Interferon proproteins and uses thereof

Abstract

The present disclosure provides protease-activated IFN proproteins. The IFN proproteins include an IFN portion that is sterically hindered from binding to its receptor by being linked to an Fc portion via a protease-cleavable linker. In response to the action of a protease, for example, at a tumor site, the protease-cleavable linker is cleaved, thereby releasing and activating the IFN portion. The IFN proproteins optionally further include a targeting moiety, for example, a targeting moiety that recognizes a tumor-associated antigen and directs the proprotein to the 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

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent No. 63 / 399,040, filed August 18, 2022, U.S. Provisional Patent No. 63 / 383,804, filed November 15, 2022, and U.S. Provisional Patent No. 63 / 481,303, filed January 24, 2023, the contents of each of which are incorporated herein by reference in their entirety.

[0002] 2. Sequence Listing This application contains a Sequence Listing that has been submitted electronically and is incorporated herein 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. [Background technology]

[0003] 3.Background technology Type I interferons (IFNs) are thought to directly suppress tumor cell proliferation. Type I IFNs are useful in the treatment of several types of cancer, including hematological tumors (chronic myeloid leukemia, hairy cell leukemia, multiple myeloma, and non-Hodgkin's lymphoma) and solid tumors (melanoma, renal carcinoma, and Kaposi's sarcoma). See, for example, Non-Patent Document 1 and Non-Patent Document 2.

[0004] A particular advantage of type I IFN therapy is its ability to intervene at multiple points in generating an anti-tumor immune response, including stimulating innate and adaptive cytotoxic lymphocyte populations, negatively regulating suppressor cell types, and influencing tumor cells by inhibiting proliferation and modulating apoptosis, differentiation, migration, and cell surface antigen expression (Non-Patent Document 3).

[0005] One of the biggest barriers to the use of type I IFN in the clinic is the severe side effects associated with such treatment. The most frequently encountered side effects are flu-like symptoms, hematological toxicity, elevated transaminases, nausea, fatigue, and psychiatric sequelae. These side effects prevent patients from reaching and maintaining the dose required for maximum therapeutic efficacy, and their occurrence may completely outweigh the clinical benefits of type I IFN treatment (Non-Patent Document 4). Type I IFN signals through the IFNAR1 / IFNAR2 complex, which is expressed on most cells and tissues in the body. Therefore, the ability to preferentially or specifically deliver active type I IFN to tumor-reactive immune cells (see, e.g., Non-Patent Documents 5 and 6) or the tumor microenvironment is essential for the continued clinical use of type I IFN. Strategies for modifying type I IFN are needed to obtain new drug forms that preferentially exert type I IFN activity on tumor-reactive immune cells and / or tumors, and also to reduce side effects on normal IFNAR-expressing cells.

[0006] Therefore, there is a need in the art for new type I IFN therapies with improved therapeutic efficacy and safety profiles. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Zitvogel et al.,2015, Nat Rev Immunol 15:405-414 [Non-patent document 2] Antonelli et al.,2015,Cytokine Growth Factor Rev 26:121-131 [Non-patent document 3] Parker et al.,2016,Nature Reviews Cancer 16:131-144 [Non-patent document 4] Lotrich,2009,Dialogues Clin Neurosci 11:417-425 [Non-patent document 5] Diamond et al.,2011,J Exp Med.208(10):1989-2003 [Non-patent document 6] Cauwels et al.,2018,Cancer Res.78(2):463-474 Summary of the Invention

[0008] 4. Summary of the Invention The present disclosure relates to IFN proproteins that are activated by proteases, for example, proteases expressed in the tumor environment.

[0009] The IFN proprotein comprises an IFN moiety that is sterically hindered from binding to its receptor, configured such that the IFN moiety is activated by cleavage of a linker within the IFN proprotein by a protease, thereby relieving the steric hindrance of the IFN moiety. The IFN proprotein may further comprise a targeting moiety that directs the IFN proprotein to a particular tissue or cell type.

[0010] Typically, the IFN proproteins of the present disclosure comprise two polypeptide chains, each comprising, from N- to 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 (PCL). 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.

[0011] The IFN proprotein can further comprise a targeting moiety (or a component thereof, e.g., one chain of a Fab), e.g., at the N-terminus of one or both Fc domains. The targeting moiety comprises, for example, an antigen-binding domain ("ABD") that can bind to a target molecule present on the tumor surface (e.g., a tumor-associated antigen) or other component in the tumor microenvironment (e.g., the extracellular matrix ("ECM") or tumor lymphocytes).

[0012] Exemplary IFN moieties that can be used in the IFN proproteins of the present disclosure are described in Section 6.3. Protease-cleavable linkers that can be used in the IFN proproteins of the present disclosure are described in Section 6.4.

[0013] Protease-non-cleavable linkers that can be used in the IFN proproteins of the present disclosure are described in Section 6.5. Targeting moieties that can be used in the IFN proproteins of the present disclosure are described in Section 6.6, and targeting moiety formats are disclosed in Section 6.7.

[0014] Fc domains that can be incorporated into the IFN proproteins of the present disclosure are described in Section 6.8. Exemplary IFN proproteins of the present disclosure are described in Section 6.2 and in numbered embodiments 1-146.

[0015] The present disclosure further provides nucleic acids encoding the IFN proproteins of the present disclosure. The nucleic acid encoding the IFN proprotein can be a single nucleic acid (e.g., a vector encoding all 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 of producing the IFN proproteins of the present disclosure. Exemplary nucleic acids, host cells, and cell lines, as well as methods of producing the IFN proproteins of the present disclosure, are described in Section 6.9 and numbered embodiments 147-149.

[0016] 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 in numbered embodiment 150. Further provided herein are 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 embodiments 151-201.

[0017] 5. Brief description of the drawings [Brief explanation of the drawings]

[0018] [Figure 1A]

[0023] Figure 1A is a schematic diagram depicting three IFN proprotein configurations of the present disclosure with protease-cleavable linkers adjacent to the interferon moiety. Figure 1A depicts an IFN proprotein with the overall configuration: antibody-PCL-IFN-PCL-Fc, Figure 1B depicts a single-hinge IFN proprotein with the configuration: Fab-PCL-IFN-PCL-hinge-Fc, and Figure 1C depicts a double-hinge IFN proprotein with the configuration: Fab-hinge-PCL-IFN-PCL-hinge-Fc. Although shown with the targeting moiety in the form of a Fab, the VH, VL, and CL domains of the Fab are optional for non-targeting IFN proproteins or can be replaced by other targeting moieties, such as scFvs. [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 2A] FIG. 1 is a schematic diagram depicting the configuration of six IFN proproteins of the present disclosure, each having a protease-cleavable linker (PCL) on only one side of the interferon moiety and a non-cleavable linker (NCL) on the other side of the interferon moiety. FIG. 2A represents an IFN proprotein having the overall configuration: antibody-NCL-IFN-PCL-Fc, FIG. 2B represents a single-hinge IFN proprotein having the configuration: Fab-NCL-IFN-PCL-hinge-Fc, FIG. 2C represents a double-hinge IFN proprotein having the configuration: Fab-hinge-NCL-IFN-PCL-hinge-Fc, FIG. 2D represents an IFN proprotein having the overall configuration: antibody-PCL-IFN-NCL-Fc, FIG. 2E represents a single-hinge IFN proprotein having the configuration: Fab-PCL-IFN-NCL-hinge-Fc, and FIG. 2F represents a double-hinge IFN proprotein having the configuration: Fab-hinge-PCL-IFN-NCL-hinge-Fc. Although shown with the targeting moiety in the form of a Fab, the VH, VL, and CL domains of the Fab are optional for non-targeting IFN proproteins or can be replaced by other targeting moieties such as scFvs. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Figure 2E] Same as above. [Figure 2F] Same as above. [Figure 3]

[0023] Figure 1 is a table of exemplary targeting IFN proproteins and their constituent polypeptide chains according to Figure 1. TM refers to a targeting moiety, HC refers to an antibody heavy chain, LC refers to an antibody light chain, IFN refers to an interferon (IFN) moiety, ΔN and ΔC refer to N- and C-terminal truncations, respectively, in the IFN sequence of the IFN moiety (e.g., as described in Section 6.3), PCL refers to a protease-cleavable linker (e.g., as described in Section 6.4), Fc refers to an Fc domain (e.g., as described in Section 6.8), hinge refers to an antibody hinge sequence, and long hinge and short hinge refer to full-length or truncated versions of immunoglobulin hinge sequences (e.g., as described in Section 6.8.3). Additionally, although shown with protease-cleavable linkers (PCLs) adjacent to the IFN moiety, one of the PCLs in each of chain 1 and chain 2 may be replaced by, for example, a non-cleavable linker (NCL), such as those described in Section 6.5. [Figure 4A] Figure 4A illustrates the SEC profile of an IFN molecule, Fc-IFNα1, having an N-terminal Fc domain and a C-terminal IFN portion, Fc-IFNα2b, and Figure 4C illustrates the SEC profile of an IFN molecule, IFNα2b-Fc, having an N-terminal IFN portion and a C-terminal Fc domain. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 5A]Figure 5A shows the in vitro activity of exemplary IFN molecules linked to an Fc molecule at either the N- or C-terminus. The schematic diagram in Figure 5A depicts N- and C-terminal Fc fusions of IFN. Fc-IFN is a general representation of an IFN molecule having an N-terminal Fc domain and a C-terminal IFN portion, and IFN-Fc is a general representation of an IFN molecule having an N-terminal IFN portion 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. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 6A] 6A and 6B illustrate SE-UPLC profiles of exemplary mutant IFN molecules that can be incorporated into the IFN proprotein constructs of the present disclosure. Figure 6A illustrates the SE-UPLC profile of a mutant IFN molecule, Fc-IFNα2bR33A. Figure 6B illustrates the SE-UPLC profile of a mutant IFN molecule, Fc-IFNα2bR149A. Figure 6C illustrates the SE-UPLC profile of a mutant IFN molecule, Fc-IFNα2bR120A. Figure 6D illustrates the SE-UPLC profile of a mutant IFN molecule, Fc-IFNα2bS152A. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 7A] Figure 7 shows the in vitro activity of exemplary mutant IFN molecules that can be incorporated into the IFN proprotein constructs of the present disclosure. The schematic diagram in Figure 7A depicts the overall structure of a wild-type (WT) or mutant (Mut) Fc-IFN molecule. Figure 7B is a graph showing the in vitro activity of Fc-IFNα2b molecules with mutations affecting either the IFNAR1 or IFNAR2 interface. [Figure 7B] Same as above. [Figure 8A]8A and 8B illustrate the SE-UPLC profiles of exemplary IFN proprotein constructs. Figure 8A illustrates the SE-UPLC profile of a single-hinge full-length IFN proprotein, aPD1-single-hinge-FLIFN. Figure 8B illustrates the SE-UPLC profile of a double-hinge IFN proprotein, aPD1-double-hinge-long-IFN. Figure 8C illustrates the SE-UPLC profile of an IFN proprotein with two Fc regions. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 9A] Figure 9 shows in vitro enzymatic cleavage of single-hinge and double-hinge IFN proproteins. Figure 9A is a gel image illustrating cleavage of three single-hinge proproteins, two double-hinge IFN proproteins, and a positive control by uPA. Similarly, Figure 9B is a gel image illustrating cleavage of the same single- and double-hinge IFN proproteins by MMP2 and MMP9. [Figure 9B] Same as above. [Figure 10A] Figure 10 shows the in vitro activity of exemplary IFN proproteins compared to IFNα2b and Fc-IFNα2b. Figure 10A is a graph showing the in vitro activity of two single-hinge IFN proproteins (full-length and truncated) and one double-hinge IFN proprotein. Figure 10B is a graph illustrating the change in activity of the same IFN proproteins in Figure 10A upon addition of MMP buffer alone (dashed line) or a mixture of MMP buffer and enzyme (dotted line). [Figure 10B] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0019] 6. MODE FOR CARRYING OUT THE INVENTION 6.1.Definition As used herein, the following terms are intended to have the following meanings:

[0020] ABD chain, targeting moiety chain: The targeting moiety and antigen-binding site (ABD) therein may be present as a single polypeptide chain (e.g., in the case of an scFv or scFab) or as a form via the association of two or more polypeptide chains (e.g., in the case of an Fab or Fv). As used herein, the terms "ABD chain" and "targeting moiety chain" refer to all or a portion of an ABD or targeting moiety present on a single polypeptide chain. The use of the term "ABD chain" or "targeting moiety chain" is intended for convenience and descriptive purposes only and does not imply a particular configuration or method of production. Furthermore, reference to an ABD or targeting moiety when describing an IFN proprotein encompasses the ABD chain or targeting moiety chain unless the context dictates otherwise. Thus, when describing an IFN proprotein in which an Fc domain is operably linked to a targeting moiety, the Fc domain can be covalently linked via a peptide bond, for example, to (1) a first ABD or targeting moiety chain of a Fab or Fv (with other components of the Fab or Fv on a second associated ABD or targeting moiety chain), or (2) an ABD or targeting moiety chain comprising an scFv or scFab, either directly or indirectly (e.g., via a linker).

[0021] About, Approximately: The terms "about," "approximately," and the like are used throughout the specification before a numerical value to indicate that the numerical value is not necessarily exact (e.g., to account for fractions, variations in measurement precision and / or accuracy, timing, etc.). A disclosure of "about X" or "approximately X," where X is a numerical value, should be understood to also be a disclosure of "X." Thus, for example, disclosure of embodiments in which a sequence has "about X% sequence identity" with another sequence is also a disclosure of embodiments in which the sequence has "X% sequence identity" with the other sequence.

[0022] Activate, activation: The terms "activate," "activation," and the like, in conjunction with the IFN proproteins of the present disclosure, refer to the protease-mediated enzymatic cleavage of a protease-cleavable linker, resulting in release of the IFN portion from the sterically hindering constant domain.

[0023] And / or: Unless otherwise indicated, the conjunction "or" is intended to be used in its proper sense as a Boolean logic operator, encompassing both the selection of features in an alternative (A or B, where the selection of A is mutually exclusive of B) and the selection of conjunctive features (A or B, where both A and B are selected). In several places in the text, the term "and / or" is used interchangeably and should not be interpreted to mean that "or" is used in reference to mutually exclusive alternatives.

[0024] Antibody: As used herein, the term "antibody" refers to a polypeptide (or set of polypeptides) of the immunoglobulin family that can non-covalently, reversibly, and specifically bind to an antigen. For example, a naturally occurring "antibody" of the IgG type is a tetramer comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody may 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 (Clq) of the classical complement system. 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-idiotype (anti-id) antibodies. Antibodies may 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 regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be understood that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity.Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, placental transport, Fc receptor binding, complement fixation, etc. By convention, the numbering of constant region domains increases as they become more distant from the antigen-binding domain or amino-terminus of the antibody. The N-terminus is the variable region, the C-terminus is the constant region, and the CH3 and CL domains represent the carboxy-terminus of the heavy and light chains of a native antibody, respectively. For convenience, and unless the context dictates otherwise, reference to an antibody also refers to antibody fragments and engineered antibodies containing non-native antigen-binding domains and / or antigen-binding domains with non-native configurations.

[0025] Antigen-binding domain: As used herein, the term "antigen-binding domain" or "ABD" refers to a portion of an antibody or antibody fragment (e.g., a 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 the VL, VH, CL, and CH1 domains, F(ab)2 fragments, bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region, Fd fragments consisting of the VH and CH1 domains, Fv fragments consisting of the VL and VH domains of a single antibody arm, dAb fragments consisting of the VH domain (Ward et al., 1989, Nature 341:544-546), and isolated complementarity-determining regions (CDRs). Thus, the term "antibody fragment" encompasses both proteolytic fragments of antibodies (e.g., Fab fragments and F(ab)2 fragments) and engineered proteins comprising one or more portions of an antibody (e.g., scFv). Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology 23:1126-1136).

[0026] Associated: The term "associated" in the context of an IFN proprotein refers to a functional relationship between two or more polypeptide chains. Specifically, the term "associated" means that two or more polypeptides are associated with one another, e.g., noncovalently through molecular interactions or covalently through one or more disulfide or chemical bridges, to produce a functional IFN proprotein. Examples of associations that may be present in the IFN proproteins of the present disclosure include, but are not limited to, associations between Fc domains that form the Fc region (homodimers or heterodimers as described in Section 6.8), associations between the VH and VL regions in a Fab or Fv, and associations between the CH1 and CL in a Fab.

[0027] Cancer: The term "cancer" refers to a disease characterized by the uncontrolled (and often rapid) growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Various examples of cancer are described herein, including, but not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, adrenal gland cancer, autonomic ganglion cancer, biliary tract cancer, bone cancer, endometrial cancer, eye cancer, fallopian tube cancer, reproductive tract cancer, colon 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, thymus cancer, thyroid cancer, upper aerodigestive tract cancer, urinary tract cancer, vaginal cancer, vulvar cancer, lymphoma, leukemia, lung cancer, etc., for example, TAA-positive cancer of any of the aforementioned types.

[0028] Complementarity-Determining Region: The term "complementarity-determining region" or "CDR," as used herein, refers to the sequence of amino acids within an antibody variable region that confers antigen specificity and binding affinity. For example, generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, and CDR-H3), and each light chain variable region has three CDRs (CDR-L1, CDR-L2, and CDR-L3). The precise amino acid sequence boundaries of a given CDR can be determined using the "Kabat" numbering scheme, 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 ImMunoGenTics (IMGT) numbering scheme (Lefranc, 1999, The Immunologist 7:132-136; Lefranc et al., 1999, The Immunologist 7:132-136). The CDR numbering scheme can be determined using any of several well-known schemes, including those described by Kabat et al., 2003, Dev. Comp. Immunol. 27:55-77 ("IMGT" numbering scheme). For example, for the classical format, 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 (VH) are numbered 31-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3). The CDR amino acid residues in a VL are numbered 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3). Under Chothia, the CDR amino acids in a VH are numbered 26-32 (CDR-H1), 52-56 (CDR-H2), and 95-102 (CDR-H3), and the amino acid residues in a VL are numbered 26-32 (CDR-L1), 50-52 (CDR-L2), and 91-96 (CDR-L3).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 residues in VH are numbered approximately 26-35 (CDR-H1), 51-57 (CDR-H2), and 93-102 (CDR-H3), and the CDR amino acid residues in VL are numbered approximately 27-32 (CDR-L1), 50-52 (CDR-L2), and 89-97 (CDR-L3) ("Kabat" numbering). Under IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0029] Constant domain: The term "constant domain" refers to a CH1, CH2, CH3, or CL domain of an immunoglobulin. The term "CH1 domain" refers to the heavy chain constant region that connects the variable domain to the hinge within the heavy chain constant domain. In some embodiments, the term "CH1 domain" refers to the region of an immunoglobulin molecule spanning amino acids 118-215 (EU numbering). The term "CH1 domain" encompasses wild-type CH1 domains as well as variants thereof (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 variants thereof having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5, and / or up to 5, 4, 3, 2, or 1 mutation, e.g., substitution, deletion, and / or addition. Exemplary CH1 domains include CH1 domains with mutations that modify the biological activity of the antibody, such as ADCC, CDC, or half-life.

[0030] The term "CH2 domain" refers to the heavy chain constant region that connects the hinge to the CH3 domain within the heavy chain constant domain. In some embodiments, the term "CH2 domain" refers to a region of an immunoglobulin molecule spanning amino acids 238-340 (EU numbering). The term "CH2 domain" encompasses wild-type CH2 domains as well as variants thereof (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 variants thereof having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5, and / or up to 5, 4, 3, 2, or 1 mutation, e.g., substitution, deletion, and / or addition. Exemplary CH2 domains include CH2 domains with mutations that modify antibody biological activity, such as ADCC, CDC, purification, dimerization, or half-life.

[0031] The term "CH3 domain" refers to the heavy chain constant region C-terminal to the CH2 domain within the heavy chain constant domain. In some embodiments, the term "CH3 domain" refers to a region of an immunoglobulin molecule spanning amino acids 341-447 (EU numbering). The term "CH3 domain" encompasses wild-type CH3 domains as well as 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, 1-3, 1-5, 3-5, and / or up to 5, 4, 3, 2, or 1 mutation, e.g., substitution, deletion, and / or addition. Exemplary CH3 domains include CH3 domains having mutations that modify antibody biological activity, such as ADCC, CDC, purification, dimerization, or half-life.

[0032] The term "CL domain" refers to the constant region of an immunoglobulin light chain. The term "CL domain" encompasses wild-type CL domains (e.g., kappa or lambda light chain constant regions) as well as variants thereof (e.g., non-naturally occurring CL domains or modified CL domains). For example, the term "CL domain" includes wild-type kappa and lambda constant domains and variants thereof having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5, and / or up to 5, 4, 3, 2, or 1 mutations, e.g., substitutions, deletions, and / or additions.

[0033] Effector function: The term "effector function" refers to an activity of an antibody molecule that is typically mediated by binding of an effector molecule through a domain of the antibody other than the antigen-binding domain. Effector function includes, for example, complement-mediated effector function, which is mediated by the binding of the C1 component of complement to an antibody. Complement activation is important for opsonization and lysis of cellular pathogens. Complement activation also stimulates inflammatory responses and may be involved in autoimmune hypersensitivity. Effector function also includes Fc receptor (FcR)-mediated effector function, which can be triggered upon binding of the constant domain of an antibody to an Fc receptor (FcR). Binding of an antibody to an Fc receptor on the cell surface triggers a number of important and diverse biological responses, including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and regulation of immunoglobulin production. The effector function of an antibody can be altered by changing, e.g., increasing or decreasing, the affinity of the antibody for an effector molecule, such as an Fc receptor or a complement component. Binding affinity is generally altered by modifying the effector molecule binding site; in this case, it is appropriate to locate the site of interest and modify at least a portion of that site in a suitable manner. It is also contemplated that altering the binding site on an antibody for an effector molecule need not significantly alter the overall binding affinity but may alter the shape of the interaction, such as in non-productive binding, to disable the effector mechanism. It is further contemplated that effector function can also be altered by modifying sites not directly involved in effector molecule binding but that are otherwise involved in the performance of the effector function.

[0034] Epitope: An epitope, or antigenic determinant, is the portion of an antigen that is recognized by an antibody or other antigen-binding moiety described herein. Epitopes can be linear or conformational.

[0035] Fab: The term "Fab" refers to a pair of polypeptide chains, where the first polypeptide chain comprises an antibody variable heavy (VH) domain operably linked (typically N-terminally) to a first constant domain (referred to herein as C1), and the second polypeptide chain comprises an antibody N-terminal variable light (VL) domain operably linked (typically N-terminally) to a second constant domain (referred to herein as C2) that can pair with the first constant domain. In a native antibody, the VH is N-terminal to the first constant domain (CH1) of the heavy chain, and the VL is N-terminal to the constant domain (CL) of the light chain. The Fabs of the present disclosure can be oriented according to their native orientation or can include domain substitutions or swaps 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 modified correct Fab-chain pairing in a heterodimeric molecule. It is also possible to reverse the CH1 and CL, so that CH1 is attached to VL and CL is attached to VH, a configuration generally known as a Crossmab. The term "Fab" encompasses single-chain Fab.

[0036] Fc domain and Fc region: The term "Fc domain" refers to the portion of a heavy chain that pairs with the corresponding portion of another heavy chain. In some embodiments, an Fc domain comprises a CH2 domain followed by a CH3 domain, with or without a hinge region N-terminal to 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 an Fc region may be identical to one another or different from one another. In native antibodies, the Fc domains are typically identical, but one or both Fc domains may be engineered to allow heterodimerization, for example, via knob-in-hole interactions.

[0037] Fv: The term "Fv" refers to the smallest antibody fragment derivable from an immunoglobulin that contains a complete target recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association (VH-VL dimer). In this configuration, the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer. Often, the six CDRs confer target binding specificity to the antibody. However, in some instances, even a single variable domain (or half of an Fv comprising only three CDRs specific for a target) may be capable of recognizing and binding to a target. Reference herein to a VH-VL dimer is not intended to convey any particular configuration. When present on a single polypeptide chain (e.g., scFv), the VH and VL may be N- or C-terminal.

[0038] Half antibody: The term "half antibody" refers to a molecule that contains at least one Fc domain and can associate with another molecule that contains an Fc domain, for example, through a disulfide bridge or molecular interaction. A half antibody can be composed of one polypeptide chain or two or more polypeptide chains (e.g., the two polypeptide chains of a Fab). An example of a half antibody is a molecule that contains the heavy and light chains of an antibody (e.g., an IgG antibody). Another example of a half antibody is a molecule that contains a first polypeptide that contains a VL domain and a CL domain, and a second polypeptide that contains a VH domain, a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, where the VL domain and the VH domain form an ABD. Yet another example of a half antibody is a polypeptide that contains an scFv domain, a CH2 domain, and a CH3 domain.

[0039] The IFN proproteins of the present disclosure typically comprise two half antibodies each comprising an IFN moiety adjacent to a protease-cleavable linker, with the Fc domain C-terminal to the C-terminal protease-cleavable linker and the constant domain N-terminal to the N-terminal protease-cleavable linker. One or both half antibodies within the IFN proprotein may further comprise a targeting moiety.

[0040] The term "half antibody" is intended for descriptive purposes only and does not imply a particular composition or method of production. Description of half antibodies as a "first" half antibody, a "second" half antibody, a "left" half antibody, a "right" half antibody, etc. is solely for convenience and descriptive purposes.

[0041] Host cell or recombinant host cell: The terms "host cell" or "recombinant host cell" refer to cells that have been genetically engineered, for example, through the introduction of heterologous nucleic acid. It is understood that such terms are intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur later, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. A host cell may harbor heterologous nucleic acid transiently, for example, on an extrachromosomal heterologous expression vector, or stably, for example, by integration of the heterologous nucleic acid into the host cell genome. For purposes of expressing the IFN proproteins of the present disclosure, host cells are preferably cell lines 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 higher densities than the original cell line, and / or derivatives with modified glycan profiles and / or site-specific integration sites.

[0042] Interferon: As used herein, the term "interferon" refers to a full-length interferon or a modified interferon, e.g., a truncated and / or mutated 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 is attenuated by a range bounded 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%, etc. In other embodiments, the modified interferons substantially retain the biological activity of the corresponding wild-type interferons (e.g., retain 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).

[0043] Linker: As used herein, the term "linker" refers to a protease-cleavable linker or a non-cleavable linker. Non-cleavable linker: As used herein, a non-cleavable linker refers to a peptide whose amino acid sequence lacks a specific sequence motif, e.g., a substrate sequence for a protease, e.g., a protease described in Section 6.4.1, that recognizes and cleaves a substrate described in Section 6.4.2.

[0044] 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 segments are joined to produce a functional polypeptide. For example, in the context of the IFN proproteins of the present disclosure, separate components (e.g., the Fc domain and IFN moiety) 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 within the IFN proprotein of the present disclosure, "operably linked" means that the 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 of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.

[0045] Polypeptide, Peptide, and Protein: The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.

[0046] Proprotein: A "proprotein" is a protein precursor that is inactive and can be activated by proteolysis with a protease. A proprotein is therefore "protease-activatable."

[0047] 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, such as those described in Section 6.4.1, recognize and cleave specific sequence motifs, e.g., substrates such as those described in Section 6.4.2. Preferably, the protease is expressed at higher levels in cancer tissue compared to normal tissue.

[0048] Protease-cleavable linker: As used herein, the term "protease-cleavable linker" or "PCL" refers to a peptide whose amino acid sequence includes one or more (e.g., two, three, or more) substrate sequences for 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.

[0049] Recognize: As used herein, the term "recognize" refers to an antibody or antibody fragment (e.g., a targeting moiety) finding and interacting with (e.g., binding to) the epitope.

[0050] 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 aforementioned domains and linker are arranged in one of the following orders, from N- to C-terminus: (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 suitably a non-cleavable linker of at least 30 amino acids, preferably 32-50 amino acids. Single-chain Fab fragments are typically stabilized via a native disulfide bond between the CL and CH1 domains. In addition, these single-chain Fab molecules can be further stabilized by the creation of interchain disulfide bonds through the insertion of cysteine ​​residues (e.g., at position 44 in the VH domain and position 100 in the VL domain according to the Kabat numbering system).

[0051] Single-chain Fv or scFv: As used herein, the term "single-chain Fv" or "scFv" refers to an antibody comprising the VH and VL domains, 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 that enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Plückthun, *The Pharmacology of Monoclonal Antibodies*, vol. 113, Rosenburg and Moore eds. (1994), Springer-Verlag, New York, pp. 269-315. Typically, the VH and VL are arranged N-terminally to C-terminally in VH-VL or VL-VH order, separated by a linker, e.g., a linker described in Table E.

[0052] Spacer: As used herein, the term "spacer" refers to a peptide of an amino acid sequence that is not a substrate for a protease, incorporated into a linker that includes a substrate. A spacer can be used to separate the substrate from other domains within a molecule, e.g., an ABD. In some embodiments, residues within the spacer minimize the action of aminopeptidases and / or exopeptidases to prevent cleavage of the N-terminal amino acid.

[0053] Specifically (or selectively) binds: The term "specifically (or selectively) binds" to an antigen or epitope refers to a binding reaction that determines the presence of the cognate antigen or epitope in a heterogeneous population of proteins and other molecules. The binding reaction can be, but need not be, mediated by an antibody or antibody fragment. The term "specifically binds" 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 does not in itself alter 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 Macaca fascicularis, Macaca mulatta, and Macaca nemestrina), or rodent species, e.g., Mus musculus.

[0054] Subject: The term "subject" includes human 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.

[0055] Substrate: The term "substrate" refers to a peptide sequence on which a protease acts and in which the protease cleaves a peptide bond. Target molecule: As used herein, the term "target molecule" refers to any biomolecule (e.g., a protein, carbohydrate, lipid, or combination thereof) expressed on a cell surface or in the extracellular matrix that can be specifically bound by the targeting moiety of an IFN proprotein of the present disclosure.

[0056] Targeting moiety: As used herein, the term "targeting moiety" refers to any molecule or binding portion thereof (e.g., an immunoglobulin or antigen-binding fragment) that can bind to a cell surface or extracellular matrix molecule at a site where an IFN proprotein of the present disclosure is to be localized, e.g., on a tumor cell or lymphocyte in the tumor microenvironment. In some embodiments, the targeting moiety binds to a TAA. In other embodiments, the targeting moiety binds to a TCA. A targeting moiety may also have functional activity in addition to localizing an IFN proprotein to a particular site. For example, a targeting moiety that binds to a checkpoint inhibitor such as PD1 may also exhibit anti-tumor activity or enhance IFN-mediated anti-tumor activity, e.g., by inhibiting PD1 signaling.

[0057] T cell antigen, TCA: The term "T cell antigen" or "TCA" refers to a molecule (typically a protein, carbohydrate, lipid, or combination thereof) that is expressed on the surface of a T lymphocyte and is useful for preferential targeting of an agent to a specific site. In some embodiments, the site is cancerous tissue and / or the T cell antigen is a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or viral lymphocyte, or a checkpoint inhibitor expressed on a T lymphocyte.

[0058] Tumor: The term "tumor" is used interchangeably herein with the term "cancer", e.g., both terms encompass solid and liquid, e.g., diffuse or circulating, tumors. As used herein, the term "cancer" or "tumor" includes pre-cancerous and malignant cancers and tumors.

[0059] Tumor-associated antigen, TAA: The term "tumor-associated antigen" or "TAA" refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) that is expressed on the surface of cancer cells, either in whole or as a fragment (e.g., MHC / peptide), and that is useful for preferential targeting of pharmacological agents to cancer cells. In some embodiments, a TAA is a marker, e.g., a lineage marker, that is expressed by both normal and cancer cells. In some embodiments, a TAA is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, e.g., by 1-fold overexpression, 2-fold overexpression, 3-fold overexpression, or more compared to normal cells. In some embodiments, a TAA is a cell surface molecule that is inappropriately synthesized in cancer cells, e.g., a molecule that contains a deletion, addition, or mutation compared to the molecule expressed on normal cells. In some embodiments, a TAA is expressed entirely or as a fragment (e.g., MHC / peptide) only on the cell surface of cancer cells and is not synthesized or expressed on the surface of normal cells. Thus, the term "TAA" encompasses antigens specific to cancer cells, sometimes known in the art as tumor-specific antigens ("TSAs").

[0060] Treat, Treatment, Treating: As used herein, the terms "treat," "treatment," and "treating" refer to a reduction or amelioration of the progression, severity, and / or duration of a disorder, or an amelioration of one or more symptoms (preferably one or more discernible symptoms) of the disorder, resulting from the administration of one or more IFN receptor agonists (e.g., IFN proproteins capable of agonizing an IFN receptor, e.g., after activation) of the present disclosure. 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 the proliferative disorder, such as tumor growth, not necessarily discernible by the patient. In other embodiments, the terms "treat," "treatment," and "treating" refer to an inhibition of the progression of the proliferative disorder, either physical, e.g., by stabilization of a discernible symptom, physiological, e.g., by stabilization of a physical parameter, or both. In other embodiments, the terms "treat," "treatment," and "treating" refer to a reduction or stabilization of tumor size or cancerous cell number.

[0061] Universal Light Chain, UCL: As used herein, the term "universal light chain" or "ULC" refers to a light chain variable region (VL) that can pair with more than one heavy chain variable region (VL). In the context of a targeting moiety, the term "universal light chain" or "ULC" refers to a light chain polypeptide that can pair with the heavy chain region of the targeting moiety and can also pair with other heavy chain regions. The ULC may also include a constant domain, e.g., the CL domain of an antibody. The universal light chain is also known as a "common light chain."

[0062] VH: The term "VH" refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, dsFv, or Fab. VL: The term "VL" refers to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv, or Fab.

[0063] IFN Proprotein The present disclosure relates to IFN proproteins containing an IFN moiety attenuated by steric hindrance of an adjacent constant domain. The IFN proprotein is configured such that upon encountering a protease, e.g., a protease overexpressed in a tumor environment, a protease-cleavable linker is cleaved to release the IFN. This is achieved by incorporating the IFN moiety between the constant domain and Fc domain of an antibody adjacent to a protease-cleavable linker (PCL). Thus, these proproteins are sometimes referred to as "internal" IFN constructs. For example, an IFN proprotein that can agonize an IFN receptor after activation is sometimes referred to herein as an "IFN receptor agonist."

[0064] Generally, an IFN proprotein is composed of two half antibodies comprising a pair of Fc domains that associate to form an Fc region (typically including a hinge sequence), a linker at the N-terminus that may be cleavable or non-cleavable, an IFN moiety, and an additional linker that may be cleavable or non-cleavable. In some embodiments, each half antibody comprises two protease-cleavable linkers flanking the IFN moiety. Exemplary IFN proproteins with two protease-cleavable linkers in each half antibody are illustrated in Figures 1A-1C. In other embodiments, each half antibody comprises a single protease-cleavable linker on one side of the IFN moiety, and the linker on the other side of the IFN moiety is a non-cleavable linker. Exemplary IFN proproteins with one protease-cleavable linker in each half antibody are illustrated in Figures 2A-2F. Further N-terminal to the protease-cleavable linker is an antibody constant domain, which may be either an entire constant domain including the Fc domain that associates to form a separate Fc region, or only a portion of the constant domain (e.g., the CH1 domain).

[0065] Exemplary configurations of IFN proproteins of the present disclosure are shown in Figures 1A-1C and 2A-2F. As illustrated, the IFN moieties and surrounding linkers can include hinge domains only at their C-termini (e.g., the "single hinge" illustrated in Figures 1A, 1B, 2A, 2B, 2D, and 2E) or at both their N- and C-termini (e.g., the "double hinge" illustrated in Figures 1C, 2C, and 2F).

[0066] Generally, IFN proproteins: a) a first polypeptide chain, i) a first immunoglobulin constant domain; ii) a first linker; iii) a first type I interferon (IFN) moiety; iv) a second linker, and v) a first polypeptide chain comprising a first Fc domain; and b) a second polypeptide chain, i) a second immunoglobulin constant domain; ii) a third linker; iii) a second type I interferon (IFN) moiety; iv) a fourth linker, and v) a second polypeptide chain comprising a second Fc domain that associates with the first Fc domain to form an Fc region.

[0067] 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.

[0068] Preferably, the IFN moiety in the IFN proprotein is sterically hindered from binding to the IFN receptor by the Fc domain and / or the constant domain. An exemplary IFN proprotein is shown in Figure 1A, where the targeting moiety (shown as a Fab domain represented by VH-CH1 paired with VL-CL) is optional. The IFN proprotein of Figure 1A comprises a first polypeptide chain, a second polypeptide chain, an optional third polypeptide chain, and an optional fourth polypeptide chain, a) the first polypeptide chain comprises: i) an optional first VH1 domain; ii) the first CH1 domain; iii) a first Fc domain comprising a hinge domain, a CH2 domain, and a CH3 domain, wherein the CH3 domain is a first immunoglobulin constant domain; iv) a first protease-cleavable linker (PCL); v) a first type I interferon (IFN) moiety; vi) a second protease-cleavable linker (PCL), and vii) comprises a second Fc domain; b) the second polypeptide chain comprises: i) an optional second VH1 domain; ii) a second CH1 domain; iii) a third Fc domain comprising a hinge domain, a CH2 domain, and a CH3 domain, wherein the CH3 domain is a second immunoglobulin constant domain; iv) a third protease-cleavable linker (PCL); v) a second type I interferon (IFN) moiety; vi) a fourth protease-cleavable linker (PCL), and vii) a fourth Fc domain; c) the optional third polypeptide chain is i) a first VL domain; ii) comprises a first CL domain; d) the fourth polypeptide chain is i) a second VL domain, and ii) comprises a second CL domain; The first polypeptide chain is associated 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.

[0069] If 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.

[0070] Thus, the first polypeptide chain (together with the third polypeptide chain, if present) represents a first half antibody, and the second polypeptide chain (together with the fourth polypeptide chain, if present) represents a second half antibody.

[0071] Another exemplary IFN proprotein is shown in Figure IB, where the targeting moiety (shown as a Fab domain represented by VH-CH1 paired with VL-CL) is optional. The IFN proprotein of Figure IB comprises a first polypeptide chain, a second polypeptide chain, an optional third polypeptide chain, and an optional fourth polypeptide chain, a) the first polypeptide chain comprises: i) an optional first VH1 domain; ii) the first CH1 domain; iii) a first protease-cleavable linker (PCL); iv) a first type I interferon (IFN) moiety; v) a second protease-cleavable linker (PCL), and vi) comprises a first Fc domain; b) the second polypeptide chain comprises: i) an optional second VH1 domain; ii) a second CH1 domain; iii) a third protease-cleavable linker (PCL); iv) a second type I interferon (IFN) moiety; v) a fourth protease-cleavable linker (PCL), and vi) comprises a second Fc domain; c) the optional third polypeptide chain is i) a first VL domain; ii) comprises a first CL domain; d) the fourth polypeptide chain is i) a second VL domain, and ii) comprises a second CL domain; 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.

[0072] If 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.

[0073] Thus, the first polypeptide chain (together with the third polypeptide chain, if present) represents a first half antibody, and the second polypeptide chain (together with the fourth polypeptide chain, if present) represents a second half antibody.

[0074] A variant of the IFN proprotein of Figure 1B is shown in Figure 1C. The IFN proprotein of Figure 1C 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 third protease-cleavable linker.

[0075] Another exemplary IFN proprotein is shown in Figure 2A, where the targeting moiety (shown as a Fab domain represented by VH-CH1 paired with VL-CL) is optional. The IFN proprotein of Figure 2A comprises a first polypeptide chain, a second polypeptide chain, an optional third polypeptide chain, and an optional fourth polypeptide chain, a) the first polypeptide chain comprises: i) an optional first VH1 domain; ii) the first CH1 domain; iii) a first Fc domain comprising a hinge domain, a CH2 domain, and a CH3 domain, wherein the CH3 domain is a first immunoglobulin constant domain; iv) a first non-cleavable linker (NCL); v) a first type I interferon (IFN) moiety; vi) a first protease-cleavable linker (PCL), and vii) comprises a second Fc domain; b) the second polypeptide chain comprises: i) an optional second VH1 domain; ii) a second CH1 domain; iii) a third Fc domain comprising a hinge domain, a CH2 domain, and a CH3 domain, wherein the CH3 domain is a second immunoglobulin constant domain; iv) a second non-cleavable linker (NCL); v) a second type I interferon (IFN) moiety; vi) a second protease-cleavable linker (PCL), and vii) a fourth Fc domain; c) the optional third polypeptide chain is i) a first VL domain; ii) comprises a first CL domain; d) the fourth polypeptide chain is i) a second VL domain, and ii) comprises a second CL domain; The first polypeptide chain is associated 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.

[0076] If 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.

[0077] Thus, the first polypeptide chain (together with the third polypeptide chain, if present) represents a first half antibody, and the second polypeptide chain (together with the fourth polypeptide chain, if present) represents a second half antibody.

[0078] Another exemplary IFN proprotein is shown in Figure 2B, where the targeting moiety (shown as a Fab domain represented by VH-CH1 paired with VL-CL) is optional. The IFN proprotein of Figure 2B comprises a first polypeptide chain, a second polypeptide chain, an optional third polypeptide chain, and an optional fourth polypeptide chain, a) the first polypeptide chain comprises: i) an optional first VH1 domain; ii) the first CH1 domain; iii) a first non-cleavable linker (NCL); iv) a first type I interferon (IFN) moiety; v) a first protease-cleavable linker (PCL), and vi) comprises a first Fc domain; b) the second polypeptide chain comprises: i) an optional second VH1 domain; ii) a second CH1 domain; iii) a second non-cleavable linker (NCL); iv) a second type I interferon (IFN) moiety; v) a second protease-cleavable linker (PCL), and vi) comprises a second Fc domain; c) the optional third polypeptide chain is i) a first VL domain; ii) comprises a first CL domain; d) the fourth polypeptide chain is i) a second VL domain, and ii) comprises a second CL domain; 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.

[0079] If 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.

[0080] Thus, the first polypeptide chain (together with the third polypeptide chain, if present) represents a first half antibody, and the second polypeptide chain (together with the fourth polypeptide chain, if present) represents a second half antibody.

[0081] A variant of the IFN proprotein of Figure 2B is shown in Figure 2C. The IFN proprotein of Figure 2C further comprises a first hinge domain between the first CH1 domain and the first protease non-cleavable linker, and a second hinge domain between the second CH1 domain and the second protease non-cleavable linker.

[0082] Another exemplary IFN proprotein is shown in Figure 2D, where the targeting moiety (shown as a Fab domain represented by VH-CH1 paired with VL-CL) is optional. The IFN proprotein of Figure 2D comprises a first polypeptide chain, a second polypeptide chain, an optional third polypeptide chain, and an optional fourth polypeptide chain, a) the first polypeptide chain comprises: i) an optional first VH1 domain; ii) the first CH1 domain; iii) a first Fc domain comprising a hinge domain, a CH2 domain, and a CH3 domain, wherein the CH3 domain is a first immunoglobulin constant domain; iv) a first protease-cleavable linker (PCL); v) a first type I interferon (IFN) moiety; vi) a first non-cleavable linker (NCL), and vii) comprises a second Fc domain; b) the second polypeptide chain comprises: i) an optional second VH1 domain; ii) a second CH1 domain; iii) a third Fc domain comprising a hinge domain, a CH2 domain, and a CH3 domain, wherein the CH3 domain is a second immunoglobulin constant domain; iv) a second protease-cleavable linker (PCL); v) a second type I interferon (IFN) moiety; vi) a second non-cleavable linker (NCL), and vii) a fourth Fc domain; c) the optional third polypeptide chain is i) a first VL domain; ii) comprises a first CL domain; d) the fourth polypeptide chain is i) a second VL domain, and ii) comprises a second CL domain; The first polypeptide chain is associated 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.

[0083] If 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.

[0084] Thus, the first polypeptide chain (together with the third polypeptide chain, if present) represents a first half antibody, and the second polypeptide chain (together with the fourth polypeptide chain, if present) represents a second half antibody.

[0085] Another exemplary IFN proprotein is shown in Figure 2E, where the targeting moiety (shown as a Fab domain represented by VH-CH1 paired with VL-CL) is optional. The IFN proprotein of Figure 2E comprises a first polypeptide chain, a second polypeptide chain, an optional third polypeptide chain, and an optional fourth polypeptide chain, a) the first polypeptide chain comprises: i) an optional first VH1 domain; ii) the first CH1 domain; iii) a first protease-cleavable linker (PCL); iv) a first type I interferon (IFN) moiety; v) a first non-cleavable linker (NCL), and vi) comprises a first Fc domain; b) the second polypeptide chain comprises: i) an optional second VH1 domain; ii) a second CH1 domain; iii) a second protease-cleavable linker (PCL); iv) a second type I interferon (IFN) moiety; v) a second non-cleavable linker (NCL), and vi) comprises a second Fc domain; c) the optional third polypeptide chain is i) a first VL domain; ii) comprises a first CL domain; d) the fourth polypeptide chain is i) a second VL domain, and ii) comprises a second CL domain; 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.

[0086] If 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.

[0087] Thus, the first polypeptide chain (together with the third polypeptide chain, if present) represents a first half antibody, and the second polypeptide chain (together with the fourth polypeptide chain, if present) represents a second half antibody.

[0088] A variant of the IFN proprotein of Figure 2E is shown in Figure 2F. The IFN proprotein of Figure 2E 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.

[0089] Typically, the Fc domain in the IFN proproteins shown in Figures 1A-1C and 2A-2F includes a hinge domain. Thus, IFN proproteins typically contain two to four protease-cleavable linkers. Cleavage of all protease-cleavable linkers in an IFN proprotein with four protease-cleavable linkers results in the release of an activated IFN protein containing the IFN moiety and lacking the C-terminal Fc moiety, the N-terminal constant domain, and, if present, the targeting moiety. Cleavage of all protease-cleavable linkers in an IFN proprotein with two protease-cleavable linkers removes one of the sterically hindering moieties and yields an IFN molecule containing a targeting moiety (antibody-IFN) or an Fc domain (IFN-Fc).

[0090] In some embodiments, this configuration is advantageously utilized for IFN proproteins that include a targeting moiety that binds to a TAA or ECM target molecule expressed in the tumor environment. Without intending to be bound by theory, the inventors believe that in this configuration, the targeting moiety targets the IFN proprotein to the tumor environment, where a protease cleaves the protease-cleavable linker, resulting in the release of the IFN protein that includes the IFN moiety and the linker sequence. This locally activated IFN protein then induces an immune response against cancer cells.

[0091] The sequence and length of the hinge and linker sequences can vary, as can the sequence of the IFN moiety (including either a full-length IFN sequence or an N-terminally and / or C-terminally truncated IFN sequence). Exemplary IFN moieties are described in Section 6.3, including the IFNα-based moieties and IFNβ-based moieties 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 moieties are disclosed in Section 6.6.

[0092] 6.3. IFN part There are two major classes of IFNs: 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.

[0093] The IFN moiety of the present disclosure can include any wild-type or modified (e.g., truncated and / or mutated) IFN or IFN-like cytokine sequence, but is preferably a type I IFN moiety. Type I IFNs bind to IFNAR, a heterodimeric plasma membrane receptor composed of IFNAR1 and IFNAR2, which is ubiquitously 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 IFNs can act on virtually all cells in the body. Sixteen type I interferon subtypes have been identified, which differ in their inherent variability in affinity and activity for IFNAR2.

[0094] 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.

[0095] In other embodiments, the type I IFN moiety is an interferon-ω (IFNω), interferon-ε (IFNε), or interferon-κ (IFNκ) moiety. The type I IFN portion may contain one or more mutations, e.g., substitutions, deletions, or insertions, that differ from the wild-type IFN sequence. Substitutions that reduce receptor binding and thereby attenuate IFN activity may be suitably used. N- or C-terminal deletions (or truncations), e.g., amino acids having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids truncated from the N- and / or C-terminus of mature type I IFN, may also be used. Without being bound by theory, the inventors believe that the truncations impose additional steric constraints on the IFN portion, reducing IFN activity until cleavage of the protease-cleavable linker within the IFN proprotein.

[0096] Further details of exemplary type I IFN moieties are provided below. 6.3.1. Interferon-α Moiety The IFNα gene is a member of the alpha interferon gene cluster on chromosome 9. The encoded cytokine is a member of the type I interferon family, produced in response to viral infection as an important part of the innate immune response with potent antiviral, antiproliferative, and immunomodulatory properties. IFNα refers to a family of proteins with at least 15 known subtypes of human IFNα. The major identified subtypes are IFNα1, IFNα2, IFNα8, IFNα10, IFNα14, and IFNα21.

[0097] The IFNα1 gene has two allelic variants: IFNα1a and IFNα1b. The amino acid sequence of human IFNα1a has been assigned UniProtKB accession number P01562 and is reproduced below, with the signal peptide underlined.

[0098] [ka]

[0099] The human IFNα1b gene differs from the IFNα1a allelic variant by a single base change in the coding region that results 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).

[0100] The IFNα2 allele has three allelic variants: IFNα2a, IFNα2b, and IFNα2c. The IFNα2b allele is the predominant allele, while the IFNα2a allele is less predominant and the IFNα2c allele is only a minor allelic variant. The amino acid sequence of human IFNα2 has been assigned UniProtKB accession number P01563. The sequence of the IFNα2b allele is reproduced below, with the signal peptide underlined.

[0101] [ka]

[0102] IFNα2b has an arginine (R) at position 23 of the mature protein, and IFNα2a has a lysine (K). Thus, in some embodiments, the IFNα2 portion has an arginine at the position corresponding to position 23 of the mature protein. In other embodiments, the IFNα2 portion has a lysine at the position corresponding to position 23 of the mature protein.

[0103] In various aspects, the IFNα portion 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 IFNα1a, IFNα1b, and / or IFNα2b, IFNα2a, or IFNα2c, or a fragment thereof, having a truncation of up to 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).

[0104] In some embodiments, the IFNα portion has one or more amino acid substitutions, e.g., substitutions that alter IFNAR binding and / or agonism. Exemplary substitutions are found in WO2013 / 107791, U.S. Patent No. 8,258,263, WO2007 / 000769A2, WO2008 / 124086, WO2010 / 030671, WO2018 / 144999A1, and WO2015 / 007520, WO2013 / 059885, WO2020156467A1, WO2021 / 126929A1. In some embodiments, the IFNα portion comprises: a)L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K or R33A or R33Q, H34A, D35A, Q40A, H57Y, E58N, Q61S, F64A, N65A, T69A, L80A, D82E, Y85A, T86I, Y89A, D one or more substitutions selected from 114R or D114A, L117A, R120A or R120E or R120K, K121E, R125A, K133A, K134A, R144A, A145G or A145M, M148A, R149A, R149K, S152A, L153A, N156A, and / or b) one or more substitutions at amino acids 57 to 89 and 159 to 165 as described in WO2007 / 000769A2, and / or c) One or more amino acid substitutions at 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157 to alanine, glycine, or threonine as described in WO202 / 1126929A1.

[0105] The amino acid positions of the above substitutions are given with reference to mature IFNα2b. In further embodiments, the IFNα portion comprises one or more amino acid substitutions set forth in Table 1. Table 1 sets forth the IFNα substitutions identified with reference to the amino acid position within the sequence of IFNα2.

[0106] [Table 1-1]

[0107] [Table 1-2]

[0108] [Table 1-3]

[0109] In some embodiments, the IFNα portion comprises an amino acid sequence including 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, for example, Q90A+R120A or A145M+R149K.

[0110] Exemplary IFNα moiety sequences that can be utilized in the IFN proproteins of the present disclosure are set forth in Table 2 below.

[0111] [Table 2-1]

[0112] [Table 2-2]

[0113] 6.3.2. Interferon 1-β Moiety 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 that is produced in large quantities by fibroblasts, and is therefore also known as fibroblast interferon. IFN1β binds to the IFNAR receptor, which is composed of IFNAR1 and IFNAR2 dimers, and induces signal transduction through the JAK / STAT pathway and other pathways. IFN1β can also function by binding only to IFNAR1 and signaling independently of the JAK-STAT pathway (see, e.g., Stanifer et al., 2019, Int. J Mol. Sci. 20(6):1445).

[0114] IFN-1β contains five α-helices designated 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)). These five α-helices are interconnected by loops of 2 to 28 residues designated AB, BC, CD, and DE loops. The A helix of the AB loop and the E helix of the DE loop have been reported to be involved in the binding of IFN-1β to the IFN-α receptor.

[0115] Two types of IFN1β have been described: interferon 1-β1 (IFN1β1) and interferon 1-β3 (IFN1β3). The amino acid sequence of the human IFN-β precursor is listed under GenBank accession number AAA36040.1 and is reproduced below (with the signal peptide underlined).

[0116] [ka]

[0117] In various aspects, the IFNβ portion 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 IFN1β1 or a fragment thereof having a truncation of up to 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).

[0118] In various embodiments, the IFNβ portion comprises one or more amino acid substitutions and / or deletions relative to IFN1β1. In some embodiments, the substitution is C17S (with reference to mature IFN1β1) and the deletion is one of the C-terminal truncations described in US 2009 / 0025106 A1 as IFN-ΔI, IFNA2, IFNA3, IFNA4, IFNA5, IFNA6, IFN-Δ7, IFN-Δδ, IFNA9, and IFN-ΔIO. Other Type I Interferons In certain embodiments, the type I IFN moiety is other than an IFNα or IFNβ moiety, eg, an interferon-ω (IFNω), interferon-ε (IFNε), or interferon-κ (IFNκ) moiety.

[0119] Human IFNω is identified by UniProt accession number P05000, and the IFNω1 allele has the amino acid sequence set forth below, with the signal sequence underlined.

[0120] [ka]

[0121] In various embodiments, the IFNω portion 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 IFN1ω1 or a fragment thereof having a truncation of up to 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).

[0122] Human IFNε is identified by UniProt accession number Q86WN2 and has the amino acid sequence set forth below, with the signal sequence underlined.

[0123] [ka]

[0124] In various embodiments, the IFNε portion 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 IFNε or a fragment thereof having a truncation of up to 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 IFNε).

[0125] Human IFNκ is identified by UniProt accession number Q9P0W0 and has the amino acid sequence set forth below, with the signal sequence underlined.

[0126] [ka]

[0127] In various embodiments, the IFNκ portion 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 IFNκ or a fragment thereof having a truncation of up to 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 IFNκ).

[0128] 6.4. Protease-Cleavable Linkers The IFN proproteins of the present disclosure typically include four linkers, referred to in the numbered embodiments below as a first linker, a second linker, a third linker, and a fourth linker, with the first and second linkers on one polypeptide chain and the third and fourth linkers on another polypeptide chain. Two to four of these linkers are protease-cleavable linkers. In some embodiments, the first, second, third, and fourth linkers are all protease-cleavable linkers. In other embodiments, only two of the first, second, third, and fourth linkers are protease-cleavable linkers. For example, in certain embodiments, the first and third linkers are protease-cleavable linkers, and the second and fourth linkers 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.

[0129] Protease-cleavable linkers can range from 8 to 100 or more amino acids, in various embodiments, protease-cleavable linkers range from 8 to 15, 10 to 20, or 20 to 80 amino acids, and in certain aspects, non-cleavable peptide linkers range from 20 to 60, 20 to 40, 30 to 50, 20 to 80, or 30 to 70 amino acids in length.

[0130] A protease-cleavable linker comprises one or more substrate sequences for one or more proteases, e.g., one or more of the proteases described in Section 6.4.1. The one or more substrate sequences, e.g., one or more of the substrate sequences described in Section 6.4.2, are typically (but not necessarily) flanked by one or more spacer sequences, e.g., spacer sequences described in Section 6.4.3. Each protease-cleavable linker can comprise one, two, three, or more substrate sequences. The spacer sequences may be adjacent, overlapping, or separated by spacer sequences. Preferably, the C-terminus and N-terminus of the protease-cleavable linker comprise spacer sequences.

[0131] In various aspects of the IFN proprotein comprising four protease-cleavable linkers, the first and third protease-cleavable linkers are cleavable by the same protease, and / or the second and fourth protease-cleavable linkers are cleavable by the same protease. In some embodiments, the protease is a protease listed in Table A.

[0132] In further aspects of IFN proproteins comprising four protease-cleavable linkers, the first and third protease-cleavable linkers comprise the same substrate sequence(s), and / or the second and fourth protease-cleavable linkers comprise the same substrate sequence(s). In some embodiments, the substrate sequence(s) are set forth in Table B. In further embodiments, the first and third protease-cleavable linkers also comprise the same spacer sequence(s), and / or the second and fourth protease-cleavable linkers also comprise the same spacer sequence(s). In some embodiments, the spacer sequence(s) are set forth in Table C.

[0133] In further aspects of the IFN proprotein comprising four protease-cleavable linkers, the first and third linkers comprise the same linker sequence(s) and / or the second and fourth linkers comprise the same linker sequence(s). In some embodiments, the linker sequence(s) are set forth in Table D.

[0134] In some embodiments of the IFN proprotein comprising four protease-cleavable linkers, the first and third protease-cleavable linkers are the same as the second and fourth protease-cleavable linkers.

[0135] In other embodiments, the first and third protease-cleavable linkers are different from the second and fourth protease-cleavable linkers. In the foregoing aspects and embodiments, the different linkers may be cleavable by the same protease, may be cleavable by different proteases, or, if the linkers comprise multiple substrate sequences, may be cleavable by multiple proteases, one or more of which are in common and one or more of which are different.

[0136] Exemplary protease-cleavable linker sequences are described in Section 6.4.4. Proteases Exemplary proteases whose substrate sequences can be incorporated into a protease-cleavable linker are set forth in Table A below.

[0137] [Table 3]

[0138] In certain embodiments, the protease is selected from the group consisting of matrix metalloproteinase (MMP)-2, MMP-9, legman asparaginyl endopeptidase, thrombin, fibroblast-activating 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 proteases, serine (TMPRSS-3 / 4), cathepsin, and the like. 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 uPA, a disintegrin and metalloprotease (ADAM) 10, ADAM12, ADAM17, ADAMs with thrombospondin motifs (ADAMTS), ADAMTS5, beta-secretase (BACE), granzyme A, granzyme B, guanidinobenzoatase, hepsin, matriptase, matriptase 2, meprin, neprilysin, prostate-specific membrane antigen (PSMA), tumor necrosis These include factor-converting enzyme (TACE), kallikrein-related peptidase (KLK) 3, KLK5, KLK7, KLK11, hepatitis C virus NS3 / 4 protease (HCV-NS3 / 4), tissue plasminogen activator (tPA), calpain, calpain 2, glutamate carboxypeptidase II, plasma kallikrein, AMSH-like protease, AMSH, gamma-secretase component, antiplasmin cleaving enzyme (APCE), decysin 1, apoptosis-associated cysteine ​​peptidase, or N-acetylated alpha-linked acidic dipeptidase-like 1.

[0139] 6.4.2. Substrate Exemplary substrate sequences that are cleavable by tumor proteases and that can be incorporated into protease-cleavable linkers are set forth in Table B below.

[0140] [Table 4-1]

[0141] [Table 4-2]

[0142] [Table 4-3]

[0143] [Table 4-4]

[0144] [Table 4-5]

[0145] Spacer Exemplary spacer sequences that can be incorporated into a protease-cleavable linker are set forth below in Table C. In addition to the spacer sequences set forth in Table C, any of the non-cleavable linker sequences set forth in Section 6.5, such as the non-cleavable linker sequences set forth in Table E, or portions thereof, can be used as spacer sequences. In some embodiments, no spacer sequences are present in the protease-cleavable linker at all.

[0146] [Table 5]

[0147] In some embodiments, when used in Table C above, n is an integer between 1 and 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. 6.4.4. Exemplary Protease-Cleavable Linkers Exemplary protease-cleavable linkers comprising one or more substrate sequences and a spacer sequence are set forth in Table D below.

[0148] [Table 6-1]

[0149] [Table 6-2]

[0150] [Table 6-3]

[0151] [Table 6-4]

[0152] In certain aspects, the protease-cleavable linker comprises an amino acid sequence having at most 5, at most 4, at most 3, at most 2, or at most 1 amino acid substitution(s) compared to the sequence set forth in Table D. Thus, in some embodiments, the protease-cleavable linker comprises or consists of any of the amino acid sequences of Table D having from 1 to 5 amino acid substitutions compared to the sequence set forth in Table D.

[0153] 6.5. Non-Cleavable Linkers In certain aspects, the present disclosure provides IFN proproteins in which two or more components of the IFN proprotein are connected to each other by a peptide linker. By way of example and not limitation, a linker can be used to connect the Fc domain and the targeting moiety, or different domains within the targeting moiety (e.g., the VH and VL domains in an scFv).

[0154] Preferably, all linkers in the IFN proprotein other than the protease-cleavable linker, the cleavage of which results in activation of the IFN proprotein, are non-cleavable linkers (NCLs).

[0155] The non-cleavable linker can range from 2 to 60 or more amino acids, and in certain embodiments, the non-cleavable linker is in the range of 3 to 50 amino acids in length, 4 to 30 amino acids in length, 5 to 25 amino acids in length, 10 to 25 amino acids in length, 10 to 60 amino acids in length, 12 to 20 amino acids in length, 20 to 50 amino acids in length, or 25 to 35 amino acids in length.

[0156] In certain embodiments, 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 up to 30 amino acids in length, up to 40 amino acids in length, up to 50 amino acids in length, or up to 60 amino acids in length.

[0157] In some of the foregoing embodiments, the non-cleavable linker is between 5 and 50 amino acids in length, e.g., between 5 and 50, 5 and 45, 5 and 40, 5 and 35, 5 and 30, 5 and 25, or 5 and 20 amino acids in length. In other of the foregoing embodiments, the non-cleavable linker is between 6 and 50 amino acids in length, e.g., between 6 and 50, 6 and 45, 6 and 40, 6 and 35, 6 and 30, 6 and 25, or 6 and 20 amino acids in length. In still other of the foregoing embodiments, the non-cleavable linker is between 7 and 50 amino acids in length, e.g., between 7 and 50, 7 and 45, 7 and 40, 7 and 35, 7 and 30, 7 and 25, or 7 and 20 amino acids in length.

[0158] Charged (eg, charged hydrophilic linkers) and / or flexible non-cleavable linkers are particularly preferred. Examples of flexible linkers that can be used in the IFN proproteins of the present disclosure include those disclosed in 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 those consisting of repeats of glycine and serine, e.g., G n S (SEQ ID NO: 299) or SG n (SEQ ID NO: 300), where n is an integer between 1 and 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the non-cleavable linker is, or comprises, a monomer or multimer of, for example, (GGGGS) n (SEQ ID NO: 301).

[0159] Polyglycine non-cleavable linkers are suitable for use in the IFN proproteins of the present disclosure. In some embodiments, the peptide non-cleavable linker comprises two consecutive glycines (2 Gly), three consecutive glycines (3 Gly), four consecutive glycines (4 Gly (SEQ ID NO: 302)), five consecutive glycines (5 Gly (SEQ ID NO: 303)), six consecutive glycines (6 Gly (SEQ ID NO: 304)), seven consecutive glycines (7 Gly (SEQ ID NO: 305)), eight consecutive glycines (8 Gly (SEQ ID NO: 306)), or nine consecutive glycines (9 Gly (SEQ ID NO: 307)).

[0160] Exemplary non-cleavable linker sequences are listed in Table E below.

[0161] [Table 7-1]

[0162] [Table 7-2]

[0163] In certain aspects, the IFN proproteins of the present disclosure can comprise a polypeptide chain comprising, in N-terminal to C-terminal orientation, a targeting moiety (or targeting moiety chain), a hinge domain, a CH2 domain, and a CH3 domain (e.g., as shown in Figure 1A), or a polypeptide chain comprising a targeting moiety (or targeting moiety chain), a hinge domain, followed by a protease-cleavable linker (e.g., as shown in Figure 1C). Thus, the hinge domain can be said to constitute a type of linker. Exemplary hinge domains are described in Section 6.8.3.

[0164] 6.6. Targeting part Incorporation of a targeting moiety into the IFN proproteins of the present disclosure allows for the delivery of high concentrations of IFN to the tumor microenvironment while simultaneously reducing systemic exposure and causing fewer side effects than those obtained with non-targeted IFN.

[0165] It is anticipated that any type of target molecule that is present in a particular locality or tissue or that can drive an IFN proprotein can be targeted by the IFN proproteins of the present disclosure. In some embodiments, the IFN proproteins are 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 moiety of the IFN proproteins of the present disclosure are generally found, for example, on the surface of activated T cells, tumor cells, dendritic cells or other antigen-presenting cells, natural killer (NK) cells, virus-infected cells, other diseased cells, free in serum, in the extracellular matrix (ECM), or in immune cells present at the target site, such as tumor-reactive lymphocytes.

[0166] 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 viral lymphocyte, a T-cell antigen ("TCA"), or a checkpoint inhibitor, a tumor-associated antigen ("TAA"), a dendritic cell (DC) antigen or other antigen-presenting cell (APC) antigen, or a natural killer (NK) cell antigen. Those skilled in the art will recognize that the foregoing categories of target molecules are not mutually exclusive, and thus, a given target molecule may fall into more than one of the foregoing categories of target molecule. For example, some molecules may be considered both a TAA and an ECM protein, and other molecules may be considered both a TCA and a checkpoint inhibitor.

[0167] Exemplary types of cancer that can be targeted include acute lymphoblastic leukemia, acute myeloid leukemia, biliary tract cancer, B-cell leukemia, B-cell lymphoma, biliary tract cancer, bone cancer, brain cancer, breast cancer, triple-negative breast cancer, cervical cancer, Burkitt's lymphoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gallbladder cancer, stomach cancer, gastrointestinal tract cancer, glioma, hairy cell leukemia, head and neck cancer, Hodgkin's lymphoma, liver cancer, lung cancer, medullary thyroid cancer, melanoma, multiple myeloma, ovarian cancer, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, lung duct cancer, renal cancer, sarcoma, skin cancer, testicular cancer, urothelial cancer, and other bladder cancer. However, those skilled in the art will recognize that TAAs and other target molecules associated with the tumor microenvironment are known for virtually any type of cancer.

[0168] Non-limiting examples of ECM antigens include syndecans, heparanase, integrins, osteopontin, link, cadherins, laminins, laminin-type EGFs, lectins, fibronectin, notch, nectins (e.g., nectin-4), tenascins, collagens (e.g., type X collagen), and matrixins.

[0169] Other target molecules are cell surface molecules of tumor or viral lymphocytes, for example, T cell costimulatory 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.

[0170] In certain embodiments, the target molecule is a checkpoint inhibitor, e.g., CTLA-4, PD1, PDL1, PDL2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, or CHK2. In certain embodiments, the target molecule is PD1. In other embodiments, the target molecule is LAG3. In yet other embodiments, the target molecule is PDL1.

[0171] In certain embodiments, the target molecule is present on the surface of a dendritic cell or other antigen-presenting cell, such as XCR1, Clec9a, CD1c, CD11c, CD14, PDL1, macrophage mannose receptor (CD206), and DEC-205.

[0172] In further embodiments, the target molecule is present on the surface of natural killer (NK) cells, such as CD335, CD38, CD2, NKG2D, NKp44, NKp30, CD16, LFA-1, CD27, KIR, NKH1A, and NKp46.

[0173] Antibodies and antigen-binding moieties generally bind to a specific antigenic determinant and can direct the IFN proprotein to a target site, for example, a specific tumor cell type or tumor stroma that bears the antigenic determinant. In certain embodiments, the targeting moiety 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 (TNCA2), fibronectin extra domain B (EDB), melanoma-associated chondroitin sulfate proteoglycan (MCSP), MART-1 / Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin 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-tumor antigen family (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7), MAGE-A8, MAGE-A9, MAGE-B10, MAGE-B20, MAGE-B30, MAGE-B40, MAGE-B50, MAGE-B60, MAGE-B70, MAGE-B80, MAGE-B90, MAGE-B110, MAGE-B120, MAGE-B130, MAGE-B140, MAGE-B150, MAGE-B160, MAGE-B170, MAGE-B180, MAGE-B200, MAGE-B210, MAGE-B220, MAGE-B230, MAGE-B240, MAGE-B250, MAGE-B300, MAGE-B410, MAGE-B420, MAGE-B530, MAGE-B640, MAGE-B750, MAGE-B850, MAGE-B900 -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-tumor antigen family (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, α-fetoprotein, E-cadherin, α-catenin, β-catenin, and γ-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig-idiotypes, p15, gp75, GM2, and GD2 gangliosides, viral products such as human papillomavirus proteins, the Smad family of tumor antigens, Imp-1, P1A, EBV-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 (TCR-associated) Heteromultimers that bind to the IL-6 receptor), 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 (β-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, β-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, prostase, prostase-specific antigen (PSA), PAP, LAGA-1a, p53, prostein, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, ephrin B2, insulin 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 the A1 domain of tenascin-C (TnC A1).

[0174] Suitable targeting moiety formats are described in Section 6.7. The targeting moiety is preferably an antigen-binding portion, e.g., 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.

[0175] In some embodiments, the targeting moiety targets exemplary target molecules set forth in Table F below, along with reference to exemplary antibodies or antibody sequences on which the targeting moiety can be based.

[0176] [Table 8-1]

[0177] [Table 8-2]

[0178] [Table 8-3]

[0179] [Table 8-4]

[0180] [Table 8-5]

[0181] [Table 8-6]

[0182] [Table 8-7]

[0183] [Table 8-8]

[0184]

Table 8-9

[0185]

Table 8-10

[0186]

Table 8-11

[0187]

Table 8-12

[0188]

Table 8-13

[0189]

Table 8-14

[0190]

Table 8-15

[0191]

Table 8-16

[0192]

Table 8-17

[0193]

Table 8-18

[0194] [Table 8-19]

[0195] In some aspects, the targeting moiety competes with an antibody listed in Table F for binding to the target molecule. In further aspects, the targeting moiety comprises a CDR having the CDR sequences of an antibody listed in Table F. In some embodiments, the targeting moiety comprises all six CDR sequences of an antibody 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 an antibody listed in Table F and the light chain CDR sequences of a universal light chain. In further aspects, the targeting moiety comprises a VH comprising the amino acid sequence of the VH of an antibody listed in Table F. In some embodiments, the targeting moiety further comprises a VL comprising the amino acid sequence of the VL of an antibody listed in Table F. In other embodiments, the targeting moiety further comprises a universal light chain VL sequence.

[0196] In some embodiments, the target molecule is PDL1. Table F-1 below provides exemplary anti-PDL1 antibodies and / or antibody sequences upon which targeting moieties can be based, for example, that can be incorporated into targeting moieties for use in the interferon proproteins of the present disclosure.

[0197] [Table 9-1]

[0198] [Table 9-2]

[0199] In some aspects, the targeting moiety competes with an anti-PDL1 antibody listed in Table F-1 for binding to PDL1. In further aspects, the targeting moiety comprises a CDR having the CDR sequence of an anti-PDL1 antibody listed in Table F-1. In some embodiments, the targeting moiety comprises all six CDR sequences of an anti-PDL1 antibody 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 an anti-PDL1 antibody listed in Table F-1 and the light chain CDR sequence of a universal light chain. In further aspects, the targeting moiety comprises a VH comprising the amino acid sequence of the VH of an anti-PDL1 antibody listed in Table F-1. In some embodiments, the targeting moiety further comprises a VL comprising the amino acid sequence of the VL of an anti-PDL1 antibody listed in Table F-1. In other embodiments, the targeting moiety further comprises a universal light chain VL sequence.

[0200] In some embodiments, the target molecule is PD1. Table F-2 below provides exemplary anti-PD1 antibodies and / or antibody sequences upon which targeting moieties can be based, for example, that can be incorporated into targeting moieties for use in the interferon proproteins of the present disclosure.

[0201] [Table 10-1]

[0202] [Table 10-2]

[0203] [Table 10-3]

[0204] [Table 10-4]

[0205] In some aspects, the targeting moiety competes for binding to PD1 with an anti-PD1 antibody listed in Table F-2. In further aspects, the targeting moiety comprises a CDR having the CDR sequence of an anti-PD1 antibody listed in Table F-2. In some embodiments, the targeting moiety comprises all six CDR sequences of an anti-PD1 antibody 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 an anti-PD1 antibody listed in Table F-2 and the light chain CDR sequence of a universal light chain. In further aspects, the targeting moiety comprises a VH comprising the amino acid sequence of the VH of an anti-PD1 antibody listed in Table F-2. In some embodiments, the targeting moiety further comprises a VL comprising the amino acid sequence of the VL of an anti-PD1 antibody listed in Table F-2. In other embodiments, the targeting moiety further comprises a universal light chain VL sequence.

[0206] When the targeting molecule is a checkpoint inhibitor, in some embodiments, the checkpoint inhibitor targeting moiety is non-blocking or poorly blocking of ligand-receptor binding. Examples of non-blocking or poorly blocking anti-PD1 antibodies include antibodies having the VH / VL amino acid sequences of SEQ ID NOs: 2 / 10 in PCT Publication No. WO2015 / 112800A1, SEQ ID NOs: 16 / 17 in U.S. Patent No. 11,034,765B2, and SEQ ID NOs: 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 in U.S. Patent No. 10,294,299B2. Examples of non-blocking or low-blocking anti-LAG3 antibodies include antibodies having the VH / VL amino acid sequences of SEQ ID NOs: 23 / 24, 3 / 4, and 11 / 12 of US Publication No. 2022 / 0056126A1.

[0207] Additional target molecules that can be targeted by IFN proproteins are disclosed below in Table I and are disclosed, for example, in Hafeez et al., 2020, Molecules 25:4764, doi:10.3390 / molecules25204764, particularly Table 1. Table 1 of Hafeez et al. is incorporated herein by reference in its entirety.

[0208] 6.7. Targeting Part Format In certain aspects, the targeting moiety of the IFN proprotein of the present disclosure can be any type of antibody or fragment thereof that retains specific binding to an antigenic determinant. In one embodiment, the antigen-binding moiety is an immunoglobulin molecule or fragment thereof, particularly an IgG class immunoglobulin molecule, more particularly an IgG1 or IgG4 immunoglobulin molecule. Antibody fragments include VH (or V H ) fragment, VL (or V L ) fragments, Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, minibodies, diabodies, triabodies, and tetrabodies.

[0209] 6.7.1.Fab Fab domains have traditionally been generated by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. Fab domains can comprise constant and variable region sequences from any suitable species and thus may be murine, chimeric, human, or humanized.

[0210] A Fab domain typically comprises a CH1 domain attached to a VH domain, which pairs with a CL domain attached to a 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. Disulfide bonds 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 at the N-terminus of the IFN moiety, for example, as shown in Figures 1B and 1C.

[0211] For the IFN proproteins of the present disclosure, particularly when the light chains of the targeting moieties are not common or universal light chains, it is advantageous to use a Fab heterodimerization strategy to allow correct association of Fab domains belonging to the same targeting moiety and 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.

[0212] [Table 11]

[0213] 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 one another, or by exchanging the CH1 and CL domains with one another, as described, for example, in WO2009 / 080251.

[0214] Correct Fab pairing can also be promoted by introducing one or more amino acid modifications in the CH1 domain and one or more amino acid modifications in the CL domain of the Fab, and / or by introducing one or more amino acid modifications in the VH domain and one or more amino acid modifications in the VL domain of the Fab. 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 other Fab components.

[0215] In one embodiment, the one or more amino acid modifications are limited to conserved framework residues of the variable (VH, VL) and constant (CH1, CL) domains, 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.

[0216] 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 in terms of lock and key fit, knob into hole, protrusion and cavity, donor and acceptor, etc., all of which suggest the nature of the structural and chemical match between two interacting surfaces.

[0217] In one embodiment, one or more of the introduced modifications introduce new hydrogen bonds across the interface of the Fab component. In one embodiment, one or more of the introduced modifications introduce new salt bridges across the interface of the Fab component. Exemplary substitutions are described in WO2014 / 150973 and WO2014 / 082179, the contents of which are incorporated herein by reference.

[0218] In some embodiments, the Fab domain comprises a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain, which introduces a salt bridge between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0219] 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 hydrophobic and polar contact regions between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0220] In some embodiments, the Fab domain can include modifications in some or all of the VH, CH1, VL, and CL domains to introduce an orthogonal Fab interface that promotes correct assembly of the Fab domain (Lewis et al., 2014 Nature Biotechnology 32:191-198). In embodiments, a 39K, 62E modification is introduced in the VH domain, an H172A, F174G modification is introduced in the CH1 domain, a 1R, 38D, (36F) modification is introduced in the VL domain, and an L135Y, S176W modification is 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.

[0221] Fab domains can also be modified to replace the native CH1:CL disulfide bond with an engineered disulfide bond, thereby increasing the efficiency of pairing of the Fab components. For example, an engineered disulfide bond can be introduced by introducing 126C into the CH1 domain and 121C into the CL domain (see, e.g., Mazor et al., 2015, Mabs 7:377-89).

[0222] Fab domains 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 a T cell receptor constant domain and the CL domain with a T cell receptor b domain, 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.

[0223] Alternatively, or in addition to using a Fab heterodimerization strategy to promote correct VH-VL pairing, a 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, using a common light chain as described herein reduces the number of incorrect 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 comprises a 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 in mouse B cells previously engineered to express a limited human light chain repertoire or a single human light chain cognate to a human heavy chain, generating an antibody repertoire containing multiple human VHs cognate to one of one or two possible human VLs in response to challenge with an antigen of interest, the antibody repertoire being 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, including somatically mutated (e.g., affinity matured) forms. See, e.g., U.S. Patent No. 10,412,940.

[0224] 6.7.2.scFv Single-chain Fv or "scFv" antibody fragments comprise the VH and VL domains of an antibody in a single polypeptide chain, can be expressed as single-chain polypeptides, and retain the specificity of the intact antibody from which they are derived. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for target binding. Examples of linkers suitable for connecting the VH and VL chains of an scFv are the non-cleavable linkers identified in Section 6.5.

[0225] As used herein, unless otherwise specified, an scFv may have a VL variable region and a VH variable region in either order, e.g., with respect to the N-terminus and C-terminus of the polypeptide, and may comprise a VL-linker-VH or a VH-linker-VL.

[0226] The scFv can comprise VH and VL sequences from any suitable species, such as murine, human, or humanized VH and VL sequences. To generate a nucleic acid encoding an scFv, DNA fragments encoding the VH and VL are operably linked to another fragment encoding a linker, for example, a fragment encoding any of the linkers described in Section 6.5 (typically, a repeat of a 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 contiguous single-chain protein with the VL and VH regions connected 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).

[0227] 6.8.Fc area The IFN proproteins of the present disclosure typically comprise a pair of Fc domains that associate to form an Fc region. In natural antibodies, Fc regions include a hinge region at their N-terminus to form a constant domain. Throughout this disclosure, unless otherwise specified, reference to an Fc domain includes an Fc domain having a hinge domain at its N-terminus.

[0228] The Fc domain can be derived from any suitable species operably linked to the 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, C-terminus, or both, of the IgG Fc domain.

[0229] The Fc domain can be derived from any suitable class of antibody, 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.

[0230] [Table 12]

[0231] In some embodiments, 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. When an Fc domain has at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 410 (e.g., 90%-99% sequence identity to SEQ ID NO: 410), the Fc domain may also include one or more amino acid substitutions described herein, e.g., one or more substitutions that reduce effector function (e.g., those described in Section 6.8.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., those described in Section 6.8.2).

[0232] In some embodiments, 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. When an Fc domain has at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 411 (e.g., 90%-99% sequence identity to SEQ ID NO: 411), the Fc domain may also include one or more amino acid substitutions described herein, e.g., one or more substitutions that reduce effector function (e.g., those described in Section 6.8.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., those described in Section 6.8.2).

[0233] In some embodiments, 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. When an Fc domain has at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 412 (e.g., 90%-99% sequence identity to SEQ ID NO: 412), the Fc domain can also include one or more amino acid substitutions described herein, e.g., one or more substitutions that reduce effector function (e.g., those described in Section 6.8.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., those described in Section 6.8.2).

[0234] In some embodiments, 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. When an Fc domain has at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 413 (e.g., 90%-99% sequence identity to SEQ ID NO: 413), the Fc domain can also include one or more amino acid substitutions described herein, e.g., one or more substitutions that reduce effector function (e.g., those described in Section 6.8.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., those described in Section 6.8.2).

[0235] The two Fc domains within an Fc region may be identical or different from one another. In natural antibodies, the Fc domains are typically identical, but for purposes of generating multispecific binding molecules, such as the IFN proproteins of the present disclosure and MBMs generated by their activation, the Fc domains may advantageously differ to allow heterodimerization, as described in Section 6.8.2 below.

[0236] In natural antibodies, the heavy chain Fc domain of IgA, IgD, and IgG consists of two heavy chain constant domains (CH2 and CH3), while the domain of IgE and IgM consists of three heavy chain constant domains (CH2, CH3, and CH4), which dimerize to create the Fc region.

[0237] In the IFN proproteins of the present disclosure, the Fc region, and / or Fc domains therein, can comprise heavy chain constant domains from one or more different classes of antibodies, for example, from one, two, or three different classes.

[0238] In one embodiment, the Fc region comprises a CH2 domain and a CH3 domain derived from IgG1. In one embodiment, the Fc region comprises a CH2 domain and a CH3 domain derived from IgG2.

[0239] In one embodiment, the Fc region comprises a CH2 domain and a CH3 domain derived from IgG3. In one embodiment, the Fc region comprises a CH2 domain and a CH3 domain derived from IgG4.

[0240] In one embodiment, the Fc region comprises a CH4 domain from IgM. The IgM CH4 domain is typically located C-terminal to the CH3 domain. In one embodiment, the Fc region comprises a CH2 domain and a CH3 domain derived from an IgG and a CH4 domain derived from an IgM.

[0241] It will be understood that heavy chain constant domains for use in generating Fc regions for the IFN proproteins of the present disclosure can include variants of the naturally occurring constant domains described above. Such variants can include one or more amino acid mutations compared to the wild-type constant domain. In one example, the Fc region of the present disclosure includes at least one constant domain that differs in sequence from the wild-type constant domain. It will be understood that the variant constant domain can 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.

[0242] IgM and IgA naturally occur in humans as covalently linked multimers of a common H2L2 antibody unit. IgM exists as a pentamer when a J chain is incorporated and as a hexamer when the J chain is absent. IgA exists in both monomeric and dimeric forms. The heavy chains of IgM and IgA have an 18-amino acid extension to the C-terminal constant domain known as the tail. The tail contains cysteine ​​residues that form disulfide bonds between heavy chains within the polymer and is thought to play an important role in polymerization. The tail also contains glycosylation sites. In certain embodiments, the IFN proproteins of the present disclosure do not include a tailpiece.

[0243] The Fc domain incorporated into the IFN proproteins of the present disclosure can include one or more modifications that alter the functional properties of the protein, for example, binding to an Fc receptor such as FcRn or a leukocyte receptor, 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.

[0244] The Fc domain can also be engineered to contain modifications that improve the manufacturability of asymmetric IFN proproteins, for example, by allowing heterodimerization, the preferential pairing of non-identical Fc domains with identical Fc domains. Heterodimerization allows for the generation of IFN proproteins in which different polypeptide components are connected to each other by Fc regions that contain Fc domains that differ in sequence. Examples of heterodimerization strategies are illustrated in Section 6.8.2.

[0245] It will be appreciated that any of the above modifications can 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.

[0246] 6.8.1. Fc Domains with Altered Effector Function In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and / or effector function.

[0247] In a specific embodiment, 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 activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more particularly human FcγRIIIa, FcγRI, or FcγRIIa, most particularly 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 a specific embodiment, the effector function is ADCC.

[0248] In one embodiment, the Fc domain (e.g., the Fc domain of an IFN proprotein half antibody) or Fc region (e.g., one or both Fc domains of an IFN proprotein that can associate to form an Fc region) comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331, and P329 (numbering according to Kabat EU index). In a more specific embodiment, the Fc domain or Fc region comprises an amino acid substitution at a position selected from the group of L234, L235, and P329 (numbering according to Kabat EU index). In some embodiments, the Fc domain or region comprises the amino acid substitutions L234A and L235A (numbering according to 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 comprises an amino acid substitution at position P329. In more specific embodiments, 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 comprises 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 more specific embodiments, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a particular embodiment, the Fc domain or Fc region comprises amino acid substitutions at positions P329, L234 and L235 (numbering according to the Kabat EU index). In a more specific embodiment, the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA,” “PGLALA,” or “LALAPG”).

[0249] Typically, the same one or more amino acid substitutions are present in each of the two Fc domains of the Fc region. Thus, in certain embodiments, each Fc domain of the Fc region comprises the amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering), i.e., the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A), and the proline residue at position 329 is replaced with a glycine residue (P329G) in each of the first and second Fc domains of the Fc region (Kabat EU index numbering).

[0250] 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 containing D265A, N297A mutations (EU numbering) to reduce effector function.

[0251] In another embodiment, the Fc domain is an IgG4 Fc domain with reduced binding to Fc receptors. Exemplary IgG4 Fc domains with reduced binding to Fc receptors may comprise an amino acid sequence selected from Table H below. In some embodiments, the Fc domain comprises only the bolded portion of the sequence shown below:

[0252] [Table 13-1]

[0253] [Table 13-2]

[0254] [Table 13-3]

[0255] [Table 13-4]

[0256] [Table 13-5]

[0257] In a particular embodiment, the IgG4 with reduced effector function comprises the bolded portion of the amino acid sequence of SEQ ID NO: 31 of WO2014 / 121087 having the following amino acid sequence, and is sometimes referred to herein as IgG4 or hIgG4: ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 396).

[0258] For heterodimeric Fc regions, it is possible to incorporate combinations of the above-mentioned variant IgG4 Fc sequences, for example an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 30 of WO2014 / 121087 (or the bolded portion thereof) and an Fc domain comprising 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 comprising the amino acid sequence of SEQ ID NO: 31 of WO2014 / 121087 (or the bolded portion thereof) and an Fc domain comprising the amino acid sequence of SEQ ID NO: 38 of WO2014 / 121087 (or the bolded portion thereof).

[0259] Fc Heterodimerization Variants Certain IFN proproteins, unlike native immunoglobulins, involve dimerization between two Fc domains operably linked to non-identical N- or C-terminal regions. Inefficient heterodimerization of two Fc domains to form an Fc region can be an obstacle to increasing the yield of the desired heterodimeric molecule and presents a purification challenge. Various approaches available in the art can be used to enhance the dimerization of Fc domains that may be present in the IFN proproteins of the present disclosure, as disclosed, for example, in EP 1870459A1, U.S. Pat. No. 5,582,996, U.S. Pat. No. 5,731,168, U.S. Pat. No. 5,910,573, U.S. Pat. No. 5,932,448, U.S. Pat. No. 6,833,441, U.S. Pat. No. 7,183,076, U.S. Patent Application Publication No. 2006 / 204493A1, and PCT Publication No. WO2009 / 089004A1.

[0260] In some embodiments, the present disclosure provides IFN proproteins comprising Fc heterodimers, i.e., Fc regions comprising heterologous, non-identical Fc domains. Typically, each Fc domain in the Fc heterodimer comprises an antibody CH3 domain. The CH3 domain is derived from the constant region of an antibody of any isotype, class, or subclass, preferably the IgG (IgG1, IgG2, IgG3, and IgG4) class, as described in the preceding section.

[0261] Heterodimerization of two different heavy chains at their CH3 domains results in the desired IFN proprotein, whereas homodimerization of identical heavy chains reduces the yield of the desired IFN proprotein. Thus, in preferred embodiments, polypeptides that associate to form the IFN proproteins of the present disclosure comprise a CH3 domain with modifications that favor heterodimeric association compared to an unmodified Fc domain.

[0262] In certain embodiments, the modification that promotes Fc heterodimer formation is a so-called "knob-into-hole" or "knob-in-hole" modification, which comprises a "knob" modification in one of the Fc domains and a "hole" modification in the other Fc domain. Knob-into-hole technology is described, for example, in U.S. Pat. No. 5,731,168, U.S. Pat. No. 7,695,936, Ridgway et al., 1996, Prot Eng 9:617-621, and Carter, 2001, Immunol Meth 248:7-15. Generally, this method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, allowing the protrusion to be positioned within the cavity, to promote heterodimer formation and prevent homodimer formation. The protrusions are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protrusions are created in the interface of the second polypeptide by replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine).

[0263] Thus, in some embodiments, amino acid residues in the CH3 domain of a first subunit of an Fc domain are replaced with amino acid residues having a larger side chain volume, thereby creating a protrusion in the CH3 domain of the first subunit that can be positioned within a cavity in the CH3 domain of a second subunit, and amino acid residues in the CH3 domain of a second subunit of an Fc domain are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity in the CH3 domain of the second subunit into which the protrusion in 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 protrusions and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. An exemplary substitution is Y470T.

[0264] In particular 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) (numbering according to the Kabat EU index). In further embodiments, the first Fc domain additionally has a serine residue at position 354 replaced with a cysteine ​​residue (S354C) or a glutamic acid residue at position 356 replaced with a cysteine ​​residue (E356C) (particularly, the serine residue at position 354 is replaced with a cysteine ​​residue). The second Fc domain additionally has the tyrosine residue at position 349 replaced with a cysteine ​​residue (Y349C) (Kabat EU index numbering). In a specific embodiment, the first Fc domain comprises amino acid substitutions S354C and T366W, and the second Fc domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (Kabat EU index numbering).

[0265] 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 promote association of a first Fc domain and a second Fc domain of an Fc region.

[0266] Alternatively, or in addition to using an Fc domain modified to promote heterodimerization, the Fc domain can be modified to enable a purification strategy that allows for the selection of Fc heterodimers. In one such embodiment, one polypeptide contains a modified Fc domain that abrogates its binding to Protein A, thus enabling a purification method that results in a heterodimeric protein. See, e.g., U.S. Pat. No. 8,586,713. Thus, an 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 from each other by at least one amino acid, and the at least one amino acid difference reduces 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 to Protein A and the second CH3 domain contains a mutation / modification, e.g., an H95R modification (according to IMGT exon numbering, H435R according to EU numbering), that reduces or eliminates Protein A binding. The second CH3 may further comprise a Y96F modification (by IMGT, Y436F by EU). This class of modifications is referred to herein as "star" mutations.

[0267] In some embodiments, the Fc may contain one or more mutations to promote heterodimerization (e.g., knob and hole mutations) and a star mutation to facilitate purification. Hinge Domain The IFN proproteins of the present disclosure may comprise an Fc domain comprising a hinge domain at its N-terminus. The hinge region may be a natural hinge region or a modified hinge region. The hinge region is typically found at the N-terminus of the Fc region. The term "hinge domain," unless otherwise indicated by context, refers to a naturally occurring or non-natural 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 on separate polypeptide chains (e.g., a homodimeric or heterodimeric IFN proprotein formed by association of two Fc domains). The two associated hinge sequences may also be referred to as "hinge regions." In certain embodiments of IFN proproteins, additional repeats of the hinge region may be incorporated into the polypeptide sequence.

[0268] A native hinge region is typically the hinge region found between the Fab and Fc domains of naturally occurring antibodies. A modified hinge region is any hinge that differs in length and / or composition from the native hinge region. Such hinges can include hinge regions from other species, such as human, mouse, rat, rabbit, shark, pig, hamster, camel, llama, or goat. Other modified hinge regions can include a complete hinge region derived from an antibody of a different class or subclass than that of the heavy chain Fc domain or Fc region. Alternatively, the modified hinge region can include a portion or repeat units of a native hinge, with each repeat unit derived from a native hinge region. In yet another approach, the native hinge region can 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. By such means, the number of cysteine ​​residues in the hinge region can be increased or decreased. Other modified hinge regions may be entirely synthetic and may be designed to have desired properties such as length, cysteine ​​composition, and flexibility.

[0269] Several modified hinge regions have been previously described, for example, in U.S. Pat. No. 5,677,425, WO99 / 15549, WO2005 / 003170, WO2005 / 003169, WO2005 / 003170, WO98 / 25971, and WO2005 / 003171, which are incorporated herein by reference.

[0270] In one embodiment, the IFN proprotein of the present disclosure comprises an Fc region in which one or both Fc domains have an intact hinge domain at their N-terminus. In various embodiments, positions 233-236 in the hinge region can be G, G, G, and empty; G, G, empty, and empty; G, empty, empty, and empty; or all empty, with positions numbered according to EU numbering.

[0271] In some embodiments, the IFN proproteins of the disclosure comprise a modified hinge region that reduces binding affinity to an Fcγ receptor compared to a wild-type hinge region of the same isotype (e.g., human IgG1 or human IgG4).

[0272] In one embodiment, the IFN proprotein of the present disclosure comprises an Fc region, wherein each Fc domain has an intact hinge domain at its N-terminus, and the Fc domain and hinge domain are each derived from IgG4, and each hinge domain comprises the modified sequence CPPC (SEQ ID NO: 375). The core hinge region of human IgG4 comprises the sequence CPSC (SEQ ID NO: 376), compared to IgG1, which comprises the sequence CPPC (SEQ ID NO: 375). The serine residues present in the IgG4 sequence provide increased flexibility in this region, and therefore a proportion of the molecules form disulfide bonds within the same protein chain (intrachain disulfides) rather than cross-linking to other heavy chains within an IgG molecule to form interchain disulfides. (Angel et al., 1993, Mol Immunol 30(1):105-108). Changing the serine residues to prolines to obtain the same core sequence as IgG1 allows for the complete formation of interchain disulfides within the IgG4 hinge region, thus reducing heterogeneity in the purified product. This altered isotype is called IgG4P.

[0273] The hinge sequence incorporated into the IFN proproteins 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, a truncated hinge can have a C-terminal deletion of 1, 2, 3, 4, 5, or 6 amino acids compared to a full-length hinge sequence, e.g., any of the full-length hinge sequences disclosed herein. Without being bound by theory, it is believed that a truncated hinge sequence may impart improved steric constraints to the IFN moiety. In IFN proproteins that include two hinge domains in each half antibody (e.g., IFN proproteins having the configurations shown in Figures 1C, 2C, and 2F), either of these two hinge domains can 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 and C-terminal hinge domains can be truncated.

[0274] 6.8.3.1. Chimeric Hinge Sequences The hinge domain can be a chimeric hinge domain. For example, a chimeric hinge may comprise an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region.

[0275] In certain embodiments, the chimeric hinge region comprises the amino acid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 380) (previously disclosed as SEQ ID NO: 8 of WO2014 / 121087, incorporated herein by reference in its entirety) or ESKYGPPCPPCPAPPVA (SEQ ID NO: 377) (previously disclosed as SEQ ID NO: 9 of WO2014 / 121087). Such chimeric hinge sequences may be suitably linked to an IgG4 CH2 region (e.g., by incorporation into an IgG4 Fc domain, e.g., a human or mouse Fc domain, which may be further modified in the CH2 and / or CH3 domains to reduce effector function, e.g., as described in Section 6.8.1).

[0276] 6.8.3.2. Hinge sequences with reduced effector function In further embodiments, the hinge region can be modified to reduce effector function, for example, as described in WO2016 / 161010A2, which is incorporated herein by reference in its entirety. In various embodiments, positions 233-236 of the modified hinge region can be G, G, G, and empty; G, G, empty, and empty; G, empty, empty, and empty; or all empty, with positions numbered according to EU numbering (as shown in Figure 1 of WO2016 / 161010A2). These segments can be represented as GGG-, GG--, G---, or ----, where "-" represents an empty position.

[0277] Position 236 is vacant in canonical human IgG2 but occupied in other canonical human IgG isotypes. Positions 233-235 are occupied by residues other than G in all four human isotypes (as shown in Figure 1 of WO2016 / 161010A2).

[0278] Hinge modifications within positions 233-236 can be combined with position 228 being occupied by P. Position 228 is naturally occupied by P in human IgG1 and IgG2, but is occupied by S in human IgG4 and by R in human IgG3. The S228P mutation in IgG4 antibodies is advantageous for stabilizing IgG4 antibodies and reducing heavy-light chain pair exchange between exogenous and endogenous antibodies. Preferably, positions 226-229 are occupied by C, P, P, and C, respectively.

[0279] 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 comprises CPPCPAPGGG-GPSVF (SEQ ID NO: 381) (previously disclosed as SEQ ID NO: 1 in WO2016 / 161010A2), CPPCPAPGG--GPSVF (SEQ ID NO: 382) (previously disclosed as SEQ ID NO: 2 in WO2016 / 161010A2), CPPCPAPG---GPSVF (SEQ ID NO: 383) (previously disclosed as SEQ ID NO: 3 in WO2016 / 161010A2), or CPPCPAP----GPSVF (SEQ ID NO: 384) (previously disclosed as SEQ ID NO: 4 in WO2016 / 161010A2).

[0280] The modified hinge regions described above can be incorporated into heavy chain constant regions, which typically include a CH2 domain and a CH3 domain and may have additional hinge segments (e.g., upper hinges) flanking the designated regions. Such additional constant region segments are typically of the same isotype, preferably a human isotype, but may also be hybrids of different isotypes. The isotype of such additional human constant region segments is preferably human IgG4, but may also be human IgG1, IgG2, or IgG3, or hybrids thereof, in which the domains are of different isotypes. Exemplary sequences of human IgG1, IgG2, and IgG4 are shown in Figures 2 to 4 of WO2016 / 161010A2.

[0281] In certain embodiments, a modified hinge sequence can be linked to an IgG4 CH2 region (e.g., by incorporation into an IgG4 Fc domain, e.g., a human or mouse Fc domain, which can be further modified in the CH2 and / or CH3 domain to reduce effector function, e.g., as described in Section 6.8.1).

[0282] 6.9. Nucleic Acids and Host Cells In another aspect, the disclosure provides nucleic acids encoding the IFN proproteins of the disclosure. In some embodiments, the IFN proprotein is encoded by a single nucleic acid. In other embodiments, the IFN proprotein can be encoded by multiple (e.g., two, three, four, or more) nucleic acids.

[0283] A single nucleic acid can encode an IFN proprotein comprising a single polypeptide chain, an IFN proprotein comprising two or more polypeptide chains, or a portion of an IFN proprotein comprising three or more polypeptide chains (e.g., a single nucleic acid can encode two polypeptide chains of an IFN proprotein comprising three, four, or more polypeptide chains, or three polypeptide chains of an IFN proprotein comprising four or more polypeptide chains). To separately control expression, 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). Open reading frames encoding two or more polypeptides can also be controlled by the same transcriptional regulatory elements and separated by an internal ribosome entry site (IRES) sequence, allowing translation into separate polypeptides.

[0284] In some embodiments, an IFN proprotein comprising two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding the IFN proprotein can be equal to or less than the number of polypeptide chains in the IFN proprotein (e.g., when two or more polypeptide chains are encoded by a single nucleic acid).

[0285] The nucleic acids of the present disclosure can be DNA or RNA (e.g., mRNA). In another aspect, the disclosure provides host cells and vectors comprising the nucleic acids of the disclosure. The nucleic acids may be present in a single vector or may be present in separate vectors that are present in the same host cell or in separate host cells, as described in more detail herein below.

[0286] Vectors The present disclosure provides vectors comprising nucleotide sequences encoding one or two of the polypeptide chains of an IFN proprotein or a component thereof, e.g., a half antibody of an IFN proprotein, as described herein. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).

[0287] A number of vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (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 flaviviruses.

[0288] Additionally, cells that have stably integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow for the selection of transfected host cells. Markers may provide, for example, prototropy to auxotrophic hosts, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can either be directly linked to the DNA sequence to be expressed or introduced into the same cell by cotransformation. Additional elements may also be required for optimal synthesis of mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.

[0289] Once the DNA sequence comprising the expression vector or construct is prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. To achieve this, various techniques can be used, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. The methods and conditions for culturing the resulting transfected cells and recovering the expressed polypeptide are known to those skilled in the art and can be varied or optimized based on the present specification depending on the specific expression vector and mammalian host cell used.

[0290] 6.9.2.Cells The present disclosure also provides a host cell comprising a nucleic acid of the present disclosure. In one embodiment, the host cell is genetically engineered to contain one or more nucleic acids described herein.

[0291] In one embodiment, the host cell is genetically engineered using an expression cassette. The term "expression cassette" refers to a nucleotide sequence capable of affecting the expression of a gene in a host compatible with such a sequence. Such a cassette may include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or helpful in effecting expression, such as an inducible promoter, may also be used.

[0292] The present disclosure also provides host cells comprising the vectors described herein. The cell may be, but is not limited to, a eukaryotic cell, a bacterial cell, an insect cell, or a human cell. 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.

[0293] Pharmaceutical Compositions The IFN proproteins of the present disclosure may be in the form of a composition comprising the IFN proprotein and one or more carriers, excipients, and / or diluents. The composition may be formulated for a particular use, such as veterinary use or pharmaceutical use in humans. The form of the composition (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents, and / or carriers used will depend on the intended use of the IFN proprotein and, in the case of therapeutic applications, the mode of administration.

[0294] For therapeutic use, the composition may be supplied as part of a sterile pharmaceutical composition containing a pharmaceutically acceptable carrier. This composition may be in any suitable form (depending on the desired method of administration to a patient). The pharmaceutical composition may be administered to a patient by a variety of routes, including oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, local, or topical. The most suitable route for administration in any given case will depend on the particular IFN proprotein, the subject, and the nature and severity of the disease, as well as the physical condition of the subject. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

[0295] Pharmaceutical compositions can be conveniently presented in unit dosage form, containing a predetermined amount of the IFN proprotein of the present disclosure per dose. 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 dosages can be in the form of a lyophilized dry powder containing an amount of IFN proprotein suitable for a single administration, or in liquid form. Dry powder unit dosage forms can be packaged in a kit with a syringe, a suitable amount of diluent, and / or other components useful for administration. Liquid unit dosages can conveniently be supplied in the form of a syringe pre-filled with an amount of IFN proprotein suitable for a single administration.

[0296] The pharmaceutical compositions may also be supplied in bulk, containing an amount of IFN proprotein suitable for multiple administration. Pharmaceutical compositions can be prepared for storage as lyophilized formulations or aqueous solutions by mixing IFN proproteins of the desired purity with any pharmaceutically acceptable carriers, excipients, or stabilizers (all of which are referred to herein as "carriers") typically used in the art, i.e., buffers, stabilizers, preservatives, tonicity agents, non-ionic detergents, antioxidants, and various other additives. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives should be nontoxic to recipients at the dosages and concentrations employed.

[0297] Buffering agents help maintain pH in a range close to physiological conditions. They can be present in a wide variety of concentrations, but will typically be present at concentrations ranging from about 2 mM to about 50 mM. Suitable buffering agents for use in the present disclosure include both organic and inorganic acids and their salts, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixtures, citric acid-trisodium citrate mixtures, citric acid-monosodium citrate mixtures, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-disodium succinate mixtures, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixtures, tartaric acid-potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixtures, disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, etc.), gluconate buffers (e.g., gluconate-sodium glyconate mixtures, gluconate-sodium hydroxide mixtures, gluconate-potassium glyconate mixtures, etc.), Examples of buffers include oxalic acid buffers (e.g., oxalic acid-sodium oxalate mixtures, oxalic acid-sodium hydroxide mixtures, oxalic acid-potassium oxalate mixtures, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixtures, lactic acid-sodium hydroxide mixtures, lactic acid-potassium lactate mixtures, etc.), and acetate buffers (e.g., acetic acid-sodium acetate mixtures, acetic acid-sodium hydroxide mixtures, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts (e.g., Tris) can be used.

[0298] Preservatives may be added to retard microbial growth and may be added in amounts ranging from about 0.2% to 1% (w / v). Suitable preservatives for use in the present disclosure include phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalconium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, alkylparabens (e.g., methyl or propylparaben), catechol, resorcinol, cyclohexanol, and 3-pentanol. Tonicity adjusting agents, sometimes known as "stabilizers," may be added to ensure the isotonicity of the liquid compositions of the present disclosure and include polyhydric sugar alcohols, such as trihydric or higher sugar alcohols (e.g., glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol). Stabilizers refer to a broad category of excipients that can range in function from bulking agents to additives, and help to solubilize the therapeutic agent or prevent it from denaturing or adhering to the container wall.Typical stabilizers include polyhydric sugar alcohols (as listed above), amino acids (e.g., arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.), organic sugars or sugar alcohols (e.g., lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, etc., including cyclitols such as inositol), polyethylene glycol, amino acid polymers, sulfur-containing reducing agents (e.g., urea, glutathione, Stabilizers can be selected from a wide variety of polymers, including but not limited to: thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides of 10 residues or less); proteins (e.g., human serum albumin, bovine serum albumin, gelatin, or immunoglobulins); hydrophilic polymers (e.g., polyvinylpyrrolidone); monosaccharides (e.g., xylose, mannose, fructose, glucose); disaccharides (e.g., lactose, maltose, sucrose, and trehalose); trisaccharides (e.g., raffinose); and polysaccharides (e.g., dextran). Stabilizers can be present in an amount ranging from 0.5 to 10% by weight based on the weight of the IFN proprotein.

[0299] Non-ionic surfactants or detergents (also known as "wetting agents") can be added to aid in solubilizing the glycoprotein and to protect it from agitation-induced aggregation, allowing the formulation to be exposed to stressful shear surfaces without denaturing the protein. Suitable non-ionic surfactants include polysorbates (e.g., 20, 80), poloxamers (e.g., 184, 188), and pluronic polyols. The non-ionic surfactant may 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).

[0300] Additional miscellaneous excipients include bulking agents (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), and cosolvents.

[0301] The IFN proproteins of the present disclosure can be formulated as pharmaceutical compositions 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.

[0302] For example, formulations of IFN proproteins can be prepared by mixing the IFN proprotein with a physiologically acceptable carrier, excipient, or stabilizer, for example, in the form of a lyophilized powder, a slurry, an aqueous solution, a lotion, or a suspension (see, e.g., Hardman et al., 2001, Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro, 2000, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; 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 See Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie, 2000; Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

[0303] The effective amount for a particular subject may vary depending on factors such as the condition being treated, the subject's overall health, the route and dose of administration, 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).

[0304] Compositions of the present disclosure may also be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. The route of administration selected for the IFN proprotein may include, for example, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other common routes of administration, e.g., by injection or infusion. Common administration may refer to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrapleural injection and infusion. Alternatively, compositions of the present disclosure may be administered via unconventional routes, such as topical, epithelial, or mucosal routes of administration, e.g., intranasal, oral, intravaginal, rectal, sublingual, or topical. In one embodiment, the IFN proprotein is administered by injection. In another embodiment, the IFN proprotein of the present disclosure is administered subcutaneously.

[0305] 6.10.1. Pharmaceutical Compositions for Delivery of Nucleic Acids Encoding IFN Proproteins The IFN proproteins (e.g., IFN receptor agonists) of the present disclosure can be delivered by any method useful for gene therapy, for example, as mRNA or via a viral vector encoding the IFN proprotein (e.g., IFN receptor agonist) under the control of a suitable promoter.

[0306] Exemplary viral vectors include recombinant adenovirus and adeno-associated virus vectors (rAAV). rAAV vectors are based on the defective, non-pathogenic parvovirus adeno-associated type 2 virus. Most such vectors are derived from plasmids that carry only AAV inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery via integration into the genome of transduced cells are key features of this vector system. AAV serotypes useful for delivering the IL27 transgene include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV8.2, AAV9, and AAV rh10, as well as pseudotyped AAVs such as AAV2 / 8, AAV2 / 5, and AAV2 / 6.

[0307] AAV can be produced on a clinical scale by several 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 in Penaud-Budloo et al., 2018, Mol Ther Methods Clin Dev. 8:166-180).

[0308] Replication-deficient recombinant adenoviral vectors (Ad) can be produced at high titers and easily infect several different cell types. Most adenoviral vectors are engineered so that a transgene replaces the Ad Ela, Elb, and / or E3 genes; the replication-defective vector is then propagated in human 293 cells, supplying the deleted gene function in trans. Ad vectors can transduce multiple tissue types in vivo, including non-dividing, differentiated cells found in liver, kidney, and muscle. Conventional Ad vectors have high carrying capacity.

[0309] Packaging cells are used to form viral particles capable of infecting host cells. Examples of such cells include 293 cells, which package adenovirus, and W2 or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producer cell lines that package nucleic acid vectors into viral particles. The vector typically contains minimal viral sequences necessary for packaging and subsequent integration into the host (if applicable), with other viral sequences replaced by expression cassettes encoding the proteins to be expressed. Missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically contain only the inverted terminal repeat (ITR) sequences from the AAV genome necessary for packaging and integration into the host genome. Viral DNA is packaged into a cell line containing a helper plasmid encoding other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus promotes AAV vector replication and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to the lack of ITR sequences. Adenovirus contamination can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV.

[0310] The 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 the particular disorder being treated. Optionally, other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents can be included in the compositions provided herein. For example, any one or more of wetting agents, emulsifying agents, and lubricating agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweeteners, flavorings, and perfuming agents, preservatives, antioxidants, chelating agents, and inert gases can also be present in the composition. Exemplary other agents and excipients that may be included in the compositions include, for example, water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid, and the like.

[0311] 6.11. Therapeutic Indications and Methods of Use The present disclosure provides methods for using and applying the IFN proproteins of the present disclosure. The IFN proproteins (eg, IFN receptor agonists) of the present disclosure can be used to stimulate immune responses in a variety of applications.

[0312] In certain aspects, the present disclosure provides methods of treating cancer, comprising administering to a subject in need thereof an IFN proprotein or pharmaceutical composition described herein. In some embodiments, an activated IFN protein comprising an IFN moiety is generated by cleavage of one or more protease-cleavable linkers in the IFN proprotein by one or more proteases expressed by the cancer tissue. Thus, the IFN proprotein is selectively activated in the cancer tissue.

[0313] In some embodiments, the present disclosure provides methods of treating cancer with an IFN protein that is selectively activated in cancer tissue, comprising administering to a subject in need thereof an IFN proprotein or pharmaceutical composition described herein, wherein the IFN proprotein comprises one or more protease-cleavable linkers, each comprising one or more substrates for one or more proteases expressed by the cancer tissue to which the IFN protein is targeted. Thus, an activated IFN protein comprising an IFN moiety is generated by cleavage of the one or more protease-cleavable linkers in the IFN proprotein by one or more proteases in the cancer tissue.

[0314] The present disclosure further provides a method for localized delivery of an IFN protein, comprising administering to a subject an IFN proprotein or pharmaceutical composition described herein, wherein the IFN proprotein has one or more protease-cleavable linkers, each of which comprises one or more substrates for one or more proteases expressed by the tissue to which the IFN protein is to be locally delivered. As used herein, the term "locally delivered" does not require local administration, but rather indicates activation of the protein at a desired location by a protease active at the intended site, optionally in conjunction with targeting of the active component of the IFN proprotein to the desired location using a targeting moiety that recognizes a target molecule expressed by the tissue.

[0315] The present disclosure further provides a method of administering IFN therapy to a subject with reduced systemic exposure and / or reduced systemic toxicity, comprising administering IFN therapy to the subject in the form of an IFN proprotein or pharmaceutical composition described herein, wherein the IFN proprotein has one or more protease-cleavable linkers each comprising one or more substrates for one or more proteases expressed by the tissue for which IFN therapy is desired and / or intended.

[0316] Thus, the above-described method allows for IFN therapy with reduced off-target side effects by preferentially activating IFN proproteins in the area where IFN treatment is intended.

[0317] In some embodiments of the foregoing methods, the IFN proprotein is also targeted and comprises one or more targeting moieties that recognize target molecules expressed locally (e.g., by the tissue) intended for treatment.

[0318] Thus, the present disclosure provides a method for targeted delivery of activated IFN protein to a local area where treatment is intended, e.g., cancer tissue, comprising administering to a subject an IFN proprotein or pharmaceutical composition described herein, wherein the IFN comprises one or more targeting moieties that recognize target molecules expressed in the local area or by the tissue where treatment is intended (e.g., cancer tissue), and has one or more protease-cleavable linkers each comprising one or more substrates for one or more proteases expressed by the tissue where IFN therapy is desired and / or intended.

[0319] The present disclosure further provides a method for locally inducing an immune response in a target tissue, comprising administering to a subject an IFN proprotein or pharmaceutical composition described herein 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 of which comprises one or more substrates for one or more proteases expressed in the target tissue. An activated IFN protein comprising the IFN moiety can then be generated by cleavage of the 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.

[0320] In some embodiments, administration is not local to the tissue, for example, when the target tissue is cancerous tissue, administration can be systemic or subcutaneous. The IFN proproteins of the present disclosure can be used to treat any proliferative disorder (e.g., cancer) that expresses the target molecule (either on tumor cells or in the tumor microenvironment, e.g., extracellular matrix or tumor lymphocytes). In certain embodiments, the cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, Burkitt's lymphoma, cancer of unknown primary, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasm, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma, thyroid cancer ... Cancer, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing's 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's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumor, Kaposi's 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, occult primary metastatic squamous cell neck cancer, midline carcinoma involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma , parathyroid cancer, 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, gastric cancer, T-cell lymphoma, teratoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, or Wilms' tumor.

[0321] Table I below shows exemplary indications in which IFN proproteins targeted to specific target molecules can be used.

[0322] [Table 14-1]

[0323] [Table 14-2]

[0324] [Table 14-3]

[0325] Additional target molecules and corresponding indications are disclosed, for example, in Hafeez et al., 2020, Molecules 25:4764, doi:10.3390 / molecules25204764, particularly Table 1, which is incorporated by reference in its entirety.

[0326] In further embodiments, an IFN proprotein (e.g., an IFN receptor agonist) can be used to enhance an immune response elicited by another agent. Thus, in some embodiments, an IFN proprotein (e.g., an IFN receptor agonist) of the present disclosure is administered as an adjuvant therapy with 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, bacteria, or parasite.

[0327] 7. Numbered Embodiments While various specific embodiments have been illustrated and described, it will be understood that various changes can be made without departing from the spirit and scope of the present disclosure(s), which is exemplified by the numbered embodiments set forth below.

[0328] In the following numbered embodiments, the targeting moiety preferably binds to a mammalian target molecule, the IFN moiety preferably is derived from a mammalian IFN, the Fc domain preferably is derived from a mammalian antibody, and the subject is preferably a mammal. More preferably, the mammal is a human.

[0329] 1. A type I interferon (IFN) proprotein, (a) a first polypeptide chain, (i) a first immunoglobulin constant domain; (ii) a first linker; (iii) a first type I interferon (IFN) moiety; (iv) a second linker, and (v) a first polypeptide chain comprising a first Fc domain; and (b) a second polypeptide chain, (i) a second immunoglobulin constant domain; (ii) a third linker; (iii) a second type I interferon (IFN) moiety; (iv) a fourth linker, and (v) a second polypeptide chain comprising a second Fc domain that associates with the first Fc domain to form an Fc region; A type I interferon (IFN) proprotein, wherein at least two of the first linker, the second linker, the third linker, and the fourth linker are protease-cleavable linkers (PCL), and optionally, the IFN moiety in the IFN proprotein is sterically hindered from binding to an IFN receptor by the Fc domain.

[0330] 2. The IFN proprotein of embodiment 1, wherein the first IFN moiety and the second IFN moiety each comprise an amino acid sequence having at least about 90% sequence identity with (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ, or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

[0331] 3. The IFN proprotein of embodiment 1, wherein the first IFN moiety and the second IFN moiety each comprise an amino acid sequence having about 95% sequence identity with (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ, or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

[0332] 4. The IFN proprotein of embodiment 1, wherein the first IFN moiety and the second IFN moiety each comprise an amino acid sequence having about 98% sequence identity with (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ, or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

[0333] 5. The IFN proprotein of any one of embodiments 1 to 4, wherein the first IFN moiety and the second IFN moiety each comprise an amino acid sequence having one or more attenuating mutations compared to mature human IFNα1 or IFNα2b.

[0334] 6. The IFN proprotein of any one of embodiments 1 to 5, having one or more mutations selected from 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.

[0335] 7. The IFN proprotein of any one of embodiments 1 to 6, comprising the amino acid substitution R33A. 8. The IFN proprotein of any one of embodiments 1 to 6, comprising the amino acid substitution R33K.

[0336] 9. The IFN proprotein of any one of embodiments 1 to 6, comprising the amino acid substitution Q90A. 10. The IFN proprotein of any one of embodiments 1 to 6, comprising the amino acid substitution E96A.

[0337] 11. The IFN proprotein of any one of embodiments 1 to 6, comprising the amino acid substitution R120A. 12. The IFN proprotein of any one of embodiments 1 to 6, comprising the amino acid substitution A145M.

[0338] 13. The IFN proprotein of any one of embodiments 1 to 6, comprising the amino acid substitution R149A. 14. The IFN proprotein of any one of embodiments 1 to 6, comprising the amino acid substitution R149K.

[0339] 15. The IFN proprotein of any one of embodiments 1 to 6, comprising the amino acid substitution S152A. 16. The IFN proprotein of any one of embodiments 1-6, comprising the amino acid substitutions R33A, H57Y, E58N, and Q61S.

[0340] 17. The IFN proprotein of any one of embodiments 1-6, comprising the amino acid substitutions H57Y, E58N, Q61S, and R144A. 18. The IFN proprotein of any one of embodiments 1 to 6, comprising the amino acid substitutions Q90A and R120A.

[0341] 19. The IFN proprotein of any one of embodiments 1 to 6, comprising the amino acid substitutions A145M and R149K. 20. The IFN proprotein of any one of embodiments 1-19, wherein the first linker, the second linker, the third linker, and the fourth linker are protease-cleavable linkers (PCL).

[0342] 21. The IFN proprotein of any one of embodiments 1-19, wherein the first linker and the third linker are protease-cleavable linkers (PCL), and optionally, the second linker and the fourth linker are non-cleavable linkers (NCL).

[0343] 22. The IFN proprotein of any one of embodiments 1-19, wherein the second linker and the fourth linker are protease-cleavable linkers (PCL), and optionally, the first linker and the third linker are non-cleavable linkers (NCL).

[0344] 23. The IFN proprotein of any one of embodiments 1-22, wherein said PCLs each comprise a substrate sequence cleavable by any of the proteases listed in Table A.

[0345] 24. The IFN proprotein of any one of embodiments 1-23, wherein said PCLs each comprise one or more substrate sequences selected from the substrate sequences set out in Table B. 25. The IFN proprotein of any one of embodiments 1-24, wherein said PCLs each comprise one or more spacer sequences selected from the substrate sequences listed in Table C.

[0346] 26. The IFN proprotein of any one of embodiments 1-25, wherein each PCL comprises the amino acid sequence of any of the PCL sequences set forth in Table D, or a variant thereof having up to five amino acid substitutions, such as a variant thereof having one amino acid substitution, two amino acid substitutions, three amino acid substitutions, four amino acid substitutions, or five amino acid substitutions.

[0347] 27. The IFN proprotein according to any one of embodiments 1 to 26, comprising two or four PCLs comprising or consisting of the amino acid sequence ISSGLLSGRSDNH. 28. The IFN proprotein according to any one of embodiments 1 to 26, comprising two or four PCLs comprising or consisting of the amino acid sequence GGGISSGLLSGRSDNHGGGISSGLLSGRSDNHGGS.

[0348] 29. The IFN proprotein according to any one of embodiments 1 to 26, comprising two or four PCLs comprising or consisting of the amino acid sequence GGSGGSIPVSLRSGGGISSGLLSGRSDNHGGSGGS.

[0349] 30. The IFN proprotein of any one of embodiments 1-26, comprising two or four PCLs comprising or consisting of the amino acid sequence GGSGGSVPLSLYSGGGISSGLLSGRSDNHGGSGGS.

[0350] 31. The IFN proprotein of any one of embodiments 1-26, comprising two or four PCLs comprising or consisting of the amino acid sequence GGSHPVGLLARGGGHPVGLLARGGGHPVGLLARGS.

[0351] 32. The IFN proprotein of any one of embodiments 1-26, comprising two or four PCLs comprising or consisting of the amino acid sequence GGSHPVGLLARGGGHPVGLLARGGSGRSAGGSGRSA.

[0352] 33. The IFN proprotein of any one of embodiments 1-32, wherein the first linker and the third linker are identical, and / or the third linker and the fourth linker are identical.

[0353] 34. The IFN proprotein of embodiment 33, wherein the first linker, the second linker, the third linker, and the fourth linker are identical. 35. The IFN proprotein of 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.

[0354] 36. The IFN proprotein of embodiment 35, wherein the non-cleavable linker comprises or consists of any of the NCL sequences listed in Table E. 37. The IFN proprotein of any one of embodiments 1-33, wherein the first Fc domain and / or the second Fc domain comprises a hinge domain.

[0355] 38. The IFN proprotein of any one of embodiments 1-37, further comprising one or more targeting moieties that bind to one or more target molecules. 39. The IFN proprotein of embodiment 38, comprising a first targeting moiety and a second targeting moiety.

[0356] 40. The IFN proprotein of embodiment 39, wherein the first targeting moiety and the second targeting moiety are antibodies or antigen-binding fragments thereof. 41. The IFN proprotein of embodiment 40, wherein the first targeting moiety and the second targeting moiety are Fabs.

[0357] 42. The IFN proprotein of embodiment 40 or embodiment 41, wherein the first targeting moiety and the second targeting moiety comprise Fab domains. 43. The IFN proprotein of embodiment 42, wherein the first targeting moiety and the second targeting moiety further comprise a hinge sequence.

[0358] 44. The IFN proprotein of embodiment 43, wherein the first targeting moiety and the second targeting moiety further comprise an Fc domain comprising a CH2 domain and a CH3 domain, respectively.

[0359] 45. The IFN proprotein of embodiment 44, wherein the Fc domain of the first targeting moiety and the Fc domain of the second targeting moiety are associated with each other. 46. ​​The IFN proprotein of any one of embodiments 39-45, wherein the first targeting moiety and the second targeting moiety are N-terminal to the first linker and the third linker, respectively.

[0360] 47. The IFN proprotein of any one of embodiments 38-46, 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.

[0361] 48. The IFN proprotein of embodiment 40, wherein said first immunoglobulin constant domain is a CH3 domain. 49. The IFN proprotein of embodiment 40, wherein said first immunoglobulin constant domain is a CH1 domain.

[0362] 50. The IFN proprotein of any one of embodiments 1 to 49, configured as illustrated in Figure 1A, Figure 2A, or Figure 2D. 51. A polypeptide comprising the first polypeptide chain, the second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain; (a) the first polypeptide chain (i) a first VH1 domain; (ii) the first CH1 domain; (iii) a third Fc domain comprising a hinge domain, a CH2 domain, and a CH3 domain, wherein the CH3 domain is the first immunoglobulin constant domain; (iv) said first linker; (v) said first type I interferon (IFN) moiety; (vi) the second linker, and (vii) comprises the first Fc domain; (b) the second polypeptide chain: (i) a second VH1 domain; (ii) a second CH1 domain; (iii) a fourth Fc domain comprising a hinge domain, a CH2 domain, and a CH3 domain, wherein the CH3 domain is the second immunoglobulin constant domain; (iv) said third linker; (v) said second type I interferon (IFN) moiety; (vi) the fourth linker, and (vii) comprising the third Fc domain; (c) the third polypeptide chain: (i) a first VL domain; (ii) comprises a first CL domain; (d) the fourth polypeptide chain: (i) a second VL domain, and (ii) comprises a second CL domain; The IFN proprotein of embodiment 50, wherein the first polypeptide chain associates with the third polypeptide chain such that the first VH, the CH1, the VL, and the CL form the first targeting moiety, and the second polypeptide chain associates with the fourth polypeptide chain such that the second VH, the CH1, the VL, and the CL form the second targeting moiety.

[0363] 52. The IFN proprotein of embodiment 51, wherein the first linker, the second linker, the third linker, and the fourth linker are protease-cleavable linkers (PCL).

[0364] 53. The IFN proprotein of 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).

[0365] 54. The IFN proprotein of 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).

[0366] 55. The IFN proprotein of any one of embodiments 1 to 49, configured as illustrated in Figure 1B, Figure 2B, or Figure 2E. 56. (a) the first polypeptide chain: (i) a first VH1 domain; (ii) the first CH1 domain; (iii) the first linker; (iv) said first type I interferon (IFN) moiety; (v) the second linker, and (vi) comprising the first Fc domain; (b) the second polypeptide chain: (i) a second VH1 domain; (ii) a second CH1 domain; (iii) the third linker; (iv) said second type I interferon (IFN) moiety; (v) the fourth linker, and (vi) comprising the second Fc domain; (c) the third polypeptide chain: (i) a first VL domain; (ii) comprises a second CL domain; (d) the fourth polypeptide chain: (i) a second VL domain; (ii) comprises a second CL domain; 56. The IFN proprotein of embodiment 55, wherein the first polypeptide chain associates with the third polypeptide chain such that the first VH, the CH1, the VL, and the CL form the first targeting moiety, and the second polypeptide chain associates with the fourth polypeptide chain such that the second VH, the CH1, the VL, and the CL form the second targeting moiety.

[0367] 57. The IFN proprotein of embodiment 56, wherein the first linker, the second linker, the third linker, and the fourth linker are protease-cleavable linkers (PCL).

[0368] 58. The IFN proprotein of embodiment 56, 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).

[0369] 59. The IFN proprotein of embodiment 56, 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).

[0370] 60. The IFN proprotein of any one of embodiments 1 to 49, configured as illustrated in Figure 1C, Figure 2C, or Figure 2F. 61. A polypeptide comprising the first polypeptide chain, the second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain; (a) the first polypeptide chain (i) a first VH1 domain; (ii) the first CH1 domain; (iii) the first hinge domain; (iv) said first linker; (v) said first type I interferon (IFN) moiety; (vi) the second linker, and (vii) comprises the first Fc domain; (b) the second polypeptide chain: (i) a second VH1 domain; (ii) a second CH1 domain; (iii) a second hinge domain; (iv) said third linker; (v) said second type I interferon (IFN) moiety; (vi) the fourth linker, and (vii) comprising the second Fc domain; (c) the third polypeptide chain: (i) a first VL domain; (ii) comprises a first CL domain; (d) the fourth polypeptide chain: (i) a second VL domain, and (ii) comprises a second CL domain; 61. The IFN proprotein of embodiment 60, wherein the first polypeptide chain associates with the third polypeptide chain such that the first VH, the CH1, the VL, and the CL form the first targeting moiety, and the second polypeptide chain associates with the fourth polypeptide chain such that the second VH, the CH1, the VL, and the CL form the second targeting moiety.

[0371] 62. The IFN proprotein of embodiment 61, wherein the first linker, the second linker, the third linker, and the fourth linker are protease-cleavable linkers (PCL).

[0372] 63. The IFN proprotein of embodiment 61, 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).

[0373] 64. The IFN proprotein of embodiment 61, 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).

[0374] 65. The IFN proprotein of any one of embodiments 38 to 61, wherein the first targeting moiety and / or the second targeting moiety are 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) antigen or other antigen-presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0375] 66. The IFN proprotein of any one of embodiments 38 to 65, wherein the first targeting moiety and / or the second targeting moiety are capable of binding to any of the target molecules identified in section 6.7.

[0376] 67. The IFN proprotein of any one of embodiments 38-66, wherein the first targeting moiety and / or the second targeting moiety (a) comprises (i) the CDR sequences or (ii) the VH and VL sequences of an antibody described in Table F, or (b) competes with an antibody described in Table F for binding to the target molecule.

[0377] 68. The IFN proprotein of any one of embodiments 38 to 66, wherein the first targeting moiety and / or the second targeting moiety are capable of binding to an ECM antigen optionally selected from syndecan, heparanase, integrin, osteopontin, link, cadherin, laminin, laminin-type EGF, lectin, fibronectin, notch, nectin (e.g., nectin-4), tenascin, collagen (e.g., type X collagen), and matrixin.

[0378] 69. The IFN proprotein of embodiment 68, wherein the first targeting moiety and / or the second targeting moiety are capable of binding to a nectin, such as nectin 4. 70. The IFN proprotein of embodiment 68, wherein the first targeting moiety and / or the second targeting moiety are capable of binding to collagen, for example type X collagen.

[0379] 71. The IFN proprotein of any one of embodiments 38 to 66, wherein the first targeting moiety and / or the second targeting moiety are capable of binding to a cell surface molecule of a tumor or viral lymphocyte.

[0380] 72. The IFN proprotein of embodiment 71, wherein the antigen is a T cell co-stimulatory protein. 73. The IFN proprotein of 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.

[0381] 74. The IFN proprotein of embodiment 73, wherein the T cell costimulatory protein is B7-H3. 75. The IFN proprotein of any one of embodiments 38-66, wherein the first targeting moiety and / or the second targeting moiety are capable of binding to a checkpoint inhibitor.

[0382] 76. The IFN proprotein of 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.

[0383] 77. The IFN proprotein of embodiment 76, wherein the checkpoint inhibitor is PDL1. 78. The IFN proprotein of embodiment 76, wherein the checkpoint inhibitor is PD1.

[0384] 79. The IFN proprotein of embodiment 76, wherein the checkpoint inhibitor is LAG3. 80. The IFN proprotein of any one of embodiments 38 to 66, wherein the first targeting moiety and / or the second targeting moiety are capable of binding to a tumor-associated antigen (TAA).

[0385] 81. The first targeting moiety and / or the second targeting moiety is selected from the group consisting of 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, and 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 / M AGE-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, P 81. The IFN proprotein of embodiment 80, which is capable of binding to CTA-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.

[0386] 82. The IFN proprotein of embodiment 81, wherein the TAA is EGFR. 83. The IFN proprotein of embodiment 81, wherein said TAA is HER2. 84. The IFN proprotein of embodiment 81, wherein said TAA is EPCAM.

[0387] 85. The IFN proprotein of embodiment 81, wherein said TAA is CEACAM5. 86. The IFN proprotein of embodiment 81, wherein the TAA is CD20.

[0388] 87. The IFN proprotein of any one of embodiments 38-66, wherein the first targeting moiety and / or the second targeting moiety are capable of binding to a dendritic cell (DC) antigen optionally selected from XCR1, Clec9a, CD1c, CD11c, CD14, PDL1, macrophage mannose receptor (CD206), and DEC-205.

[0389] 88. The IFN proprotein of embodiment 87, wherein the dendritic cell antigen is XCR1. 89. The IFN proprotein of embodiment 87, wherein the dendritic cell antigen is Clec9a.

[0390] 90. The IFN proprotein of embodiment 87, wherein the dendritic cell antigen is DEC-205. 91. The IFN proprotein of any one of embodiments 38-66, wherein the first targeting moiety and / or the second targeting moiety are capable of binding to a natural killer (NK) cell antigen.

[0391] 92. The IFN proprotein according to any one of embodiments 1 to 37, further comprising one or more targeting moieties, each comprising means for binding to one or more target molecules. 93. The IFN proprotein of embodiment 92, comprising a first targeting moiety and a second targeting moiety, each comprising a means for binding to a target molecule.

[0392] 94. The IFN proprotein of embodiment 93, wherein the first targeting moiety and the second targeting moiety are antibodies or antigen-binding fragments thereof. 95. The IFN proprotein of embodiment 94, wherein the first targeting moiety and the second targeting moiety are Fabs.

[0393] 96. The IFN proprotein of embodiment 94, wherein the first targeting moiety and the second targeting moiety comprise Fab domains. 97. The IFN proprotein of any one of embodiments 92-96, wherein the first targeting moiety and the second targeting moiety further comprise a hinge sequence.

[0394] 98. The IFN proprotein of embodiment 97, wherein the first targeting moiety and the second targeting moiety further comprise an Fc domain comprising a CH2 domain and a CH3 domain, respectively.

[0395] 99. The IFN proprotein of embodiment 98, wherein the Fc domain of the first targeting moiety and the Fc domain of the second targeting moiety are associated with each other. 100. The IFN proprotein of any one of embodiments 92-99, wherein the first targeting moiety and the second targeting moiety are N-terminal to the first linker and the third linker, respectively.

[0396] 101. The IFN proprotein of any one of embodiments 92-100, 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.

[0397] 102. The IFN proprotein of embodiment 101, wherein said first immunoglobulin constant domain is a CH3 domain. 103. The IFN proprotein of embodiment 101, wherein said first immunoglobulin constant domain is a CH1 domain.

[0398] 104. The IFN proprotein of any one of embodiments 92-103, configured as illustrated in Figure 1A, Figure 2A, or Figure 2D. 105. A polypeptide comprising the first polypeptide chain, the second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain; (a) the first polypeptide chain (i) a first VH1 domain; (ii) the first CH1 domain; (iii) a third Fc domain comprising a hinge domain, a CH2 domain, and a CH3 domain, wherein the CH3 domain is the first immunoglobulin constant domain; (iv) said first linker; (v) said first type I interferon (IFN) moiety; (vi) the second linker, and (vii) comprises the first Fc domain; (b) the second polypeptide chain: (i) a second VH1 domain; (ii) a second CH1 domain; (iii) a fourth Fc domain comprising a hinge domain, a CH2 domain, and a CH3 domain, wherein the CH3 domain is the second immunoglobulin constant domain; (iv) said third linker; (v) said second type I interferon (IFN) moiety; (vi) the fourth linker, and (vii) comprising the third Fc domain; (c) the third polypeptide chain: (i) a first VL domain; (ii) comprises a first CL domain; (d) the fourth polypeptide chain: (i) a second VL domain, and (ii) comprises a second CL domain; 105. The IFN proprotein of embodiment 104, wherein the first polypeptide chain associates with the third polypeptide chain such that the first VH, the CH1, the VL, and the CL form the first targeting moiety, and the second polypeptide chain associates with the fourth polypeptide chain such that the second VH, the CH1, the VL, and the CL form the second targeting moiety.

[0399] 106. The IFN proprotein of embodiment 105, wherein the first linker, the second linker, the third linker, and the fourth linker are protease-cleavable linkers (PCL).

[0400] 107. The IFN proprotein of 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).

[0401] 108. The IFN proprotein of 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).

[0402] 109. The IFN proprotein of any one of embodiments 92 to 103, configured as illustrated in Figure 1B, Figure 2B, or Figure 2E. 110. (a) the first polypeptide chain: (i) a first VH1 domain; (ii) the first CH1 domain; (iii) the first linker; (iv) said first type I interferon (IFN) moiety; (v) the second linker, and (vi) comprising the first Fc domain; (b) the second polypeptide chain: (i) a second VH1 domain; (ii) a second CH1 domain; (iii) the third linker; (iv) said second type I interferon (IFN) moiety; (v) the fourth linker, and (vi) comprising the second Fc domain; (c) the third polypeptide chain: (i) a first VL domain; (ii) comprises a second CL domain; (d) the fourth polypeptide chain: (i) a second VL domain; (ii) comprises a second CL domain; The IFN proprotein of embodiment 109, wherein the first polypeptide chain associates with the third polypeptide chain such that the first VH, the CH1, the VL, and the CL form the first targeting moiety, and the second polypeptide chain associates with the fourth polypeptide chain such that the second VH, the CH1, the VL, and the CL form the second targeting moiety.

[0403] 111. The IFN proprotein of embodiment 110, wherein the first linker, the second linker, the third linker, and the fourth linker are protease-cleavable linkers (PCL).

[0404] 112. The IFN proprotein of 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).

[0405] 113. The IFN proprotein of 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).

[0406] 114. The IFN proprotein of any one of embodiments 92 to 103, configured as illustrated in Figure 1C, Figure 2C, or Figure 2F. 115. A polypeptide comprising the first polypeptide chain, the second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain; (a) the first polypeptide chain: (i) a first VH1 domain; (ii) the first CH1 domain; (iii) the first hinge domain; (iv) said first linker; (v) said first type I interferon (IFN) moiety; (vi) the second linker, and (vii) comprises the first Fc domain; (b) the second polypeptide chain: (i) a second VH1 domain; (ii) a second CH1 domain; (iii) a second hinge domain; (iv) said third linker; (v) said second type I interferon (IFN) moiety; (vi) the fourth linker, and (vii) comprising the second Fc domain; (c) the third polypeptide chain: (i) a first VL domain; (ii) comprises a first CL domain; (d) the fourth polypeptide chain: (i) a second VL domain, and (ii) comprises a second CL domain; 115. The IFN proprotein of embodiment 114, wherein the first polypeptide chain associates with the third polypeptide chain such that the first VH, the CH1, the VL, and the CL form the first targeting moiety, and the second polypeptide chain associates with the fourth polypeptide chain such that the second VH, the CH1, the VL, and the CL form the second targeting moiety.

[0407] 116. The IFN proprotein of embodiment 115, wherein the first linker, the second linker, the third linker, and the fourth linker are protease-cleavable linkers (PCL).

[0408] 117. The IFN proprotein of embodiment 115, 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).

[0409] 118. The IFN proprotein of embodiment 115, 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).

[0410] 119. The IFN proprotein of any one of embodiments 92 to 119, wherein the first targeting moiety and / or the second targeting moiety comprises a 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) antigen or other antigen-presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0411] 120. The IFN proprotein of 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 target molecule identified in section 6.6.

[0412] 121. The IFN proprotein of 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 optionally selected from syndecan, heparanase, integrin, osteopontin, link, cadherin, laminin, laminin-type EGF, lectin, fibronectin, notch, nectin (e.g., nectin-4), tenascin, collagen (e.g., type X collagen), and matrixin.

[0413] 122. The IFN proprotein according to embodiment 121, wherein the first targeting moiety and / or the second targeting moiety comprises a means for binding to a nectin, such as nectin 4.

[0414] 123. The IFN proprotein of embodiment 121, wherein the first targeting moiety and / or the second targeting moiety comprises a means for binding to collagen, for example type X collagen.

[0415] 124. The IFN proprotein of 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 cell surface molecule of a tumor or viral lymphocyte.

[0416] 125. The IFN proprotein of embodiment 124, wherein the cell surface molecule is a T cell costimulatory protein. 126. The IFN proprotein of 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.

[0417] 127. The IFN proprotein of embodiment 126, wherein the T cell costimulatory protein is B7-H3. 128. The IFN proprotein of any one of embodiments 92-119, wherein the first targeting moiety and / or the second targeting moiety comprises a means for binding to a checkpoint inhibitor.

[0418] 129. The IFN proprotein of 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.

[0419] 130. The IFN proprotein of embodiment 129, wherein the checkpoint inhibitor is PDL1. 131. The IFN proprotein of embodiment 129, wherein the checkpoint inhibitor is PD1.

[0420] 132. The IFN proprotein of embodiment 129, wherein the checkpoint inhibitor is LAG3. 133. The IFN proprotein of 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).

[0421] 134. 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 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, or uroplakin-3.

[0422] 135. The IFN proprotein of embodiment 134, wherein the TAA is EGFR. 136. The IFN proprotein of embodiment 134, wherein the TAA is HER2.

[0423] 137. The IFN proprotein of embodiment 134, wherein said TAA is EPCAM. 138. The IFN proprotein of embodiment 134, wherein said TAA is CEACAM5.

[0424] 139. The IFN proprotein of embodiment 134, wherein the TAA is CD20. 140. The IFN proprotein of any one of embodiments 92-119, wherein the first targeting moiety and / or the second targeting moiety comprises means for binding to a dendritic cell (DC) antigen optionally selected from XCR1, Clec9a, CD1c, CD11c, CD14, PDL1, macrophage mannose receptor (CD206), and DEC-205.

[0425] 141. The IFN proprotein of embodiment 140, wherein the dendritic cell antigen is XCR1. 142. The IFN proprotein of embodiment 140, wherein the dendritic cell antigen is Clec9a.

[0426] 143. The IFN proprotein of embodiment 140, wherein the dendritic cell antigen is DEC-205. 144. The IFN proprotein of 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 natural killer (NK) cell antigen.

[0427] 145. The IFN proprotein of any one of embodiments 1-144, wherein the Fc region is a homodimer. 146. The IFN proprotein of any one of embodiments 1-144, wherein the Fc region is a heterodimer.

[0428] 147. A nucleic acid or nucleic acids encoding an IFN proprotein according to any one of embodiments 1 to 146. 148. A host cell that has been engineered to express an IFN proprotein according to any one of embodiments 1 to 146 or a nucleic acid(s) according to embodiment 147.

[0429] 149. A method for producing an IFN proprotein according to any one of embodiments 1 to 146, comprising culturing a host cell according to embodiment 148 and recovering the IFN proprotein expressed thereby.

[0430] 150. A pharmaceutical composition comprising an IFN proprotein according to any one of embodiments 1 to 146 and an excipient. 151. A method for treating cancer, comprising 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.

[0431] 152. The method of embodiment 151, wherein said IFN proprotein comprises at least one targeting moiety capable of binding to a target molecule. 153. The method of embodiment 151, wherein said IFN proprotein comprises at least one means for binding to a target molecule.

[0432] 154. The method of any one of embodiments 150-153, wherein the cancer is associated with expression of a target molecule listed in Table I, e.g., a TAA and associated cancers. 155. The method of any one of embodiments 150-154, 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.

[0433] 156. The method of embodiment 155, wherein the IFN protein is selectively activated in the cancer tissue. 157. A method for local delivery of an IFN protein, comprising administering to a subject an IFN proprotein according to any one of embodiments 1-146 (or a pharmaceutical composition comprising the IFN proprotein and an excipient) having one or more protease-cleavable linkers each comprising one or more substrates for one or more proteases expressed by the tissue to which the IFN protein is to be locally delivered.

[0434] 158. The method of embodiment 157, wherein said IFN proprotein comprises one or more targeting moieties that recognize target molecules expressed by said tissue. 159. The method of embodiment 158, wherein said IFN proprotein comprises two targeting moieties, each of which recognizes a target molecule expressed by said tissue.

[0435] 160. The method of embodiment 157, wherein said IFN proprotein comprises one or more means for binding to a target molecule expressed by said tissue. 161. The method of embodiment 160, wherein said IFN proprotein comprises two means for binding to a target molecule expressed by said tissue.

[0436] 162. The method of any one of embodiments 157-161, wherein the tissue is cancer tissue. 163. The method of 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 viral lymphocyte, a T cell antigen (TCA), a checkpoint inhibitor, a tumor-associated antigen (TAA), a dendritic cell (DC) antigen or other antigen-presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0437] 164. The method of any one of embodiments 157-163, 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 in the tissue.

[0438] 165. 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 embodiments 1-146 (or a pharmaceutical composition comprising the IFN proprotein and an excipient) having one or more protease-cleavable linkers, each of which comprises one or more substrates for one or more proteases expressed by the cancer tissue to which the IFN protein is targeted.

[0439] 166. The method of embodiment 165, wherein the IFN proprotein comprises one or more targeting moieties that recognize target molecules expressed by the cancer tissue or associated immune cells. 167. The method of embodiment 166, wherein the IFN proprotein comprises two targeting moieties, each of which recognizes a target molecule expressed by the cancer tissue or associated immune cells.

[0440] 168. The method of embodiment 165, wherein said IFN proprotein comprises one or more means for binding to a target molecule expressed by said cancer tissue or associated immune cells. 169. The method of embodiment 168, wherein said IFN proprotein comprises two means for binding to a target molecule expressed by said cancer tissue or associated immune cells.

[0441] 170. The method of any one of embodiments 165 to 169, 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 viral lymphocyte, a T cell antigen (TCA), a checkpoint inhibitor, a tumor-associated antigen (TAA), a dendritic cell (DC) antigen or other antigen-presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0442] 171. The method of any one of embodiments 165-170, 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 in the cancer tissue.

[0443] 172. A method of administering to a subject 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 excipients) according to any one of embodiments 1-146 having one or more protease-cleavable linkers, each comprising one or more substrates for one or more proteases expressed by a tissue for which IFN therapy is desired and / or intended.

[0444] 173. The method of embodiment 172, wherein said IFN proprotein comprises one or more targeting moieties that recognize target molecules expressed by said tissue. 174. The method of embodiment 173, wherein said IFN proprotein comprises two targeting moieties, each of which recognizes a target molecule expressed by said tissue.

[0445] 175. The method of embodiment 172, wherein said IFN proprotein comprises one or more means for binding to a target molecule expressed by said tissue. 176. The method of embodiment 175, wherein said IFN proprotein comprises two means for binding to a target molecule expressed by said tissue.

[0446] 177. The method of any one of embodiments 172-176, wherein the tissue is cancer tissue or associated immune cells. 178. The method of 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) antigen or other antigen-presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0447] 179. The method of any one of embodiments 172-178, 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 in the tissue.

[0448] 180. 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 of any one of embodiments 1-146 (or a pharmaceutical composition comprising the IFN proprotein and an excipient) having one or more protease-cleavable linkers, each of which comprises one or more substrates for one or more proteases expressed by the cancer tissue.

[0449] 181. The method of embodiment 180, wherein said IFN proprotein comprises one or more targeting moieties that recognize target molecules expressed by said cancer tissue or associated immune cells. 182. The method of embodiment 181, wherein the IFN proprotein comprises two targeting moieties, each of which recognizes a target molecule expressed by the cancer tissue or associated immune cells.

[0450] 183. The method of embodiment 180, wherein said IFN proprotein comprises one or more means for binding to a target molecule expressed by said cancer tissue or associated immune cells. 184. The method of embodiment 183, wherein said IFN proprotein comprises two means for binding to a target molecule expressed by said cancer tissue or associated immune cells.

[0451] 185. The method of any one of embodiments 180 to 184, 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 viral lymphocyte, a T cell antigen (TCA), a checkpoint inhibitor, a tumor-associated antigen (TAA), a dendritic cell (DC) antigen or other antigen-presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0452] 186. The method of any one of embodiments 179-184, 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 in the cancer tissue.

[0453] 187. 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 embodiments 1 to 146 (or a pharmaceutical composition comprising the IFN proprotein and an excipient), wherein the IFN proprotein: (a) (i) one or more targeting moieties that recognize a target molecule expressed by the cancer tissue or associated immune cells, or (ii) a means for binding to a target molecule expressed by the cancer tissue or associated immune cells; and (b) having one or more protease-cleavable linkers each comprising one or more substrates for one or more proteases expressed in the tissue in which IFN therapy is desired and / or intended.

[0454] 188. The method of embodiment 187, wherein the IFN proprotein comprises (i) two targeting moieties, each of which recognizes a target molecule expressed by the cancer tissue or associated immune cells, or (ii) two means for binding to a target molecule expressed by the cancer tissue or associated immune cells.

[0455] 189. 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 viral lymphocyte, a T cell antigen (TCA), a checkpoint inhibitor, a tumor-associated antigen (TAA), a dendritic cell (DC) antigen or other antigen-presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0456] 190. The method of any one of embodiments 187-189, 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 in the cancer tissue.

[0457] 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 embodiments 1 to 146 (or a pharmaceutical composition comprising said IFN proprotein and an excipient), said IFN proprotein comprising (i) one or more targeting moieties capable of binding to a target molecule expressed in said target tissue, or (ii) one or more protease-cleavable linkers each comprising one or more substrates for one or more proteases expressed in said target tissue.

[0458] 192. The method of embodiment 191, wherein the IFN proprotein comprises (i) two targeting moieties, each recognizing a target molecule expressed in the target tissue or associated immune cells, or (ii) two means for binding to a target molecule expressed in the target tissue or associated immune cells.

[0459] 193. The method of embodiment 191 or 192, wherein the target tissue is cancer tissue. 194. The method of any one of embodiments 191 to 193, wherein the target molecule expressed in the target tissue or associated 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) antigen or other antigen-presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0460] 195. The method of any one of embodiments 190-194, 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 in the target tissue.

[0461] 196. The method of embodiment 195, wherein said IFN protein induces an immune response against at least one cell type in said target tissue. 197. A method for 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 (e.g., an IFN receptor agonist) described in any one of embodiments 1-146 (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., one described in Section 6.10.1.

[0462] 198. The method of embodiment 197, wherein the administration of the immunogenic agent and the IFN proprotein (e.g., IFN receptor agonist) is concurrent, separate but simultaneous, or sequential.

[0463] 199. The method of 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.

[0464] 200. The method of any one of embodiments 151-199, wherein the administration is non-topical. 201. The method of embodiment 200, wherein the administration is systemic. [Example]

[0465] 8. Working Example 8.1. Proprotein Construct Sequence Table 3 below provides the sequences of the IFN proprotein constructs and control constructs utilized in the studies described herein.

[0466] [Table 15-1]

[0467] [Table 15-2]

[0468] [Table 15-3]

[0469] [Table 15-4]

[0470] [Table 15-5]

[0471] [Table 15-6]

[0472] [Table 15-7]

[0473] [Table 15-8]

[0474] [Table 15-9]

[0475] [Table 15-10]

[0476] [Table 15-11]

[0477] 8.2. Materials and Methods 8.2.1. Generation of Type I IFN Constructs Constructs encoding the antibody and sterically attenuated IFN fusion proteins were generated in standard mammalian protein expression DNA vectors (pcDNA3.4 or similar) suitable for high-yield protein production and containing standard elements such as promoter sequences, polyA sequences, regulatory elements, and resistance genes. Where applicable, sequences were codon-optimized. A 29-amino acid signal sequence from the mouse inactive tyrosine-protein kinase transmembrane receptor ROR1 (mROR1) was added to the N-terminus of the construct to function as a signal for secretion. All IFN fusion proteins were expressed as preproteins containing a signal sequence that was cleaved by intracellular processing to produce the mature protein. Constructs were expressed in Expi293F™ cells by transient transfection (Thermo Fisher Scientific). Proteins in Expi293F supernatants were purified using the ProteinMaker system (Protein BioSolutions, Gaithersburg, MD) equipped with either a HiTrap™ Protein G HP or MabSelect SuRe pcc column (Cytiva). After single-step elution, the protein was neutralized and dialyzed into a final buffer of phosphate-buffered saline (PBS) containing 5% glycerol, aliquoted, and stored at −80° C. Samples were further analyzed by SE-UPLC to determine the presence of high or low molecular weight species relative to the species of interest.

[0478] 8.2.2. In Vitro Cleavage of IFN Constructs The protease-cleavable linker was enzymatically cleaved by incubating the proprotein construct with either uPA or MMP enzymes. For enzymatic cleavage with uPA, 8 μg of the protein construct was incubated with 100 ng of uPA enzyme in a 200 μL volume of uPA buffer (50 mM Tris pH 8.5, 0.01% (v / v) Tween 20) at 37°C for 20 hours. For enzymatic cleavage with MMP, 8 μg of the protein construct was incubated with 200 μg each of MMP2 and MMP9 in a 200 μL volume of MMP buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10 mM CaCl2, 0.05% Brij 35) at 37°C for 20 hours.

[0479] 8.2.3. Manipulation of Reporter KG-1a Cells The promyeloblast-macrophage cell line KG-1a was transduced with an ISRE-driven luciferase reporter construct and maintained in Iscove's modified Dulbecco's medium supplemented with 2 mM L-glutamine / penicillin / streptomycin, 20% FBS, and 1 μg / mL puromycin. A single cell clone highly responsive to IFNα2b was identified, renamed KG-1a / ISRE-Luc cl.2F5, and used in the assay as described.

[0480] 8.2.4. Luciferase Assay Setup RPMI1640 medium supplemented with 2 mM L-glutamine / penicillin / streptomycin plus 10% FBS was used as the assay medium to prepare cell suspensions and fusion protein dilutions.

[0481] On the day of the assay, cells were centrifuged and plated at 5 x 10 in assay medium. 5 The IFNα2b and / or IFN fusion proteins were diluted 1:5 according to an 11-point dilution range (100 nM to 10.2 fM range for recombinant interferon and 500 nM to 51.2 fM range for fusion proteins) (the 12th point did not contain recombinant protein). 4Reporter cells were added to 96-well white flat-bottom plates and incubated with serially diluted recombinant IFN or IFN fusion proteins. After incubating the plates at 37°C and 5% CO for 5 hours, 100 μL of ONE-Glo™ (Promega) reagent was added to the lysed cells to detect luciferase activity. Luminescence was captured in relative light units (RLU) on a multilabel plate reader, Envision (PerkinElmer).

[0482] 8.3. Example 1: SE-UPLC Profiles of Fc-Binding Interferon Molecules SE-UPLC was performed to evaluate IFN molecules linked to either the C- or N-terminal Fc domain. Three exemplary constructs analyzed by SE-UPLC, Fc-IFNα1 (Figure 4A), Fc-IFNα2b (Figure 4B), and IFNα2b-Fc (Figure 4C), exhibited distinct main peaks with varying levels of high molecular weight species. The main peak percent area for Fc-IFNα1 was calculated to be 37.43, whereas these percent values ​​were higher for Fc-IFNα2b and IFNα2b-Fc, calculated to be 57.66 and 56.4, respectively.

[0483] 8.4. Example 2: Activity of Interferon Molecules An interferon-stimulated response element (ISRE)-driven luciferase reporter was engineered into the promyeloblast macrophage cell line KG-1a as described in Section 8.2.3 and used as described in Section 8.2.4 to assess the ability of IFN receptor agonist constructs to induce the ISRE.

[0484] The results shown in Figure 5 indicated that recombinant proteins and Fc fusions of IFN variants exhibited varying degrees of attenuation in an in vitro luciferase assay of the interferon-stimulated response element (ISRE). The first evaluation required examining whether there were differences in the activity of IFN molecules linked to either the N- or C-terminal Fc molecule (Figure 5A). Compared to IFNα2b, both Fc-IFNα2b and IFNα2b-Fc exhibited weaker interferon signaling. However, the level of attenuation was similar for both molecules (Figure 5B). Next, the in vitro activity of two Fc-IFN constructs, Fc-IFNα2b and Fc-IFNα1, was compared with that of three IFN variants, IFNα2b, IFNα1, and IFNβ (Figure 5C). Among the IFN variants, the highest activity levels were observed for IFNβ and IFNα2b, whereas the activity of IFNα1 was relatively weak. Recombinant proteins with Fc fusions exhibited reduced levels of activity compared to IFN variants. In conclusion, Fc fusions result in attenuated interferon signaling compared to free interferon.

[0485] 8.5. Example 3: SE-UPLC Profiles of Mutant IFN Constructs We performed SE-UPLC to evaluate mutant IFN molecules linked to a C-terminal Fc domain. Four exemplary constructs analyzed by SE-UPLC, Fc-IFNα2bR33A (Figure 6A), Fc-IFNα2bR149A (Figure 6B), Fc-IFNα2bR120A (Figure 6C), and Fc-IFNα2bS152A (Figure 6D), exhibited distinct major peaks with varying levels of high molecular weight species.

[0486] 8.6. Example 4: Activity of mutant IFN constructs An ISRE-driven luciferase reporter assay was engineered into the promyeloblast macrophage cell line KG-1a as described in Section 8.2.3 and used as described in Section 8.2.4 to assess the ability of the mutant IFN receptor agonist constructs to induce the ISRE.

[0487] The activity of IFN variants correlates with their affinity for IFNAR. Therefore, mutations affecting IFN-IFNAR binding may affect the activity of Fc-IFN constructs. A series of mutations was introduced into IFNα2b at either its IFNAR1 or IFNAR2 interface (Figures 7A and 7B). Compared with wild-type Fc-IFNα2b, most mutations that disrupted IFNAR1 or IFNAR2 binding of Fc-IFNα2b attenuated ISRE-luciferase activity. Furthermore, the degree of attenuation varied, with some mutations causing only a slight attenuation in activity, while others resulted in very high levels of attenuation. Nevertheless, for these differences in attenuation levels, there was only a slight difference between mutations disrupting IFNAR1 or IFNAR2 binding.

[0488] 8.7. Example 5: SE-UPLC Profiles of Exemplary Interferon Proprotein Constructs After generating constructs encoding sterically attenuated 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 illustrates 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). While the single-hinge and double-hinge constructs showed a distinct main peak, the SEC profile of the IFN construct with two Fc domains was less clear. Therefore, subsequent analysis was performed on the single-hinge and double-hinge IFN constructs, excluding the IFN construct with two Fc domains.

[0489] 8.8. Example 6: Enzymatic Cleavage of IFN Proprotein Constructs Exemplary IFN proprotein constructs were cleaved with uPA and MMP enzymes as described in Section 8.2.2 (Figure 9). Cleavage of the proprotein constructs was assessed by the presence of bands corresponding to the Fc and Fab components, both of which were expected to appear at 38-49 kDa. The identity of the different fragments was further confirmed by Western blot detecting the Fc portion.

[0490] Cleavage of both the single-hinge and double-hinge IFN proprotein constructs with uPA resulted in an additional band in the SDS-PAGE image that appeared at a higher molecular weight than the expected band (Figure 9A), indicating incomplete in vitro cleavage of these constructs with uPA. In contrast, MMP2 / 9 completely cleaved the single-hinge IFN proprotein. However, in the lane loaded with the MMP2 / 9-cleaved double-hinge IFN proprotein, an additional high-molecular-weight band was present (Figure 9B), indicating incomplete MMP cleavage of the double-hinge construct.

[0491] 8.9. Example 7: Activity of IFN Proprotein Constructs The ISRE-luciferase activity of the three IFN proproteins was evaluated relative to IFNα2b and Fc-IFNα2b. The single-hinge full-length IFN construct and the double-hinge IFN construct showed a small level of attenuation relative to Fc-IFNα2b. However, the best attenuation was associated with the single-hinge truncated IFN (Figure 10A).

[0492] To assess whether in vitro cleavage of the IFN proprotein constructs restores IFN activity, the same three IFN proprotein constructs were first incubated with either MMP buffer control or MMP2 and MMP9 enzymes in MMP buffer as described in Section 8.2.2. Constructs incubated with MMP buffer alone attenuated the activity of the IFN proprotein constructs (Figure 10B, dashed line), 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 bioassay signal. However, when these constructs were incubated with MMP enzymes in the same buffer, IFN release was associated with increased activity compared to the uncleaved constructs (Figure 10B, dotted line). More specifically, the activity of the single-hinge full-length IFN construct and the double-hinge IFN construct was similar to that of IFNα2b, while the activity of the single-hinge cleaved IFN was between that of IFNα2b and Fc-IFNα2b.

[0493] 9. Citation of References All publications, patents, patent applications, and other documents cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes. In the event of a conflict between the teachings of one or more of the references incorporated herein and the present disclosure, the teachings of the present disclosure are intended.

Claims

1. A type I interferon (IFN) proprotein, (a) a first polypeptide chain, (i) a first immunoglobulin constant domain; (ii) a first linker; (iii) a first type I interferon (IFN) moiety; (iv) a second linker, and (v) a first polypeptide chain comprising a first Fc domain; and (b) a second polypeptide chain, (i) a second immunoglobulin constant domain; (ii) a third linker; (iii) a second type I interferon (IFN) moiety; (iv) a fourth linker, and (v) a second polypeptide chain comprising a second Fc domain that associates with the first Fc domain to form an Fc region; A type I interferon (IFN) proprotein, wherein at least two of the first linker, the second linker, the third linker, and the fourth linker are protease-cleavable linkers (PCL), and optionally the IFN moiety in the IFN proprotein is sterically hindered from binding to an IFN receptor by the Fc domain.

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

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

4. 4. The IFN proprotein of any one of claims 1 to 3, wherein the first IFN moiety and the second IFN moiety each have one or more mutations selected from 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. 5. The IFN proprotein of 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. 5. The IFN proprotein of any one of claims 1 to 4, wherein the first linker and the third linker are protease-cleavable linkers (PCL), and optionally the second linker and the fourth linker are non-cleavable linkers (NCL).

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

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

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

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

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

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

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

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

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

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

21. 20. The IFN proprotein of any one of claims 1 to 19 configured as illustrated in Figure 1B, Figure 2B, or Figure 2E.

22. 20. The IFN proprotein of any one of claims 1 to 19 configured as illustrated in Figure 1C, Figure 2C, or Figure 2F.

23. 23. The IFN proprotein of any one of claims 14 to 22, wherein the first targeting moiety and / or the second targeting moiety 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) antigen or other antigen-presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

24. 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) CDR sequences or (ii) VH and VL sequences of an antibody listed in Table F, or (b) competes with an antibody listed in Table F for binding to the target molecule.

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

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

27. 27. The IFN proprotein of claim 26, wherein the antigen is a T-cell costimulatory protein 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. IFN proprotein according to any one of claims 14 to 23, wherein the first targeting moiety and / or the second targeting moiety are capable of binding to a checkpoint inhibitor.

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

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

31. The first targeting moiety and / or the second targeting moiety are selected from the group consisting of 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 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, PS 24. The IFN proprotein of any one of claims 14 to 23, which is capable of binding to a tumor-associated antigen (TAA) optionally selected from MA (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 uroplakin-3.

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

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

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

35. A nucleic acid or nucleic acids encoding an 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(s) of claim 35.

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

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

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

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

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

42. 42. The method of any one of claims 39-41, 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.

43. 35. A method for localized 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 said IFN proprotein and an excipient), said IFN proprotein having one or more protease-cleavable linkers each comprising one or more substrates for one or more proteases expressed by the tissue to which said IFN protein is to be locally delivered.

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

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

46. 35. A method of 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 said IFN proprotein and an excipient) having one or more protease-cleavable linkers each comprising one or more substrates for one or more proteases expressed by said cancer tissue.

47. 35. 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 said IFN proprotein and an excipient), wherein said 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 comprising one or more substrates for one or more proteases expressed in the tissue in which IFN therapy is desired and / or intended.

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

49. 35. A method of enhancing an immune response to an antigen, comprising administering to a subject an immunogenic agent that elicits an immune response to said antigen, together with an IFN proprotein according to any one of claims 1 to 34 (or a pharmaceutical composition comprising said IFN proprotein and an excipient) or a nucleic acid encoding such an IFN proprotein, e.g., as described in Section 6.10.

1.

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

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