0765] (vi) the fourth linker; and

[0766] (vii) the third Fc domain;

[0767] (c) The third polypeptide chain comprises:

[0768] (i) a first VL domain;

[0769] (ii) a first CL domain; and

[0770] (d) The fourth polypeptide chain comprises:

[0771] (i) a second VL domain; and

[0772] (ii) a second CL domain;

[0773] wherein the first polypeptide chain associates with the third polypeptide chain such that the first VH, CH1, VL and CL form the first targeting moiety, and wherein the second polypeptide chain associates with the fourth polypeptide chain such that the second VH, CH1, VL and CL form the second targeting moiety.

[0774] 106. The IFN proprotein according to embodiment 105, wherein the first linker, the second linker, the third linker and the fourth linker are protease-cleavable linkers (PCLs).

[0775] 107. The IFN proprotein according to embodiment 105, wherein the first linker and the third linker are non-cleavable linkers (NCL), and the second linker and the fourth linker are protease-cleavable linkers (PCL).

[0776] 108. The IFN proprotein according to embodiment 105, wherein the first linker and the third linker are protease-cleavable linkers (PCL), and the second linker and the fourth linker are non-cleavable linkers (NCL).

[0777] 109. The IFN proprotein according to any one of embodiments 92 to 103, which is configured as Figure 1B , Figure 2B or Figure 2E shown.

[0778] 110. The IFN proprotein according to embodiment 109, wherein:

[0779] (a) The first polypeptide chain comprises:

[0780] (i) A first VH1 domain;

[0781] (ii) A first CH1 domain;

[0782] (iii) The first linker;

[0783] (iv) The first type I interferon (IFN) moiety;

[0784] (v) The second linker; and

[0785] (vi) The first Fc domain;

[0786] (b) The second polypeptide chain comprises:

[0787] (i) A second VH1 domain;

[0788] (ii) A second CH1 domain;

[0789] (iii) The third linker;

[0790] (iv) The second type I interferon (IFN) moiety;

[0791] (v) The fourth linker; and

[0792] (vi) The second Fc domain;

[0793] (c) The third polypeptide chain comprises:

[0794] (i) A first VL domain;

[0795] (ii) The second CL domain; and

[0796] (d) The fourth polypeptide chain comprises:

[0797] (i) A second VL domain;

[0798] (ii) A second CL domain;

[0799] Wherein the first polypeptide chain associates with the third polypeptide chain such that the first VH, CH1, VL, and CL form the first targeting moiety, and wherein the second polypeptide chain associates with the fourth polypeptide chain such that the second VH, CH1, VL, and CL form the second targeting moiety.

[0800] 111. The IFN proprotein according to embodiment 110, wherein the first linker, the second linker, the third linker, and the fourth linker are protease-cleavable linkers (PCL).

[0801] 112. The IFN proprotein according to embodiment 110, wherein the first linker and the third linker are non-cleavable linkers (NCL), and the second linker and the fourth linker are protease-cleavable linkers (PCL).

[0802] 113. The IFN proprotein according to embodiment 110, wherein the first linker and the third linker are protease-cleavable linkers (PCL), and the second linker and the fourth linker are non-cleavable linkers (NCL).

[0803] 114. The IFN proprotein according to any one of embodiments 92 to 103, which is constructed as Figure 1C 、 Figure 2C or Figure 2F shown.

[0804] 115. The IFN proprotein according to embodiment 114, which comprises the first polypeptide chain, the second polypeptide chain, the third polypeptide chain, and the fourth polypeptide chain, wherein:

[0805] (a) The first polypeptide chain comprises:

[0806] (i) A first VH1 domain;

[0807] (ii) A first CH1 domain;

[0808] (iii) A first hinge domain;

[0809] (iv) The first linker;

[0810] (v) The first type I interferon (IFN) moiety;

[0811] (vi) the second linker; and

[0812] (vii) the first Fc domain;

[0813] (b) The second polypeptide chain comprises:

[0814] (i) a second VH1 domain;

[0815] (ii) a second CH1 domain;

[0816] (iii) a second hinge domain;

[0817] (iv) the third linker;

[0818] (v) the second type I interferon (IFN) moiety;

[0819] (vi) the fourth linker; and

[0820] (vii) the second Fc domain;

[0821] (c) The third polypeptide chain comprises:

[0822] (i) a first VL domain;

[0823] (ii) a first CL domain; and

[0824] (d) The fourth polypeptide chain comprises:

[0825] (i) a second VL domain; and

[0826] (ii) a second CL domain;

[0827] wherein the first polypeptide chain associates with the third polypeptide chain such that the first VH, CH1, VL, and CL form the first targeting moiety, and wherein the second polypeptide chain associates with the fourth polypeptide chain such that the second VH, CH1, VL, and CL form the second targeting moiety.

[0828] 116. The IFN proprotein according to embodiment 115, wherein the first linker, the second linker, the third linker, and the fourth linker are protease-cleavable linkers (PCLs).

[0829] 117. The IFN proprotein according to embodiment 115, wherein the first linker and the third linker are non-cleavable linkers (NCLs), and the second linker and the fourth linker are protease-cleavable linkers (PCLs).

[0830] 118. The IFN proprotein according to embodiment 115, wherein the first linker and the third linker are protease-cleavable linkers (PCLs), and the second linker and the fourth linker are non-cleavable linkers (NCLs).

[0831] 119. The IFN proprotein according to any one of embodiments 92 to 119, wherein the first targeting moiety and / or the second targeting moiety comprises means for binding to an extracellular matrix (ECM) antigen, a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or viral lymphocyte, a T cell antigen (TCA), a checkpoint inhibitor, a tumor-associated antigen (TAA), a dendritic cell (DC) or other antigen-presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0832] 120. The IFN proprotein according to any one of embodiments 92 to 119, wherein the first targeting moiety and / or the second targeting moiety comprises means for binding to any target molecule identified in Section 6.6.

[0833] 121. The IFN proprotein according to any one of embodiments 92 to 119, wherein the first targeting moiety and / or the second targeting moiety comprises means for binding to an ECM antigen, which is optionally selected from syndecan, heparanase, integrin, osteopontin, tenascin, cadherin, laminin, laminin-type EGF, lectin, fibronectin, notch protein, nectin (e.g., nectin-4), tenascin, collagen (e.g., type X collagen), and matrix protein.

[0834] 122. The IFN proprotein according to embodiment 121, wherein the first targeting moiety and / or the second targeting moiety comprises means for binding to nectin, e.g., nectin 4.

[0835] 123. The IFN proprotein according to embodiment 121, wherein the first targeting moiety and / or the second targeting moiety comprises means for binding to collagen, e.g., collagen X.

[0836] 124. The IFN proprotein according to any one of embodiments 92 to 119, wherein the first targeting moiety and / or the second targeting moiety comprises means for binding to a cell surface molecule of a tumor or viral lymphocyte.

[0837] 125. The IFN proprotein according to embodiment 124, wherein the cell surface molecule is a T cell co-stimulatory protein.

[0838] 126. The IFN proprotein according to embodiment 125, wherein the T cell costimulatory protein is CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C or B7-H3.

[0839] 127. The IFN proprotein according to embodiment 126, wherein the T cell costimulatory protein is B7-H3.

[0840] 128. The IFN proprotein according to any one of embodiments 92 to 119, wherein the first targeting moiety and / or the second targeting moiety comprises a means for binding to a checkpoint inhibitor.

[0841] 129. The IFN proprotein according to embodiment 128, wherein the checkpoint inhibitor is CTLA-4, PD1, PDL1, PDL2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1 or CHK2.

[0842] 130. The IFN proprotein according to embodiment 129, wherein the checkpoint inhibitor is PDL1.

[0843] 131. The IFN proprotein according to embodiment 129, wherein the checkpoint inhibitor is PD1.

[0844] 132. The IFN proprotein according to embodiment 129, wherein the checkpoint inhibitor is LAG3.

[0845] 133. The IFN proprotein according to any one of embodiments 92 to 119, wherein the first targeting moiety and / or the second targeting moiety comprises a means for binding to a tumor-associated antigen (TAA).

[0846] 134. The IFN proprotein according to embodiment 133, wherein the TAA is AFP, ALK, BAGE protein, BIRC5 (survivin), BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CEACAM5 (also known as carcinoembryonic antigen or CEA), CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE protein (e.g., GAGE-1 or -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, LMP2, MAGE protein (e.g., MAGE-1, -2, -3, -4, -6 and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase or uroplakin-3.

[0847] 135. The IFN proprotein according to embodiment 134, wherein the TAA is EGFR.

[0848] 136. The IFN proprotein according to embodiment 134, wherein the TAA is HER2.

[0849] 137. The IFN proprotein according to embodiment 134, wherein the TAA is EPCAM.

[0850] 138. The IFN proprotein according to embodiment 134, wherein the TAA is CEACAM5.

[0851] 139. The IFN proprotein according to embodiment 134, wherein the TAA is CD20.

[0852] 140. The IFN proprotein according to any one of embodiments 92 to 119, wherein the first targeting moiety and / or the second targeting moiety comprises means for binding to a dendritic cell (DC) antigen, which DC antigen is optionally selected from XCR1, Clec9a, CD1c, CD11c, CD14, PDL1, macrophage mannose receptor (CD206), and DEC-205.

[0853] 141. The IFN proprotein according to embodiment 140, wherein the dendritic cell antigen is XCR1.

[0854] 142. The IFN proprotein according to embodiment 140, wherein the dendritic cell antigen is Clec9a.

[0855] 143. The IFN proprotein according to embodiment 140, wherein the dendritic cell antigen is DEC-205.

[0856] 144. The IFN proprotein according to any one of embodiments 92 to 119, wherein the first targeting moiety and / or the second targeting moiety comprises means for binding to a natural killer (NK) cell antigen.

[0857] 145. The IFN proprotein according to any one of embodiments 1 to 144, wherein the Fc region is homodimeric.

[0858] 146. The IFN proprotein according to any one of embodiments 1 to 144, wherein the Fc region is heterodimeric.

[0859] 147. A nucleic acid or nucleic acids encoding the IFN proprotein according to any one of embodiments 1 to 146.

[0860] 148. A host cell engineered to express the IFN proprotein according to any one of embodiments 1 to 146 or the nucleic acid according to embodiment 147.

[0861] 149. A method for producing the IFN proprotein according to any one of embodiments 1 to 146, comprising culturing the host cell according to embodiment 148 and recovering the IFN proprotein expressed thereby.

[0862] 150. A pharmaceutical composition comprising the IFN proprotein according to any one of embodiments 1 to 146 and an excipient.

[0863] 151. A method for treating cancer, which comprises administering to a subject in need thereof an IFN proprotein according to any one of embodiments 1 to 146 or a pharmaceutical composition according to embodiment 150.

[0864] 152. The method according to embodiment 151, wherein the IFN proprotein comprises at least one targeting moiety capable of binding to a target molecule.

[0865] 153. The method according to embodiment 151, wherein the IFN proprotein comprises at least one tool for binding to a target molecule.

[0866] 154. The method according to any one of embodiments 150 to 153, wherein the cancer is associated with the expression of the target molecule, for example, TAAs and related cancers listed in Table I.

[0867] 155. The method according to any one of embodiments 150 to 154, wherein the activated IFN protein comprising the IFN moiety is produced by cleavage of one or more protease-cleavable linkers in the IFN proprotein by one or more proteases expressed by the cancer tissue.

[0868] 156. The method according to embodiment 155, wherein the IFN protein is selectively activated in the cancer tissue.

[0869] 157. A method for local delivery of an IFN protein, which comprises administering to a subject an IFN proprotein according to any one of embodiments 1 to 146 (or a pharmaceutical composition comprising the IFN proprotein and an excipient), the IFN proprotein having one or more protease-cleavable linkers, each protease-cleavable linker comprising one or more substrates for one or more proteases expressed by the tissue to which the IFN protein is to be locally delivered.

[0870] 158. The method according to embodiment 157, wherein the IFN proprotein comprises one or more targeting moieties that recognize a target molecule expressed by the tissue.

[0871] 159. The method according to embodiment 158, wherein the IFN proprotein comprises two targeting moieties that each recognize a target molecule expressed by the tissue.

[0872] 160. The method according to embodiment 157, wherein the IFN proprotein comprises one or more tools for binding to a target molecule expressed by the tissue.

[0873] 161. The method according to embodiment 160, wherein the IFN proprotein comprises two tools for binding to a target molecule expressed by the tissue.

[0874] 162. A method according to any one of embodiments 157 to 161, wherein the tissue is a cancer tissue.

[0875] 163. A method according to embodiment 162, wherein the target molecule expressed by the tissue is an extracellular matrix (ECM) antigen, a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or virus lymphocyte, a T cell antigen (TCA), a checkpoint inhibitor, a tumor-associated antigen (TAA), a dendritic cell (DC) or other antigen-presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0876] 164. A method according to any one of embodiments 157 to 163, wherein the activated IFN protein comprising the IFN moiety is produced by cleavage of one or more protease-cleavable linkers in the IFN precursor protein by one or more proteases in the tissue.

[0877] 165. A method of treating cancer with an IFN protein selectively activated in cancer tissue, comprising administering to a subject in need thereof an IFN precursor protein (or a pharmaceutical composition comprising the IFN precursor protein and an excipient) according to any one of embodiments 1 to 146, the IFN precursor protein having one or more protease-cleavable linkers, each protease-cleavable linker comprising one or more substrates for one or more proteases expressed by the cancer tissue targeted by the IFN protein.

[0878] 166. A method according to embodiment 165, wherein the IFN precursor protein comprises one or more targeting moieties that recognize a target molecule expressed by the cancer tissue or associated immune cells.

[0879] 167. A method according to embodiment 166, wherein the IFN precursor protein comprises two targeting moieties each recognizing a target molecule expressed by the cancer tissue or associated immune cells.

[0880] 168. A method according to embodiment 165, wherein the IFN precursor protein comprises one or more tools for binding to a target molecule expressed by the cancer tissue or associated immune cells.

[0881] 169. A method according to embodiment 168, wherein the IFN precursor protein comprises two tools for binding to a target molecule expressed by the cancer tissue or associated immune cells.

[0882] 170. The method according to any one of embodiments 165 to 169, wherein the target molecule expressed by the cancer tissue or related immune cells is an extracellular matrix (ECM) antigen, a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or virus lymphocyte, a T cell antigen (TCA), a checkpoint inhibitor, a tumor-associated antigen (TAA), a dendritic cell (DC) or other antigen-presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0883] 171. The method according to any one of embodiments 165 to 170, wherein the activated IFN protein comprising the IFN moiety is produced by cleavage of one or more protease-cleavable linkers in the IFN precursor protein by one or more proteases in the cancer tissue.

[0884] 172. A method of administering an IFN therapy with reduced systemic exposure and / or reduced systemic toxicity to a subject, comprising administering to the subject the IFN therapy in the form of an IFN precursor protein (or a pharmaceutical composition comprising the IFN precursor protein and an excipient) according to any one of embodiments 1 to 146, the IFN precursor protein having one or more protease-cleavable linkers, each protease-cleavable linker comprising one or more substrates for one or more proteases expressed by the tissue for which IFN therapy is desired and / or anticipated.

[0885] 173. The method according to embodiment 172, wherein the IFN precursor protein comprises one or more targeting moieties that recognize a target molecule expressed by the tissue.

[0886] 174. The method according to embodiment 173, wherein the IFN precursor protein comprises two targeting moieties that each recognize a target molecule expressed by the tissue.

[0887] 175. The method according to embodiment 172, wherein the IFN precursor protein comprises one or more tools for binding to a target molecule expressed by the tissue.

[0888] 176. The method according to embodiment 175, wherein the IFN precursor protein comprises two tools for binding to a target molecule expressed by the tissue.

[0889] 177. The method according to any one of embodiments 172 to 176, wherein the tissue is cancer tissue or related immune cells.

[0890] 178. The method according to embodiment 177, wherein the target molecule expressed by the tissue is an extracellular matrix (ECM) antigen, a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or viral lymphocyte, a T cell antigen (TCA), a checkpoint inhibitor, a tumor-associated antigen (TAA), a dendritic cell (DC) or other antigen-presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0891] 179. The method according to any one of embodiments 172 to 178, wherein the activated IFN protein comprising the IFN moiety is produced by cleavage of one or more protease-cleavable linkers in the IFN precursor protein by one or more proteases in the tissue.

[0892] 180. A method of treating cancer with an IFN protein selectively activated in cancer tissue, comprising administering to a subject in need thereof an IFN precursor protein (or a pharmaceutical composition comprising the IFN precursor protein and an excipient) according to any one of embodiments 1 to 146, the IFN precursor protein having one or more protease-cleavable linkers, each protease-cleavable linker comprising one or more substrates for one or more proteases expressed by the cancer tissue.

[0893] 181. The method according to embodiment 180, wherein the IFN precursor protein comprises one or more targeting moieties that recognize a target molecule expressed by the cancer tissue or related immune cells.

[0894] 182. The method according to embodiment 181, wherein the IFN precursor protein comprises two targeting moieties that each recognize a target molecule expressed by the cancer tissue or related immune cells.

[0895] 183. The method according to embodiment 180, wherein the IFN precursor protein comprises one or more targeting moieties that recognize a target molecule expressed by the tissue.

[0896] 184. The method according to embodiment 183, wherein the IFN precursor protein comprises two tools for binding to a target molecule expressed by the cancer tissue or related immune cells.

[0897] 185. The method according to any one of embodiments 180 to 184, wherein the target molecule expressed by the cancer tissue or related immune cells is an extracellular matrix (ECM) antigen, a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or viral lymphocyte, a T cell antigen (TCA), a checkpoint inhibitor, a tumor-associated antigen (TAA), a dendritic cell (DC) or other antigen-presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0898] 186. According to the method of any one of embodiments 179 to 184, wherein the activated IFN protein comprising the IFN moiety is produced by cleavage of one or more protease-cleavable linkers in the IFN precursor protein by one or more proteases in the cancer tissue.

[0899] 187. A method of targeted delivery of an activated IFN protein to cancer tissue, comprising administering to a subject an IFN precursor protein (or a pharmaceutical composition comprising the IFN precursor protein and an excipient) according to any one of embodiments 1 to 146, wherein the IFN precursor protein:

[0900] (a) comprises (i) one or more targeting moieties that each recognize a target molecule expressed by the cancer tissue or associated immune cells, or (ii) a tool for binding to a target molecule expressed by the cancer tissue or associated immune cells; and

[0901] (b) has one or more protease-cleavable linkers, each protease-cleavable linker comprising one or more substrates for one or more proteases expressed in a tissue for which IFN therapy is desired and / or anticipated.

[0902] 188. According to the method of embodiment 187, wherein the IFN precursor protein comprises (i) two targeting moieties that each recognize a target molecule expressed by the cancer tissue or associated immune cells, or (ii) two tools for binding to a target molecule expressed by the cancer tissue or associated immune cells.

[0903] 189. According to the method of embodiment 187 or 188, wherein the target molecule expressed by the cancer tissue or associated immune cells is an extracellular matrix (ECM) antigen, a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or virus lymphocyte, a T cell antigen (TCA), a checkpoint inhibitor, a tumor-associated antigen (TAA), a dendritic cell (DC) or other antigen-presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0904] 190. According to the method of any one of embodiments 187 to 189, wherein the activated IFN protein comprising the IFN moiety is produced by cleavage of one or more protease-cleavable linkers in the IFN precursor protein by one or more proteases in the cancer tissue.

[0905] 191. A method for locally inducing an immune response in a target tissue, comprising administering to a subject an IFN proprotein according to any one of Examples 1 to 146 (or a pharmaceutical composition comprising the IFN proprotein and an excipient), the IFN proprotein having (i) one or more targeting moieties capable of binding to a target molecule expressed in the target tissue, or (ii) one or more means for binding to a target molecule expressed in the target tissue, and one or more protease-cleavable linkers, each protease-cleavable linker comprising one or more substrates for one or more proteases expressed in the target tissue.

[0906] 192. The method according to Example 191, wherein the IFN proprotein comprises (i) two targeting moieties each recognizing a target molecule expressed in the target tissue or a related immune cell, or (ii) two means for binding to a target molecule expressed in the target tissue or a related immune cell.

[0907] 193. The method according to Example 191 or 192, wherein the target tissue is a cancer tissue.

[0908] 194. The method according to any one of Examples 191 to 193, wherein the target molecule expressed in the target tissue or a related immune cell is an extracellular matrix (ECM) antigen, a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or virus lymphocyte, a T cell antigen (TCA), a checkpoint inhibitor, a tumor-associated antigen (TAA), a dendritic cell (DC) or other antigen-presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0909] 195. The method according to any one of Examples 190 to 194, wherein the activated IFN protein comprising the IFN moiety is produced by cleavage of one or more protease-cleavable linkers in the IFN proprotein by one or more proteases in the target tissue.

[0910] 196. The method according to Example 195, wherein the IFN protein induces the immune response against at least one cell type in the target tissue.

[0911] 197. A method for enhancing an immune response against an antigen, comprising administering to a subject an immunogenic agent that elicits an immune response against the antigen together with an IFN proprotein according to any one of claims 1 to 146 (e.g., an IFN receptor agonist) (or a pharmaceutical composition comprising the IFN receptor agonist and an excipient) or a nucleic acid encoding such an IFN proprotein (e.g., an IFN receptor agonist), e.g., as described in Section 6.10.1).

[0912] 198. The method according to embodiment 197, wherein the administration of the immunogenic agent and the IFN proprotein (e.g., an IFN receptor agonist) is parallel, separate but simultaneous, or sequential.

[0913] 199. The method according to embodiment 197 or 198 or embodiment 1, wherein the immunogenic agent is a vaccine, optionally wherein the vaccine is a cancer vaccine or a vaccine against an infectious agent.

[0914] 200. The method according to any one of embodiments 151 to 199, wherein the administration is non - local.

[0915] 201. The method according to embodiment 200, wherein the administration is systemic.

[0916] 8. Examples

[0917] 8.1. Proprotein construct sequences

[0918] Table 3 below provides the sequences of the IFN proproteins and control constructs used in the studies described herein.

[0919]

[0920]

[0921]

[0922]

[0923]

[0924]

[0925]

[0926]

[0927] 8.2. Materials and methods

[0928] 8.2.1. Generation of type I IFN constructs

[0929] Constructs encoding antibody and spatially attenuated IFN fusion proteins are generated in a standard mammalian protein expression DNA vector (pcDNA3.4 or similar vectors) that are suitable for high-yield protein production and contain standard elements such as promoter sequences, polyA sequences, regulatory elements, and resistance genes. Where applicable, the sequences are codon-optimized. A 29-amino acid signal sequence from the murine inactivated tyrosine protein kinase transmembrane receptor ROR1 (mROR1) is added to the N-terminus of the construct as a signal for secretion. All IFN fusion proteins are expressed as pre-proteins containing the signal sequence, which is cleaved by intracellular processing to produce the mature protein. The constructs are expressed in Expi293FTM cells by transient transfection (Thermo Fisher Scientific). Proteins in the Expi293F supernatant are purified using the ProteinMaker system (ProteinBioSolutions, Gaithersburg, MD) and HiTrapTM Protein G HP or MabSelect SuRe pcc columns (Cytiva). After single-step elution, the proteins are neutralized, dialyzed into a final buffer of phosphate-buffered saline (PBS) containing 5% glycerol, aliquoted, and stored at -80 °C. Samples are further analyzed by SE-UPLC to determine the presence of high-molecular weight or low-molecular weight species relative to the substance of interest.

[0930] 8.2.2. In vitro cleavage of IFN constructs

[0931] Alternatively, enzymatic cleavage of the protease-cleavable linker is performed by incubating the pre-protein construct with uPA or MMP enzymes. For enzymatic cleavage with uPA, 8 μg of the protein construct is incubated with 100 ng of uPA enzyme at 37 °C in a 200 μL volume of uPA buffer (50 mM Tris pH 8.5, 0.01% (v / v) Tween 20) for 20 hours. For enzymatic cleavage with MMP, 8 μg of the protein construct is incubated with 200 nM MMP2 and MMP9 separately at 37 °C in a 200 μL volume of MMP buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10 mM CaCl 2 , 0.05% Brij35) for 20 hours.

[0932] 8.2.3. Engineering of reporter gene KG-1a cells

[0933] The promyelocytic macrophage cell line KG-1a was transduced with an ISRE-driven luciferase reporter gene construct and maintained in Iscove's Modified Dulbecco's Medium supplemented with 2 mM L-glutamine / penicillin / streptomycin + 20% FBS + 1 μg / mL puromycin. Single cell clones with high responsiveness to IFNα2b were identified and renamed KG-1a / ISRE-Luc cl.2F5 and used for the assays as described above.

[0934] 8.2.4. Luciferase assay setup

[0935] RPMI1640 medium supplemented with 2 mM L-glutamine / penicillin / streptomycin + 10% FBS was used as the assay medium to prepare cell suspensions and fusion protein dilutions.

[0936] On the day of the assay, the cells were centrifuged and resuspended in the assay medium at a density of 5 x 10 5 / mL. IFNα2b and / or IFN fusion proteins were diluted 1:5 according to an 11-point dilution range (100 nM to 10.2 fM for recombinant interferons and 500 nM to 51.2 fM for fusion proteins), with the 12th point without recombinant protein. 2.5 x 10 4 reporter gene cells were added to a 96-well white flat-bottom plate and incubated with serial dilutions of recombinant IFN or IFN fusion protein. The plate was incubated at 37 °C and 5% CO 2 for 5 hours, then 100 μL of ONE-Glo TM (Promega) reagent was added to lyse the cells and detect luciferase activity. The emitted light was captured in relative light units (RLU) on a multimode plate reader Envision (PerkinElmer).

[0937] 8.3. Example 1: SE-UPLC profiles of Fc-linked interferon molecules

[0938] SE-UPLC was performed to evaluate IFN molecules with an Fc domain linked at the C-terminus or N-terminus. Three exemplary constructs analyzed by SE-UPLC, Fc-IFNα1( Figure 4A ), Fc-IFNα2b( Figure 4B ), and IFNα2b-Fc( Figure 4C ), showed discrete main peaks of different levels of high molecular weight species. The percentage of the main peak area of Fc-IFNα1 was calculated to be 37.43, while for Fc-IFNα2b and IFNα2b-Fc, these percentage values were larger, calculated to be 57.66 and 56.4, respectively.

[0939] 8.4. Example 2: Activity of Interferon Molecules

[0940] As described in Section 8.2.3, a luciferase reporter gene driven by an interferon-stimulated response element (ISRE) was incorporated into the promyelocytic macrophage cell line KG-1a and used as described in Section 8.2.4 to evaluate the ability of IFN receptor agonist constructs to induce ISRE.

[0941] The results shown in Figure 5 indicate that the Fc fusions of recombinant proteins and IFN variants exhibited varying degrees of attenuation in the in vitro luciferase assay of the interferon-sensitive response element (ISRE). The first assessment involved testing for differences in activity in IFN molecules linked to the Fc molecule at the N-terminus or C-terminus ( Figure 5A ). Relative to IFNα2b, both Fc-IFNα2b and IFNα2b-Fc exhibited weaker interferon signaling. However, for both molecules, the level of attenuation was similar ( Figure 5B ). Next, the in vitro activities of the two Fc-IFN constructs, Fc-IFNα2b and Fc-IFNα1, were compared with the activities of three IFN variants, IFNα2b, IFNα1, and IFNβ ( Figure 5C ). Among the IFN variants, the highest activity levels were observed with IFNβ and IFNα2b, while the activity of IFNα1 was relatively weak. Compared with the IFN variants, the recombinant proteins with Fc fusions exhibited attenuated activity levels. In summary, the Fc fusions resulted in attenuation of interferon signaling relative to free interferon.

[0942] 8.5. Example 3: SE-UPLC Profiles of Mutant IFN Constructs

[0943] SE-UPLC was performed to evaluate mutant IFN molecules linked to the Fc domain at the C-terminus. Four exemplary constructs analyzed by SE-UPLC, Fc-IFNα2bR33A ( Figure 6A ), Fc-IFNα2bR149A ( Figure 6B ), Fc-IFNα2bR120A ( Figure 6C ), and Fc-IFNα2bS152A ( Figure 6D ), showed discrete main peaks of high molecular weight substances at different levels.

[0944] 8.6. Example 4: Activity of Mutant IFN Constructs

[0945] As described in Section 8.2.3, an ISRE-driven luciferase reporter gene was incorporated into the promyelocytic macrophage cell line KG-1a and used as described in Section 8.2.4 to evaluate the ability of mutant IFN receptor agonist constructs to induce ISRE.

[0946] The activity of IFN variants is related to their affinity for the IFNAR. Thus, mutations that affect IFN-IFNAR binding can affect the activity of Fc-IFN constructs. A series of mutations were introduced at the IFNAR1 or IFNAR2 interface of IFNα2b ( Figure 7A and 7B ). Relative to wild-type Fc-IFNα2b, most mutations that interfere with IFNAR1 or IFNAR2 binding of Fc-IFNα2b weakened ISRE-luciferase activity. Moreover, the degree of this weakening varied; while some mutations caused only a slight weakening of activity, other mutations led to very high levels of weakening. Nevertheless, for these differences in the level of weakening, the distinction between mutations that interfere with IFNAR1 or IFNAR2 binding was minimal.

[0947] 8.7. Example 5: SE-UPLC Profiles of Exemplary Interferon Proprotein Constructs

[0948] After generating constructs encoding spatially weakened IFN fusion proteins, SE-UPLC was performed to assess the presence of high or low molecular weight species in the samples, as described in Section 8.2.1. Figure 8 depicts the profiles of three exemplary IFN proprotein constructs: a single-hinge IFN construct ( Figure 8A ), a double-hinge IFN construct ( Figure 8B ), and an IFN construct with two Fc domains ( Figure 8C ). Although the single-hinge and double-hinge constructs showed discrete major peaks, the SEC profile of the IFN construct with two Fc domains was less clear. Therefore, subsequent analyses were performed with the single- and double-hinge IFN constructs, and the IFN construct with two Fc domains was excluded.

[0949] 8.8. Example 6: Enzymatic Cleavage of IFN Proprotein Constructs

[0950] As described in Section 8.2.2, exemplary IFN proprotein constructs were cleaved with uPA and MMP enzymes. (Figure 9). Cleavage of the proprotein constructs was evaluated by the presence of bands corresponding to the Fc and Fab components, both of which were expected to have molecular weights between 38 and 49 kDa. The identity of the different fragments was further confirmed by Western blot detection of the Fc portion.

[0951] Cleavage of the single-hinge and double-hinge IFN proprotein constructs by uPA resulted in additional bands appearing at higher molecular weights than expected bands ( Figure 9A ), indicating incomplete cleavage of these constructs by uPA in vitro. In contrast, MMP2 / 9 completely cleaved the single-hinge IFN proprotein. However, in the lane loaded with the double-hinge IFN proprotein cleaved by MMP2 / 9, there were additional high molecular weight bands ( Figure 9B ), indicating incomplete MMP cleavage of the double-hinge construct.

[0952] 8.9. Example 7: Activity of IFN proprotein constructs

[0953] The ISRE-luciferase activities of three IFN proproteins were evaluated compared to IFNα2b and Fc-IFNα2b. The single-hinge full-length IFN and double-hinge IFN constructs exhibited a moderately attenuated level relative to Fc-IFNα2b. However, the best attenuation was associated with the single-hinge truncated IFN ( Figure 10A ).

[0954] To evaluate whether in vitro cleavage of the IFN proprotein constructs restored IFN activity, the same three IFN protein constructs were first incubated with an MMP buffer control or MMP2 and MMP9 enzymes in MMP buffer as described in Section 8.2.2. Constructs incubated only with MMP buffer attenuated the activity of the IFN proprotein constructs ( Figure 10B , dashed line), which was similar to the attenuation of activity by the uncleaved constructs seen in Figure 10A . The presence of MMP buffer was associated with a general decrease in the bioassay signal. However, when the constructs were incubated with MMP enzymes in the same buffer, the release of IFN was associated with an increase in the activity potency relative to the uncleaved constructs ( Figure 10B , dashed line). More specifically, the activities of the single-hinge full-length IFN and double-hinge IFN constructs were similar to the activity of IFNα2b, while the activity of the single-hinge truncated IFN was between the activities of IFNα2b and Fc-IFNα2b.

[0955] 9. Citation of references

[0956] All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the extent that each individual publication, patent, patent application, or other document is specifically indicated to be incorporated by reference for all purposes into this document. If there are inconsistencies between the teachings of one or more of the references incorporated herein and the present disclosure, the teachings of this specification shall govern.

Claims

1. A type I interferon (IFN) proprotein comprising: (a) a first polypeptide chain comprising: (i) a first immunoglobulin constant domain; (ii) a first connector; (iii) a first type I interferon (IFN) portion; (iv) a second connector; and (v) a first Fc domain; (b) a second polypeptide chain comprising: (i) a second immunoglobulin constant domain; (ii) a third joint; (iii) a second type I interferon (IFN) portion; (iv) a fourth connector; and (v) a second Fc domain, which associates with the first Fc domain to form an Fc region; wherein at least two of the first linker, the second linker, the third linker and the fourth linker are protease cleavable linkers (PCLs), optionally wherein the IFN part in the IFN proprotein is sterically hindered by the Fc domain and cannot bind to an IFN receptor.

2. The IFN proprotein of claim 1, wherein the first IFN portion and the second IFN portion each comprise an amino acid sequence having at least about 90%, at least about 95%, or at least about 98% sequence identity to: (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ, or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ having at most one truncation of 15 amino acids at the N-terminus and / or C-terminus.

3. The IFN proprotein of claim 1, wherein the first IFN portion and the second IFN portion each comprise an amino acid sequence having one or more attenuating mutations compared to mature human IFNα1 or IFNα2b.

4. The IFN proprotein of any one of claims 1 to 3, wherein the first IFN portion and the second IFN portion each comprise one or more mutations selected from the group consisting of L26A, F27A, R33A, R33K, L30A, D35E, H57Y, E58N, Q61S, H57S, E58S, H57A, E58A, Q61A, Q90A, E96A, R120A, L135A, R144A, R144S, R144T, R144Y, R144I, R144L, A145D, A145H, A145K, A145M, A145V, A145Y, R149A, R149K, S152A, R162A, and E165D.

5. The IFN proprotein according to any one of claims 1 to 4, wherein the first linker, the second linker, the third linker and the fourth linker are protease cleavable linkers (PCL).

6. The IFN proprotein according to any one of claims 1 to 4, wherein the first linker and the third linker are protease cleavable linkers (PCL), optionally wherein the second linker and the fourth linker are non-cleavable linkers (NCL).

7. The IFN proprotein according to any one of claims 1 to 4, wherein the second linker and the fourth linker are protease cleavable linkers (PCL), optionally wherein the first linker and the third linker are non-cleavable linkers (NCL).

8. The IFN proprotein according to any one of claims 1 to 7, wherein each of the PCLs comprises a substrate sequence cleavable by any protease listed in Table A.

9. The IFN proprotein according to any one of claims 1 to 8, wherein each of the PCLs comprises one or more substrate sequences selected from the substrate sequences listed in Table B.

10. The IFN proprotein according to any one of claims 1 to 9, wherein each of the PCLs comprises one or more spacer sequences selected from the sequences listed in Table C.

11. The IFN proprotein according to any one of claims 1 to 10, wherein each of the PCLs comprises the amino acid sequence of any one of the PCL sequences listed in Table D or a variant thereof having up to 5 amino acid substitutions.

12. The IFN proprotein according to any one of claims 1 to 11, wherein the first linker and the third linker are identical and / or the third linker and the fourth linker are identical.

13. The IFN proprotein according to any one of claims 1 to 12, wherein the first Fc domain and / or the second Fc domain comprises a hinge domain.

14. The IFN proprotein according to any one of claims 1 to 13, further comprising a first targeting moiety and a second targeting moiety.

15. The IFN proprotein of claim 14, wherein the first targeting moiety and the second targeting moiety are antibodies or antigen-binding fragments thereof.

16. The IFN proprotein of claim 15, wherein the first targeting moiety and the second targeting moiety comprise a Fab domain.

17. The IFN proprotein according to any one of claims 14 to 16, wherein the first targeting moiety and the second targeting moiety are located at the N-terminus of the first linker and the third linker, respectively.

18. The IFN proprotein according to any one of claims 14 to 17, wherein the first immunoglobulin constant domain is part of the first targeting moiety and the second immunoglobulin constant domain is part of the second targeting moiety.

19. The IFN proprotein of claim 18, wherein the first immunoglobulin constant domain is a CH3 domain or a CH1 domain.

20. The IFN proprotein according to any one of claims 1 to 19, which is configured as shown in Figure 1A, Figure 2A or Figure 2D.

21. The IFN proprotein according to any one of claims 1 to 19, which is configured as shown in Figure 1B, Figure 2B or Figure 2E.

22. The IFN proprotein according to any one of claims 1 to 19, which is configured as shown in Figure 1C, Figure 2C or Figure 2F.

23. The IFN preprotein according to any one of claims 14 to 22, wherein the first targeting moiety and / or the second targeting moiety herein is capable of binding to an extracellular matrix (ECM) antigen, a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or viral lymphocyte, a T cell antigen (TCA), a checkpoint inhibitor, a tumor-associated antigen (TAA), a dendritic cell (DC) or other antigen presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

24. The IFN proprotein of any one of claims 14 to 23, wherein the first targeting moiety and / or the second targeting moiety (a) comprises (i) CDRs or (ii) VH sequences and VL sequences of an antibody listed in Table F or (b) competes with the antibody listed in Table F for binding to a target molecule.

25. The IFN proprotein of any one of claims 14 to 23, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to an ECM antigen, the ECM antigen being optionally selected from syndecan, heparanase, integrin, osteopontin, connexin, cadherin, laminin, laminin-type EGF, lectin, fibronectin, notch proteins, adhesion proteins (e.g., adhesion protein-4), tenascin, collagen (e.g., type X collagen), and matrix proteins.

26. The IFN proprotein according to any one of claims 14 to 23, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to a cell surface molecule of a tumor or viral lymphocyte.

27. The IFN proprotein of claim 26, wherein the antigen is a T cell co-stimulatory protein, which is optionally selected from CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C and B7-H3.

28. The IFN pro-protein according to any one of claims 14 to 23, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to a check point inhibitor.

29. The IFN proprotein of claim 28, wherein the checkpoint inhibitor is PDL1.

30. The IFN proprotein of claim 28, wherein the checkpoint inhibitor is PD1.

31. The IFN proprotein according to any one of claims 14 to 23, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to a tumor associated antigen (TAA), the TAA being optionally selected from AFP, ALK, BAGE protein, BIRC5 (survivin), BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CEACAM5 (also known as carcinoembryonic antigen or CEA), CCR5 , CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​protein (e.g., GAGE-1 or -2), GD2, GD3, GloboH, Glypican-3, G M3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY- ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase, and uroplasmin-3.

32. The IFN proprotein according to any one of claims 14 to 23, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to a dendritic cell (DC) antigen, the DC antigen being optionally selected from XCR1, Clec9a, CD1c, CD11c, CD14, PDL1, macrophage mannose receptor (CD206) and DEC-205.

33. The IFN proprotein according to any one of claims 14 to 23, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to a Natural Killer (NK) cell antigen.

34. The IFN proprotein according to any one of claims 1 to 33, wherein the Fc region is homodimeric.

35. A nucleic acid or nucleic acids encoding the IFN proprotein according to any one of claims 1 to 34.

36. A host cell engineered to express the IFN proprotein of any one of claims 1 to 34 or the nucleic acid of claim 35.

37. A method of producing an IFN proprotein according to any one of claims 1 to 34, comprising culturing the host cell according to claim 36 and recovering the IFN proprotein expressed thereby.

38. A pharmaceutical composition comprising the IFN proprotein according to any one of claims 1 to 34 and an excipient.

39. A method of treating cancer comprising administering the IFN proprotein according to any one of claims 1 to 34 or the pharmaceutical composition according to claim 38 to a subject in need thereof.

40. The method of claim 39, wherein the IFN proprotein comprises at least one targeting moiety capable of binding to a target molecule.

41. The method of claim 39, wherein the cancer is associated with expression of the target molecule, e.g., a TAA and associated cancers as listed in Table 1.

42. The method of any one of claims 39 to 41, wherein the activated IFN protein comprising the IFN portion is produced by cleavage of one or more protease cleavable linkers in the IFN proprotein by one or more proteases expressed by the cancer tissue.

43. A method for local delivery of an IFN protein, comprising administering to a subject an IFN proprotein according to any one of claims 1 to 34 (or a pharmaceutical composition comprising the IFN proprotein and an excipient), the IFN proprotein having one or more protease cleavable linkers, each protease cleavable linker comprising one or more substrates for one or more proteases expressed by a tissue to which the IFN protein is to be locally delivered.

44. A method for treating cancer with an IFN protein that is selectively activated in cancer tissue, comprising administering to a subject in need thereof an IFN proprotein according to any one of claims 1 to 34 (or a pharmaceutical composition comprising the IFN proprotein and an excipient), the IFN proprotein having one or more protease cleavable linkers, each protease cleavable linker comprising one or more substrates for one or more proteases expressed by the cancer tissue targeted by the IFN protein.

45. A method of administering to a subject an IFN therapy with reduced systemic exposure and / or reduced systemic toxicity, comprising administering to the subject the IFN therapy in the form of an IFN proprotein (or a pharmaceutical composition comprising the IFN proprotein and an excipient) according to any one of claims 1 to 34, the IFN proprotein having one or more protease cleavable linkers, each protease cleavable linker comprising one or more substrates for one or more proteases expressed by a tissue for which IFN therapy is desired and / or anticipated.

46. ​​A method for treating cancer with an IFN protein that is selectively activated in cancer tissue, comprising administering to a subject in need thereof an IFN proprotein according to any one of claims 1 to 34 (or a pharmaceutical composition comprising the IFN proprotein and an excipient), the IFN proprotein having one or more protease cleavable linkers, each protease cleavable linker comprising one or more substrates for one or more proteases expressed by the cancer tissue.

47. A method for targeted delivery of activated IFN protein to cancer tissue, comprising administering to a subject an IFN proprotein according to any one of claims 1 to 34 (or a pharmaceutical composition comprising the IFN proprotein and an excipient), wherein the IFN proprotein: (a) comprising one or more targeting moieties that recognize a target molecule expressed by the cancer tissue or associated immune cells; and (b) having one or more protease cleavable linkers, each protease cleavable linker comprising one or more substrates for one or more proteases expressed in a tissue for which IFN therapy is desired and / or anticipated.

48. A method for locally inducing an immune response in a target tissue, comprising administering to a subject an IFN proprotein according to any one of claims 1 to 34 (or a pharmaceutical composition comprising the IFN proprotein and an excipient), the IFN proprotein having one or more targeting moieties capable of binding to a target molecule expressed in the target tissue and one or more protease cleavable linkers, each protease cleavable linker comprising one or more substrates for one or more proteases expressed in the target tissue.

49. A method of enhancing an immune response to an antigen, comprising administering to a subject an immunogenic agent that elicits an immune response to the antigen together with an IFN proprotein according to any one of claims 1 to 34 (or a pharmaceutical composition comprising the IFN proprotein and an excipient) or a nucleic acid encoding such an IFN proprotein, e.g., as described in Section 6.10.1).

50. The method of any one of claims 39 to 49, wherein the administration is non-topical.

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