Interferon receptor agonists and uses thereof

Abstract

The present disclosure provides interferon receptor agonists with improved safety profile and therapeutic index. The interferon receptor agonists are weakened by masking and / or reducing receptor binding as compared to the wild-type interferon. The IFN receptor agonist optionally further comprises a targeting moiety, e.g., a targeting moiety that recognizes tumor or immune cell associated antigens and directs the interferon receptor agonist to a tumor site and / or tumor reactive immune cells. The disclosure further provides pharmaceutical compositions comprising the interferon receptor agonists and methods of using the interferon receptor agonists in therapy, as well as nucleic acids encoding the interferon receptor agonists, recombinant cells expressing the interferon receptor agonists, and methods of producing the interferon receptor agonists.

Description

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 399,048, filed on August 18, 2022, U.S. Provisional Application No. 63 / 383,797, filed on November 15, 2022, and U.S. Provisional Application No. 63 / 481,312, filed on January 24, 2023, the contents of each of which are incorporated herein by reference in their entirety.

[0003] 2. Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically and is hereby incorporated by reference in its entirety. Said copy, created on August 15, 2023, is named RGN-024WO_SL.xml and is 386,251 bytes in size. 3. Background Technology

[0005] Type I interferon (IFN) is considered to directly inhibit tumor cell proliferation. Type I IFN has practicality in treating 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 cancer and Kaposi's sarcoma). See, for example, Zitvogel et al., 2015, Nat Rev Immunol 15:405-414 and Antonelli et al., 2015, Cytokine Growth Factor Rev 26:121-131.

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

[0007] One of the biggest obstacles to the use of type I IFNs in the clinic is the severe side effects associated with such treatments. The most common side effects are flu-like symptoms, hematotoxicity, elevated transaminases, nausea, fatigue, and psychiatric sequelae. These side effects hinder the dose required to achieve and maintain maximum therapeutic effect, and their occurrence may completely outweigh the clinical benefits of type I IFN treatment (Lotrich, 2009, Dialogues Clin Neurosci 11:417-425). Type I IFNs signal 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 IFNs to tumor-reactive immune cells (see, e.g., Diamond et al., 2011, J Exp Med. 208(10):1989–2003; Cauwels et al., 2018, Cancer Res. 78(2):463–474) or to the tumor microenvironment is crucial for the continued clinical use of type I IFNs. Strategies are needed to modify type I IFNs to obtain novel forms of drugs that preferentially target tumor-reactive immune cells and / or exert their activity at the tumor site, and also reduce side effects on normal IFNAR-expressing cells.

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

[0009] The present disclosure relates to Fc-coupled IFN receptor agonists having an IFN sequence (IFN portion) having an improved safety profile compared to a wild-type type I interferon molecule due to attenuation by: (i) masking, e.g., masking with an interferon receptor (IFNR) portion; (ii) one or more mutations in the IFN sequence, e.g., one or more amino acid substitutions and / or truncations; (iii) use of a native IFN sequence with low receptor affinity (e.g., a native IFN sequence with a lower receptor affinity than IFNα2b and / or IFNβ); or (iv) any combination of two or all three of (i), (ii), and (iii).

[0010] The IFN receptor agonist may further comprise a targeting moiety (or a component thereof, e.g., one chain of a Fab) at the N-terminus of, for example, one or both Fc domains. The targeting moiety comprises an antigen binding domain (ABD), which may, for example, bind to a target molecule (e.g., a tumor-associated antigen) present on the surface of a tumor or other components of the tumor microenvironment (e.g., extracellular matrix (ECM) or tumor lymphocytes), dendritic cells, or natural killer cells.

[0011] IFN receptor agonists, particularly IFN receptor agonists having an IFN masking moiety, can be activated by including one or more protease-cleavable linkers, cleavage of which (e.g., by proteases in the tumor environment) releases the IFN moiety from the masking moiety.

[0012] Exemplary IFN moieties that can be used in the IFN receptor agonists of the present disclosure are described in Section 6.3.

[0013] Exemplary masking moieties useful in the IFN receptor agonists of the present disclosure are described in Section 6.4.

[0014] Protease-cleavable linkers useful in the IFN receptor agonists of the present disclosure are described in Section 6.5.

[0015] Non-cleavable linkers useful in the IFN receptor agonists of the present disclosure are described in Section 6.6.

[0016] Targeting moieties useful in the IFN receptor agonists of the present disclosure are described in Section 6.7, and targeting moiety formats are disclosed in Section 6.8.

[0017] Fc domains that can be incorporated into the IFN receptor agonists of the present disclosure are described in Section 6.9.

[0018] Exemplary IFN receptor agonists of the present disclosure are described in Section 6.2 and numbered Examples 1-227 and 284-344.

[0019] The present disclosure further provides nucleic acids encoding IFN receptor agonists of the present disclosure. The nucleic acids encoding IFN receptor agonists can be single nucleic acids (e.g., vectors encoding all polypeptide chains of IFN receptor agonists) or multiple nucleic acids (e.g., two or more vectors encoding different polypeptide chains of IFN receptor agonists). The present disclosure further provides host cells and cell lines engineered to express nucleic acids and IFN receptor agonists of the present disclosure. The present disclosure further provides methods for producing IFN receptor agonists of the present disclosure. Exemplary nucleic acids, host cells and cell lines, and methods for producing IFN receptor agonists are described in Section 6.10 and numbered Examples 228 to 230 and 345 to 347.

[0020] The present disclosure further provides pharmaceutical compositions comprising the IFN receptor agonists of the present disclosure.Exemplary pharmaceutical compositions are described in Section 6.11 and in Numbered Examples 231 and 348.

[0021] Further provided herein are methods of using the disclosed IFN receptor agonists and pharmaceutical compositions, for example, to treat cancer. Exemplary methods are described in Section 6.12 and in numbered Examples 232 to 283 and 349 to 356. 5. Description of the Figures

[0022] Figure 1 is a schematic representation of the type I IFN-IFN receptor complex.

[0023] Figures 2A to 2X is an IFN receptor agonist ( Figures 2B to 2X ) and its components ( Figure 2A ) sketch. Figures 2A to 2X As used herein, "IFN" generally refers to any IFN portion, "IFNAR1" generally refers to any IFNAR1 portion, and "IFNAR2" generally refers to an IFNAR2 portion. Although IFN receptor agonists are shown without targeting moieties, targeting moieties can be incorporated into these IFN receptor agonists, e.g., Figures 3A to 3X shown.

[0024] Figures 3A to 3X is an IFN receptor agonist ( Figures 3B to 3X ) and its components ( Figure 3A ) sketch. Figures 3A to 3X As used herein, "IFN" generally refers to any IFN portion, "IFNAR1" generally refers to any IFNAR1 portion, and "IFNAR2" generally refers to an IFNAR2 portion. Although shown as a targeting moiety with the form of a Fab, Fab can be replaced by other types of targeting moieties (e.g., scFv).

[0025] Figures 4A to 4B Exemplary IFN molecules that can be incorporated into the IFN receptor agonists of the present disclosure are listed.

[0026] Figures 5A to 5D is a size exclusion ultra-performance liquid chromatography (SE-UPLC) profile of an exemplary IFN molecule that can be incorporated into the IFN receptor agonists of the present disclosure.

[0027] Figures 6A to 6C The in vitro activities of exemplary IFN molecules that can be incorporated into the IFN receptor agonists of the present disclosure are shown. Figure 6A The sketch image in represents the N- and C-terminal Fc fusions of IFN. Figure 6B is a graph showing the in vitro activity of exemplary IFN molecules, Fc-IFNα2b, IFNα2b-Fc, and Fc-IFNα2b x Fc, compared to unlinked IFNα2b. Figure 6C is a graph showing the activity of Fc-IFN molecules compared to different unlinked IFNs.

[0028] Figures 7A to 7Dis a SE-UPLC profile of an exemplary mutant IFN molecule that can be incorporated into the IFN receptor agonist constructs of the present disclosure. Figure 7A The SE-UPLC spectrum of the mutant IFN molecule Fc-IFNα2bR33A is depicted. Figure 7B The SE-UPLC spectrum of the mutant IFN molecule Fc-IFNα2bR149A is depicted. Figure 7C The SE-UPLC spectrum of the mutant IFN molecule Fc-IFNα2bR120A is depicted. Figure 7D The SE-UPLC spectrum of the mutant IFN molecule Fc-IFNα2bS152A is depicted.

[0029] Figures 8A to 8B Depicted are the in vitro activities of exemplary mutant IFN molecules that can be incorporated into the IFN receptor agonist constructs of the present disclosure. Figure 8A The sketch images in represent the overall structure of wild-type (WT) or mutant (Mut) Fc-IFN molecules. Figure 8B is a graph showing the in vitro activity of Fc-IFNα2b molecules with mutations affecting the IFNAR1 or IFNAR2 interface.

[0030] Figures 9A to 9F yes Figure 4A and 4B SE-UPLC profiles of some of the exemplary IFN receptor agonists shown in .

[0031] Figure 10 It shows Figure 4A and 4B Figure 3 is a graph of the in vitro activity of some of the exemplary IFN molecules shown in Figure 3 in reporter KG-1a cells.

[0032] Figures 11A to 11B is a graph showing the in vitro activity of exemplary IFN molecules and receptor agonists in reporter KG-1a cells. Figure 11A The effect of receptor masking on IFN activity using the homodimeric form is shown. Figure 11B Depicted are the differences in reporter gene activity between different exemplary heterodimeric knob-in-hole (KiH) Fc-IFN molecules.

[0033] Figures 12A to 12B is shown as CD8 in murine hIFNAR + Graph of in vitro activity measured by pSTAT flow cytometric analysis of exemplary IFN molecules and receptor agonists in T cells. Figure 12A The effect of receptor masking on IFN activity using the homodimeric form is shown. Figure 12B Draws the following Figure 11B Differences in activity between different exemplary heterodimeric KiH Fc-IFN molecules in FIG.

[0034] Figures 13A to 13B is shown as expressed by murine hIFNAR CD11b + Graph of in vitro activity measured by pSTAT flow cytometric analysis of exemplary IFN molecules and receptor agonists in cells. Figure 13A The effect of receptor masking on IFN activity using the homodimeric form is shown, and Figure 13B Shown Figure 11B Differences in activity between heterodimeric KiH Fc-IFN molecules of the same group.

[0035] Figures 14A to 14B is shown as CD4 in murine hIFNAR + Graph of in vitro activity measured by pSTAT flow cytometric analysis of exemplary IFN molecules and receptor agonists in T cells. Figure 14A The effect of receptor masking on IFN activity using the homodimeric form is shown, and Figure 14B Shown Figure 11B The differences in activity between the heterodimeric KiH Fc-IFN molecules are shown in .

[0036] Figures 15A to 15B is a graph showing in vitro activity as measured by pSTAT flow cytometric analysis of exemplary IFN molecules and receptor agonists in murine hIFNAR NK cells. Figure 15A The effect of receptor masking on IFN activity using the homodimeric form is shown, and Figure 15B Shown Figure 11B The differences in activity between the heterodimeric KiH Fc-IFN molecules are shown in .

[0037] Figures 16A to 16B is a graph showing the in vitro activity of exemplary IFN molecules and receptor agonists in two different types of PBMC cells as measured by pSTAT flow cytometric analysis. Figure 16A Shows that IFN molecules are expressed in PBMC CD8 + activity in cells, and Figure 16B shows the use of PBMC NK cells Figure 16A The activity of the same IFN molecules in

[0038] Figure 17 is a graph showing the in vitro activity of exemplary single- and dual-masked monovalent IFN molecules and receptor agonists in reporter KG-1a cells.

[0039] Figure 18is a graph showing the in vitro activity of exemplary single- and double-masked bivalent IFN molecules and receptor agonists in reporter KG-1a cells.

[0040] Figures 19A to 19D is a graph showing the effect of PDL1 targeting on the in vitro activity of exemplary IFN molecules and receptor agonists in reporter KG-1a cells. Figure 19A Shown are the activities of monovalent IFNAR2-masked IFN molecules and controls in PDL1 OEKG-1a cells. Figure 19B Shown are the activities of monovalent IFNAR2-masked IFN molecules and controls in PDL1 KO KG-1a cells. Figure 19C Shown are the activities of bivalent IFNAR2-masked IFN molecules and controls in PDL1 OE KG-1a cells. Figure 19D Shown are the activities of bivalent IFNAR2-masked IFN molecules and controls in PDL1 KO KG-1a cells.

[0041] Figures 20A to 20F Shown is the effect of linker length on the in vitro activity of exemplary dual-masked monovalent IFN molecules and receptor agonists in reporter KG-1a cells. Figures 20A to 20D is a schematic representation of dual masked monovalent IFN receptor agonists with linkers of varying lengths between IFNα2b and the masking moiety. Figure 20E Shown is the effect of linker length on the activity of monovalent bimasked IFN molecules and controls in PDL1 OE KG-1a cells. Figure 20F Shown is the effect of linker length on the activity of monovalent dual-masked IFN molecules and controls in PDL1 KO KG-1a cells. 6. Specific Implementation Methods

[0042] 6.1. Definitions

[0043] As used herein, the following terms have the following meanings:

[0044] ABD chain, targeting part chain: The targeting moiety and the antigen binding site (ABD) therein can exist as one (e.g., in the case of scFv or scFab) polypeptide chain or be formed by the association of more than one polypeptide chain (e.g., in the case of Fab or Fv). As used herein, the terms "ABD chain" and "targeting moiety chain" refer to all or part of the ABD or targeting moiety present on a single polypeptide chain. The use of the terms "ABD chain" or "targeting moiety chain" is intended for convenience and descriptive purposes only and does not imply a particular configuration or method of production. In addition, reference to the ABD or targeting moiety when describing an IFN receptor agonist encompasses the ABD chain or targeting moiety chain unless the context indicates otherwise. Thus, when describing an IFN receptor agonist in which an Fc domain is operably linked to a targeting moiety, the Fc domain can be covalently linked directly or indirectly via a peptide bond (e.g., via a linker) to, for example, (1) a first ABD or targeting moiety chain of a Fab or Fv (wherein the other components of the Fab or Fv are on a second associated ABD or targeting moiety chain) or (2) an ABD or targeting moiety chain containing an scFv or scFab.

[0045] approximately Throughout the specification, the terms "about," "approximately," and the like are used before numbers to indicate that the number is not necessarily exact (e.g., to account for fractions, variations in measurement accuracy and / or precision, timing, etc.). It should be understood that disclosure of "about X" or "approximately X" where X is a number also includes disclosure of "X." Thus, for example, a disclosure of an embodiment where a sequence has "about X% sequence identity" to another sequence also includes disclosure of an embodiment where the sequence has "X% sequence identity" to the other sequence.

[0046] Activate The term "activation" and the like, as used in connection with the IFN receptor agonists of the present disclosure, refers to the protease-mediated enzymatic cleavage of a protease-cleavable linker, which results in the release of the IFN portion from the masking moiety (e.g., a receptor-based masking moiety as described herein).

[0047] and, or : Unless otherwise indicated, the conjunction "or" should be used in its proper sense as a Boolean logical operator, encompassing both selection of features among alternatives (A or B, where the selection of A is mutually exclusive with B) and selection of joint features (A or B, where both A and B are selected). In some places in this text, the term "and / or" is used for the same purpose, which should not be interpreted as implying that "or" is used to refer to mutually exclusive alternatives.

[0048] AntibodyAs used herein, the term "antibody" refers to a polypeptide (or polypeptide group) 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 comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: 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 region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) 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 can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Both light and heavy chains are divided into structural and functional homologous regions. The terms "constant" and "variable" are used functionally. In this regard, it should be understood that the variable domains of both the light chain (VL) and heavy chain (VH) portions determine antigen recognition and specificity. In contrast, the constant domains of the light chain (CL) and heavy chain (CH1, CH2 or CH3) confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, complement fixation, etc. By convention, the numbering of the constant region domains increases as they become further away from the antigen-binding domain or amino terminus of the antibody. The N-terminus is the variable region, and at the C-terminus is the constant region; the CH3 and CL domains represent the carboxyl termini of the heavy and light chains of natural antibodies, respectively. For convenience, and unless the context otherwise dictates, reference to antibodies also refers to antibody fragments and engineered antibodies comprising non-naturally occurring antigen-binding domains and / or antigen-binding domains having non-natural structures.

[0049] 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 VL, VH, CL, and CH1 domains; F(ab)2 fragments, bivalent fragments comprising two Fab fragments connected by a disulfide bridge at the hinge region; Fd fragments, consisting of VH and CH1 domains; Fv fragments, consisting of the VL and VH domains of a single arm of an antibody; dAb fragments (Ward et al., 1989, Nature 341:544-546), consisting of a VH domain; and isolated complementary determining regions (CDRs). Thus, the term "antibody fragment" encompasses proteolytic fragments of antibodies (e.g., Fab and F(ab)2 fragments) and engineered proteins (e.g., scFv) comprising one or more portions of an antibody. Antibody fragments may also be incorporated into single domain antibodies, macrobodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, eg, Hollinger and Hudson, 2005, Nature Biotechnology 23: 1126-1136).

[0050] association In the context of IFN receptor agonists, the term "association" refers to a functional relationship between two or more polypeptide chains. Specifically, the term "association" means that two or more polypeptides associate with each other (e.g., non-covalently through molecular interactions or covalently through one or more disulfide bridges or chemical crosslinks) to produce a functional IFN receptor agonist. Examples of associations that may exist in the IFN receptor agonists of the present disclosure include, but are not limited to, associations between Fc domains forming an Fc region (e.g., homodimers or heterodimers as described in Section 6.9), associations between the VH and VL regions in a Fab or Fv, and associations between the CH1 and CL in a Fab.

[0051] cancer: The term "cancer" refers to a disease characterized by the uncontrolled (and usually rapid) growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers are described herein, and these examples include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, adrenal cancer, ganglion cancer, bile duct cancer, bone cancer, endometrial cancer, eye cancer, fallopian tube cancer, reproductive tract cancer, colorectal cancer, meningeal cancer, esophageal cancer, peritoneal cancer, pituitary cancer, penile cancer, placental cancer, pleural cancer, salivary gland cancer, small intestine cancer, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, upper respiratory tract digestive system cancer, urinary tract cancer, vaginal cancer, vulvar cancer, lymphoma, leukemia, lung cancer, etc., for example, any TAA-positive cancer of any type in the aforementioned types.

[0052] complementarity determining region:As used herein, the term "complementarity determining region" or "CDR" refers to the sequence of amino acids within the antibody variable region that confer antigen specificity and binding affinity. For example, typically, there are three CDRs (e.g., CDR-H1, CDR-H2, and CDR-H3) in each heavy chain variable region, and three CDRs (CDR-L1, CDR-L2, and CDR-L3) in each light chain variable region. The precise amino acid sequence boundaries of a given CDR can be determined using any of many well-known schemes, including by Kabat et al., 1991, "Sequences of Proteins of Immunological Interest," 5th Edition 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 (Lefranc, 1999, The Immunologist 7:132-136; those described in Lefranc et al., 2003, Dev. Comp. Immunol. 27:55-77 ("IMGT" numbering scheme). For example, for the classical form, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3). Under Chothia, the CDR amino acid residues in VH are numbered 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3). The amino acid residues in the VH are numbered as 26-32 (CDR-H1), 52-56 (CDR-H2) and 95-102 (CDR-H3); and the amino acid residues in the VL are numbered as 26-32 (CDR-L1), 50-52 (CDR-L2) and 91-96 (CDR-L3). By combining the CDR definitions of both Kabat and Chothia, the CDRs are composed 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) (according to "Kabat" numbering). Under IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0053] constant domain : The term "constant domain" refers to the CH1, CH2, CH3 or CL domain of an immunoglobulin.

[0054] The term "CH1 domain" refers to the heavy chain constant region of the hinge connecting the variable domain to the heavy chain constant domain. In some embodiments, the term "CH1 domain" refers to the region of the immunoglobulin molecule spanning amino acids 118 to 215 (EU numbering). The term "CH1 domain" encompasses wild-type CH1 domains and 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 to 3, 1 to 5, 3 to 5, and / or a maximum of 5, 4, 3, 2, or 1 mutation (e.g., substitution, deletion, and / or addition). Exemplary CH1 domains include CH1 domains with mutations that change the biological activity (such as ADCC, CDC, or half-life) of the antibody.

[0055] The term "CH2 domain" refers to the heavy chain constant region that connects the hinge to the CH3 domain in the heavy chain constant domain. In some embodiments, the term "CH2 domain" refers to the region of the immunoglobulin molecule spanning amino acids 238 to 340 (EU numbering). The term "CH2 domain" encompasses wild-type CH2 domains and 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 to 3, 1 to 5, 3 to 5, and / or a maximum of 5, 4, 3, 2, or 1 mutation (e.g., substitution, deletion, and / or addition). Exemplary CH2 domains include CH2 domains with mutations that alter the biological activity of the antibody (such as ADCC, CDC, purification, dimerization, and half-life).

[0056] The term "CH3 domain" refers to the heavy chain constant region located at the C-terminus of the CH2 domain in the heavy chain constant domain. In some embodiments, the term "CH3 domain" refers to the region of the immunoglobulin molecule spanning amino acids 341 to 447 (EU numbering). The term "CH3 domain" encompasses wild-type CH3 domains and variants thereof (e.g., non-naturally occurring CH3 domains or modified CH3 domains). For example, the term "CH3 domain" includes wild-type IgG1, IgG2, IgG3, and IgG4 CH3 domains and variants thereof having 1, 2, 3, 4, 5, 1 to 3, 1 to 5, 3 to 5, and / or a maximum of 5, 4, 3, 2, or 1 mutation (e.g., substitution, deletion, and / or addition). Exemplary CH3 domains include CH3 domains with mutations that alter the biological activity of the antibody (such as ADCC, CDC, purification, dimerization, and half-life).

[0057] 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 to 3, 1 to 5, 3 to 5, and / or a maximum of 5, 4, 3, 2, or 1 mutations (e.g., substitutions, deletions, and / or additions).

[0058] Effector function: The term "effector function" refers to the activity of an antibody molecule mediated by the binding of domains other than the antigen-binding domain of the antibody (usually mediated by the binding of effector molecules). Effector functions include complement-mediated effector functions, which are mediated by, for example, the C1 component of complement being bound to the antibody. Complement activation is important in the conditioning and lysis of cellular pathogens. Complement activation can also stimulate inflammatory responses and may also be involved in autoimmune hypersensitivity reactions. Effector functions also include Fc receptor (FcR)-mediated effector functions, which can be triggered when the constant domain of the antibody is bound to the Fc receptor (FcR). Binding of antibodies to Fc receptors on the cell surface can trigger many important and diverse biological responses, including phagocytosis 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, control of placental transfer, and immunoglobulin production. The effector function of an antibody can be changed by changing (e.g., enhancing or reducing) the affinity of the antibody for effector molecules such as Fc receptors or complement components. Binding affinity is usually changed by modifying the effector molecule binding site, and in this case, it is appropriate to locate the site of interest and modify at least a portion of the site in a suitable manner. It is also envisioned that changes in the binding site on the antibody for the effector molecule do not need to significantly change the overall binding affinity, but may change the geometry of the interaction, making the effector mechanism invalid as in non-productive binding. It is further envisioned that the effector function can also be changed by modifying sites that are not directly involved in effector molecule binding but otherwise participate in the execution of the effector function.

[0059] Epitope : An epitope or antigenic determinant is a portion of an antigen that is recognized by an antibody or other antigen binding moiety as described herein. An epitope can be linear or conformational.

[0060] Fab: The term "Fab" refers to a pair of polypeptide chains, the first polypeptide chain comprising the variable heavy chain (VH) domain of the antibody, which is operably connected (usually at the N-terminus) to the first constant domain (referred to herein as C1), and the second polypeptide chain comprising the variable light chain (VL) domain at the N-terminus of the antibody, which is operably connected (usually at the N-terminus) to the second constant domain (referred to herein as C2) that can be paired with the first constant domain. In natural antibodies, VH is located at the N-terminus of the first constant domain (CH1) of the heavy chain, and VL is located at the N-terminus of the constant domain of the light chain (CL). The Fab of the present disclosure can be arranged according to the natural orientation or include domain substitutions or exchanges that promote correct VH and VL pairing. For example, the CH1 and CL domain pairs in the Fab can be replaced with a CH3 domain pair to promote correct modified Fab chain pairing in heterodimeric molecules. CH1 and CL can also be reversed so that CH1 is attached to VL and CL is attached to VH, and this configuration is generally referred to as Crossmab. The term "Fab" encompasses single chain Fab.

[0061] Fc domain and Fc region : The term "Fc domain" refers to a portion of a heavy chain that pairs with the corresponding portion of another heavy chain. In some embodiments, the Fc domain comprises a CH2 domain followed by a CH3 domain, with or without a hinge region at the N-terminus of the CH2 domain. The term "Fc region" refers to the region formed by the association of two heavy chain Fc domains. The two Fc domains within an Fc region can be identical or different. In native antibodies, the Fc domains are typically identical, but one or both Fc domains may be modified to allow heterodimerization, for example, via knob-in-hole interactions.

[0062] Fv : The term "Fv" refers to the smallest antibody fragment that can be derived from an immunoglobulin containing a complete target recognition and binding site. This region is composed of a dimer (VH-VL dimer) in tight non-covalent association of a heavy chain variable domain and a light chain variable domain. It is in this configuration that the three CDRs of each variable domain interact to define the target binding site on the surface of the VH-VL dimer. Typically, six CDRs confer target binding specificity to an antibody. However, in some cases, even a single variable domain (or half of an Fv containing only three target-specific CDRs) can also have the ability to recognize and bind to a target. Mentioning VH-VL dimers herein does not imply expressing any particular configuration. When present on a single polypeptide chain (e.g., scFv), VH is located at the N-terminus or C-terminus of VL.

[0063] Half antibody: The term "half antibody" refers to a molecule that comprises at least one Fc domain and can associate with another molecule comprising an Fc domain through, for example, a disulfide bridge or a molecular interaction. A half antibody can be composed of one polypeptide chain or more than one polypeptide chain (e.g., two polypeptide chains of a Fab). An example of a half antibody is a molecule comprising the heavy and light chains of an antibody (e.g., an IgG antibody). Another example of a half antibody is a molecule comprising a first polypeptide comprising a VL domain and a CL domain and a second polypeptide comprising a VH domain, a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, wherein the VL and VH domains form an ABD. Yet another example of a half antibody is a polypeptide comprising a scFv domain, a CH2 domain, and a CH3 domain.

[0064] The IFN receptor agonists of the present disclosure generally comprise two half antibodies, each half antibody comprising an IFN portion masked by one or two receptor portions (e.g., IFNR masking moieties). One or two masking moieties can be located in the same half antibody as the IFN portion or in another half antibody, such as Figures 2B to 2X In some embodiments, the IFN portion and / or the masking moiety have an adjacent protease-cleavable linker that cleaves and releases the masking moiety from the IFN portion, thereby activating the IFN portion. Exemplary positions of protease-cleavable linkers in half antibodies are shown in Table 1. One or both half antibodies in the IFN receptor agonist may further comprise a targeting moiety, such as a scFv or Fab. Exemplary IFN receptor agonists comprising a targeting moiety are shown in FIG. Figures 3B to 3X and listed in Table 3.

[0065] The term "half antibody" is used for descriptive purposes only and does not imply a particular configuration or method of production. Describing half antibodies as the "first" half antibody, the "second" half antibody, the "left" half antibody, the "right" half antibody, etc. is merely for convenience and descriptive purposes.

[0066] Host cells or recombinant host cells: The term "host cell" or "recombinant host cell" refers to a cell that has been genetically engineered, for example, by the introduction of a heterologous nucleic acid. It should be understood that such terms refer not only to the specific subject cell, but also to the progeny of such cells. Because certain modifications may occur in subsequent generations due to mutations 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. The host cell may transiently carry the heterologous nucleic acid, for example, on an extrachromosomal heterologous expression vector, or stably carry the heterologous nucleic acid, for example, by integrating the heterologous nucleic acid into the host cell genome. For the purpose of expressing the IFN receptor agonists of the present disclosure, the host cell is preferably a cell line of mammalian origin or mammalian-like characteristics, such as monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293), baby hamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2 / 0, HeLa, Madin-Darby bovine kidney (MDBK), myeloma and lymphoma cells or derivatives thereof and / or engineered variants. Engineered variants include, for example, derivatives that grow at a higher density than the original cell line and / or derivatives with modified glycan profiles and / or site-specific integration site derivatives.

[0067] interferon : The term "interferon" as used herein refers to full-length interferon or modified interferon, such as truncated and / or mutant interferon. In certain embodiments, compared to the corresponding wild-type interferon, the modified interferon is weakened (for example, as described in Section 8.2.3, in an in vitro luciferase reporter gene assay, retain 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 certain embodiments, the modified interferon weakens the range defined by any two values ​​in the aforementioned values, for example, 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 interferon substantially retains the biological activity of the corresponding wild-type interferon (for example, as described in Section 8.2.3, in an in vitro luciferase reporter gene assay, retains at least 50% activity). Interferons include type I interferons (eg, interferon-α and interferon-β) and type II interferons (eg, interferon-γ).

[0068] connector : As used herein, the term "linker" refers to a protease cleavable linker or a non-cleavable linker.

[0069] Non-cleavable linker: As used herein, a non-cleavable linker refers to a peptide whose amino acid sequence lacks a substrate sequence for a protease (e.g., a protease as described in Section 6.5.1 that recognizes and cleaves a specific sequence motif, e.g., a substrate as described in Section 6.5.2).

[0070] operably connected : The term "operably linked" refers to the functional relationship between two or more peptides or polypeptide domains or nucleic acid (e.g., DNA) segments. In the context of fusion proteins or other polypeptides, the term "operably linked" refers to two or more amino acid fragments being connected to produce a functional polypeptide. For example, in the context of an IFN receptor agonist of the present disclosure, separate components (e.g., an Fc domain and an IFN portion) can be operably linked directly or via a peptide linker sequence. In the context of a nucleic acid encoding a fusion protein (such as a half-antibody of an IFN receptor agonist of the present disclosure), "operably linked" means that the two nucleic acids are connected so that the amino acid sequence encoded by the two nucleic acids remains in frame. In the context of transcriptional regulation, the term refers to the functional relationship between a transcriptional regulatory sequence and a transcribed sequence. For example, if a promoter or enhancer sequence stimulates or regulates the transcription of a coding sequence in an appropriate host cell or other expression system, the promoter or enhancer sequence is operably linked to the coding sequence.

[0071] Polypeptides, peptides and proteins : The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.

[0072] Preprotein A "proprotein" is an inactive protein precursor that can be activated by the proteolytic action of a protease. Thus, a proprotein is "protease-activatable."

[0073] Protease As used herein, the term "protease" refers to any enzyme that catalyzes the hydrolysis of peptide bonds. Generally speaking, proteases useful in the present disclosure, e.g., the proteases described in Section 6.5.1, recognize and cleave specific sequence motifs, e.g., substrates described in Section 6.5.2. Preferably, the protease is expressed at a higher level in cancer tissue than in normal tissue.

[0074] Protease-cleavable linker As used herein, the term "protease cleavable linker" or "PCL" refers to a peptide whose amino acid sequence contains one or more (e.g., two, three, or more) substrate sequences for one or more proteases. Exemplary protease cleavable linkers are described in Section 6.5, and exemplary protease cleavable linker sequences are disclosed in Section 6.5.4.

[0075] Identification: As used herein, the term "recognizes" refers to an antibody or antibody fragment (eg, targeting moiety) that finds and interacts with (eg, binds to) its epitope.

[0076] 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 linkers are arranged in one of the following orders in an N-terminal to C-terminal orientation: (a) VH-CH1-linker-VL-CL, (b) VL-CL-linker-VH-CH1, (c) VH-CL-linker-VL-CH1, or (d) VL-CH1-linker-VH-CL. The linker is suitably a non-cleavable linker of at least 30 amino acids, preferably between 32 and 50 amino acids. Single-chain Fab fragments are typically stabilized via a natural disulfide bond between the CL domain and the CH1 domain. In addition, these single-chain Fab molecules can be further stabilized by generating interchain disulfide bonds by inserting cysteine ​​residues (e.g., at position 44 in the VH domain and position 100 in the VL domain according to Kabat numbering).

[0077] Single-chain Fv or scFv : As used herein, the term "single-chain Fv" or "scFv" refers to an ABD comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further includes 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 scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, ed. (1994), Springer-Verlag, New York, pp. 269-315. VH and VL can be arranged in the order of N-terminus to C-terminus (i.e., VH-VL or VL-VH), typically separated by a linker, for example, as listed in Table E.

[0078] spacer As used herein, the term "spacer" refers to a peptide incorporated into a linker containing a substrate whose amino acid sequence is not a substrate for a protease. Spacers can be used to separate substrates from other domains (e.g., ABDs) in a molecule. In some aspects, the residues in the spacer minimize the action of aminopeptidases and / or exopeptidases to prevent the cleavage of the N-terminal amino acid.

[0079] Specific (or selective) binding: The term "specifically (or selectively) binds" to an antigen or epitope refers to a binding reaction that determines the presence of a homologous antigen or epitope in a heterogeneous population of proteins and other molecules. The binding reaction can, but need not, be mediated by an antibody or antibody fragment. The term "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. Therefore, this cross-species reactivity does not itself change the classification of the antigen-binding domain as a "specific" binder. In certain embodiments, the antigen-binding domain of the present disclosure that specifically binds to a human antigen has cross-species reactivity with one or more non-human mammalian species (e.g., primate species (including but not limited to one or more of cynomolgus monkeys, macaques, and pig-tailed monkeys)) or rodents (e.g., house mice).

[0080] Subjects : The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians and reptiles. In a preferred embodiment, the subject is a human.

[0081] substrate : The term "substrate" refers to the peptide sequence on which the protease will act and in which the protease will cleave peptide bonds.

[0082] target molecule As used herein, the term "target molecule" refers to any biological molecule (e.g., protein, carbohydrate, lipid, or combination thereof) expressed on the surface of a cell or in the extracellular matrix to which the targeting moiety in the IFN receptor agonists of the present disclosure can specifically bind.

[0083] Targeting moiety : The term "targeting moiety" as used herein refers to any molecule or binding portion (e.g., an immunoglobulin or antigen binding fragment) that can bind to the cell surface or extracellular matrix molecules at the site to which the IFN receptor agonist of the present invention is to be located (e.g., on a tumor cell or on a lymphocyte in a tumor microenvironment). In some embodiments, the targeting moiety binds to a TAA. In other embodiments, the targeting moiety binds to a TCA. In addition to localizing the IFN receptor agonist to a specific site, the targeting moiety can also have functional activity. For example, a targeting moiety that is bound to a checkpoint inhibitor such as PD1 can also exhibit anti-tumor activity or enhance the anti-tumor activity of IFN, for example, by inhibiting PD1 signaling.

[0084] T cell antigen, TCA: The term "T cell antigen" or "TCA" refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) expressed on the surface of a T lymphocyte and can be used to preferentially target 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.

[0085] tumor Term " tumor " is used interchangeably with term " cancer " in this article, for example, both terms encompass solid and liquid, for example, diffuse or circulating tumors.As used herein, term " cancer " or " tumor " includes precancerous as well as malignant cancers and tumors.

[0086] Tumor-associated antigens, TAAs : 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 a cancer cell, either in its entirety or as a fragment (e.g., MHC / peptide), and which can be used to preferentially target an agent to a cancer cell. In some embodiments, a TAA is a marker expressed by normal cells and cancer cells (e.g., a lineage marker). In some embodiments, a TAA is a cell surface molecule that is overexpressed in a cancer cell compared to a normal cell, e.g., 1-fold overexpression, 2-fold overexpression, 3-fold overexpression, or more compared to a normal cell. In some embodiments, a TAA is a cell surface molecule that is inappropriately synthesized in a cancer cell, e.g., a molecule that contains a deletion, addition, or mutation compared to a molecule expressed on a normal cell. In some embodiments, a TAA is expressed exclusively on the cell surface of a cancer cell, either in its entirety or as a fragment (e.g., MHC / peptide), and is not synthesized or expressed on the surface of a normal cell. Thus, the term "TAA" encompasses antigens that are specific to cancer cells, sometimes referred to in the art as tumor-specific antigens (TSAs).

[0087] treatment (treat / treatment / treating):As used herein, the term "treat / treatment / treating" refers to a reduction or improvement in the progression, severity and / or duration of a disease, or an improvement in one or more symptoms (preferably, one or more discernible symptoms) of a disease caused by the administration of one or more IFN receptor agonists of the present disclosure. In some embodiments, the disease is a proliferative disease, and the terms "treat, treatment and treating" refer to an improvement in at least one measurable physical parameter of a proliferative disease (such as the growth of a tumor) that is not necessarily discernible by the patient. In other embodiments, the terms "treat, treatment and treating" refer to physically inhibiting the progression of a proliferative disease by, for example, stabilizing discernible symptoms, physiologically by, for example, stabilizing physical parameters, or both. In other embodiments, the terms "treat, treatment and treating" refer to a reduction or stabilization of tumor size or cancer cell count.

[0088] Universal light chain, UCL As used herein, the term "universal light chain" or "ULC" refers to a light chain variable region (VL) that can be paired 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 be paired with the heavy chain region of a targeting moiety and can also be paired with other heavy chain regions. A ULC can also include a constant domain, such as the CL domain of an antibody. A universal light chain is also referred to as a "common light chain."

[0089] 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.

[0090] 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.

[0091] IFN receptor agonists

[0092] The present disclosure relates to IFN receptor agonists comprising an IFN portion that is attenuated compared to wild-type interferon. The IFN portion can be attenuated by: (i) masking by a type I interferon receptor (IFNR) portion (e.g., as described in Section 6.4); (ii) one or more mutations in the IFN portion compared to wild-type interferon, e.g., one or more amino acid substitutions and / or truncations (e.g., as described in Section 6.3); (iii) use of a natural IFN sequence with low receptor affinity; or (iv) any combination of two or all three of (i), (ii), and (iii).

[0093] Typically, IFN receptor agonists are composed of two half antibodies, which include a pair of Fc domains that associate to form an Fc region (usually including a hinge sequence).

[0094] In the IFN receptor agonists of the present disclosure, the two half antibodies together comprise at least one interferon (IFN moiety), but may comprise two or more IFN moieties.

[0095] The IFN moieties in the IFN receptor agonists can each be masked by one or two interferon receptor (IFNR) moieties, for example, interferon alpha receptor 1 (IFNAR1) and / or interferon alpha receptor 2 (IFNAR2) moieties.

[0096] In some embodiments, the IFN receptor agonist further comprises a targeting moiety, eg, an antigen binding domain of an antibody, which targets the IFN receptor agonist to a selected tissue, eg, cancer tissue.

[0097] Exemplary IFN receptor agonists are shown in Figures 2B to 2X and 3B to 3X.

[0098] Table 1 below describes exemplary half antibodies that can be incorporated into the IFN receptor agonists of the present disclosure. As is apparent from Table 1, each half antibody can comprise one or more polypeptide chains. For convenience, when describing combinations of half antibodies in the IFN receptor agonists of the present disclosure, each half antibody described in Table 1 is referred to herein as an "exemplary monomer."

[0099]

[0100]

[0101]

[0102]

[0103] IFN receptor agonist can further include one or two protease cleavable linkers (PCL) in each half antibody, while other linkers are non-cleavable. In certain embodiments, all linkers are non-cleavable. Exemplary protease cleavable linkers are described in Section 6.5, and non-cleavable linkers are described in Section 6.6. In the exemplary monomers of Table 1, the linker identified by an asterisk is optionally a protease cleavable linker, and the linker identified by two asterisks represents two linkers that can be protease cleavable linkers in a specific half antibody. In the case where two linkers in the exemplary monomer are identified as being optionally protease cleavable, in some embodiments, the N-terminal linker is protease cleavable, in other embodiments, the C-terminal linker is protease cleavable, and in yet other embodiments, both linkers are protease cleavable. The Fc domain in the polypeptide chain described in Table 1 preferably includes a hinge domain as listed in Section 6.9.3.

[0104] Table 2 below shows exemplary monomer pairs and their components that can be used in the IFN receptor agonists of the present disclosure. Additional components not specifically listed, for example, targeting moieties, can be incorporated into the IFN receptor agonists.

[0105]

[0106]

[0107] In some embodiments, this configuration is advantageously used for IFN receptor agonists comprising a targeting moiety that binds to a TAA or ECM target molecule expressed in the tumor environment. Without being bound by theory, the inventors believe that in this configuration, wherein one or more linkers are protease-cleavable linkers, the targeting moiety targets the IFN receptor agonist to the tumor environment, wherein the protease cleaves these protease-cleavable linkers, thereby resulting in the release of the IFN protein comprising the IFN portion and the linker sequence. This locally activated IFN protein subsequently induces an immune response against cancer cells. Table 3 below shows additional exemplary monomer pairings that can be used in the IFN receptor agonists of the present disclosure. The IFN receptor agonists identified in Table 3 comprise one or two targeting moieties.

[0108]

[0109]

[0110] The order and length of the hinge and linker sequences can vary, as can the order of the IFN moieties (containing full-length or N-terminal and / or C-terminal truncated IFN sequences and amino acid substitutions). Exemplary IFN moieties are described in Section 6.3 and include IFNα- and IFNβ-based moieties as described in Sections 6.3.1 and 6.3.2 below, and other type I IFN-based moieties as described in Section 6.3.3. Exemplary IFN receptor moieties are disclosed in Section 6.4. Exemplary protease-cleavable linker sequences are disclosed in Section 6.5. Exemplary non-cleavable linker and hinge sequences are disclosed in Sections 6.6 and 6.9.3, respectively. Exemplary targeting moieties are disclosed in Section 6.7. Exemplary Fc domains (including Fc domains suitable for heterodimerization when the two half antibodies of the IFN receptor agonist are not identical) are described in Section 6.9.

[0111] IFN

[0112] There are two major classes of IFNs: type I (IFN-α subtype, IFN-β, etc.) and type II (IFN-γ). Additional IFNs (IFN-like cytokines; IFN-λ subtypes) have also been identified.

[0113] The IFN portion of the present disclosure may include any wild type or modified (e.g., truncated and / or mutant) IFN or IFN-like cytokine sequence, but preferably a type I IFN portion. Type I IFN binds to the heterodimer plasma membrane receptor IFNAR consisting of IFNAR1 and IFNAR2, which is widely expressed in all nucleated cells. Ligand binding is initiated by the high-affinity receptor subunit IFNAR2 (Piehler et al., 2012, Immunological Reviews, doi.org / 10.1111 / imr.12001). Therefore, type I IFN can act on almost all cells of the body. Sixteen type I interferon subtypes have been identified, and their affinity to IFNAR2 and the intrinsic variability of activity are different.

[0114] In some embodiments, the type I IFN portion is an interferon-α (IFNα) portion. In other embodiments, the type I IFN portion is an interferon-β (IFNβ) portion.

[0115] In other embodiments, the type I IFN portion is an interferon-ω (IFNω), interferon-ε (IFNε), or interferon-κ (IFNκ) portion.

[0116] The type I IFN portion may include a sequence that differs from the wild-type IFN sequence by one or more mutations (e.g., substitutions, deletions, or insertions). Substitutions that weaken IFN activity by reducing receptor binding may be appropriately used. Amino acids with N- or C-terminal deletions (or truncations) may also be used, for example, truncations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids from the N- and / or C-termini of mature type I IFN. Without being bound by theory, the inventors believe that the terminal truncations impose additional steric constraints on the IFN portion and reduce IFN activity until the protease-cleavable linker in the IFN receptor agonist is cleaved.

[0117] Further details of exemplary type I IFN portions are provided below.

[0118] 6.3.1. Interferon-α

[0119] The IFNα gene is a member of the interferon α gene cluster on chromosome 9. The encoded cytokine is a member of the type I interferon family, produced in response to viral infection and a key component of the innate immune response, with potent antiviral, antiproliferative, and immunomodulatory properties. IFNα is a family of proteins with at least 15 known human IFNα subtypes. The major subtypes identified are IFNα1, IFNα2, IFNα8, IFNα10, IFNα14, and IFNα21.

[0120] The IFNα1 gene has two allelic variants: IFNα1a and IFNα1b. The amino acid sequence of human IFNα1a was assigned UniProtKB accession number P01562 and is reproduced below with the signal peptide underlined:

[0121] MASPFALLMVLVVLSCKSSCSLG CDLPETH SLDNRRTLML LAQMSRISPS

[0122] SCLMDRHDFG FPQEEFDGNQ FQKAPAISVL HELIQQIFNL FTTKDSSAAW

[0123] DEDLLDKFCT ELYQQLNDLE ACVMQEERVG ETPLMNADSI LAVKKYFRRI

[0124] TLYLTEKKYS PCAWEVVRAE IMRSLSLSTN LQERLRRKE(SEQ ID NO:1)

[0125] The human IFNα1b gene differs from the IFNα1a allelic variant by a single base change in the coding region, resulting in a single change in the amino acid sequence (Val114 instead of Ala114 in the mature protein, corresponding to Val137 instead of Ala137 in the full-length polypeptide).

[0126] There are three allelic variants of the IFNα2 allele: IFNα2a, IFNα2b, and IFNα2c. The IFNα2b allele is the dominant allele, while the IFNα2a allele is the subdominant, and IFNα2c is only a minor allelic variant. The amino acid sequence of human IFNα2 is assigned UniProtKB accession number P01563. The sequence of the IFNα2b allele is reproduced below, with the signal peptide underlined:

[0127] MALTFALLVALLVLSCKSSCSVG CDLPQTH SLGSRRTLML LAQMRRISLF

[0128] SCLKDRHDFG FPQEEFGNQF QKAETIPVLH EMIQQIFNLF STKDSSAAWD

[0129] ETLLDKFYTE LYQQLNDLEA CVIQGVGVTE TPLMKEDSIL AVRKYFQRIT

[0130] LYLKEKKYSP CAWEVVRAEI MRSFSLSTNL QESLRSKE(SEQ ID NO:2)

[0131] IFNα2b has an arginine (R) at position 23 of the mature protein, while IFNα2a has a lysine (K). Thus, in some embodiments, the IFNα2 portion has an arginine at a position corresponding to position 23 of the mature protein. In other embodiments, the IFNα2 portion has a lysine at a position corresponding to position 23 of the mature protein.

[0132] 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 truncation thereof having up to 15 amino acids at its N-terminus and / or C-terminus (e.g., 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).

[0133] 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 described in WO 2013 / 107791, U.S. Patent No. 8,258,263, WO2007 / 000769A2, WO2008 / 124086, WO2010 / 030671, WO2018 / 144999A1, and WO2015 / 007520, WO 2013 / 059885, WO2020156467A1, and WO2021 / 126929A1. In some embodiments, the IFNα portion comprises:

[0134] a) one or more substitutions selected from the group consisting of: 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, D82 E, Y85A, T86I, Y89A, D114R or D114A, L117A, R120A or R120E or R120K, K121E, R125A, K133A, K134A, R144A, A145G or A145M, M148A, R149A, R149K, S152A, L153A, N156A; and / or

[0135] b) one or more substitutions described in WO2007000769A2 at amino acids 57 to 89 and 159 to 165; and / or

[0136] c) Alanine, glycine or threonine described in WO2021126929A1 at 9, 17, 47, 65, 66,

[0137] One or more amino acid substitutions at positions 117, 123, 128, 147 and 157.

[0138] The amino acid positions of the aforementioned substitutions are given with reference to mature IFNα2b.

[0139] In further embodiments, the IFNα portion comprises one or more amino acid substitutions listed in Table 4. Table 4 lists IFNα substitutions identified with reference to amino acid positions within the IFNα2 sequence.

[0140]

[0141]

[0142]

[0143]

[0144] In some embodiments, the IFNα portion comprises an amino acid sequence comprising the amino acid substitutions R33A or R33K, Q90A, E96A, R120A, A145M, R149A or R149K, S152A, or any combination of two or more of the foregoing, e.g., Q90A+R120A or A145M+R149K.

[0145] The sequences of exemplary IFNα portions that can be used in the IFN receptor agonists of the present disclosure are listed below in Table 5:

[0146]

[0147]

[0148] 6.3.2. Interferon-β

[0149] Interferon-β (IFNβ) is a cytokine produced naturally by the immune system in response to biological and chemical stimuli. IFNβ is a glycosylated secretory monomer with a molecular weight of approximately 22 kDa, which is produced in large quantities by fibroblasts, and is therefore also referred to as fibroblast interferon. IFNβ binds to the IFNAR receptor composed of IFNAR1 and IFNAR2 dimers to induce signal transduction via the JAK / STAT pathway and other pathways. IFNβ can also play a role by binding to IFNAR1 alone and signals independently of the Jak-STAT pathway (Ivashkiv and Donlin, 2014, Nat Rev Immunol. 14 (1): 36-49).

[0150] IFNβ 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)). The five α-helices are interconnected by loops of 2 to 28 residues designated as the AB, BC, CD, and DE loops. It is reported that the A helix in the AB loop and the E helix in the DE loop are involved in the binding of IFNβ to the IFNAR receptor.

[0151] Two types of IFNβ have been described: interferon-β1 (IFNβ1) and interferon-β3 (IFNβ3) (Schirmer and Neumann, 2019. Cytokines. In: Nijkamp and Parnham's Principles of Immunopharmacology. Springer, Cham.).

[0152] The amino acid sequence of human IFNβ precursor is listed in GenBank under accession number AAA36040.1 and is reproduced below (with the signal peptide underlined):

[0153] MTNKCLLQIALLLCFSTTALS MSYNLLGFL QRSSNFQCQK LLWQLNGRLE YCLKDRMNFDIPEEIKQLQQ FQKEDAALTI YEMLQNIFAI FRQDSSSTGW NETIVENLLA NVYHQINHLK TVLEEKLEKEDFTRGKLMSS LHLKRYYGRI LHYLKAKEYS HCAWTIVRVE ILRNFYFINR LTGYLRN(SEQ ID NO:23)

[0154] 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 IFNβ1, 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 IFNβ1).

[0155] In various embodiments, the IFNβ portion comprises one or more amino acid substitutions and / or deletions compared to IFNβ 1. In some embodiments, the substitution is C17S (with reference to mature IFNβ 1), and the deletion is one of the C-terminal truncations described in US 2009 / 0025106A1, such as IFN-Δ1, IFN A2, IFN A3, IFN A4, IFN A5, IFN A6, IFN-Δ7, IFN-Δδ, IFN A9, and IFN-Δ10.

[0156] 6.3.3. Other type I interferons

[0157] In certain aspects, the type I IFN portion is other than the IFNα or IFNβ portion, eg, an interferon-ω (IFNω), interferon-ε (IFNε), or interferon-κ (IFNκ) portion.

[0158] Human IFNω is identified by UniProt accession number P05000, and the IFNω1 allele has the amino acid sequence listed below, with the signal sequence underlined:

[0159] MALLFPLLAALVMTSYSPVGSLGCD LPQNHGLLSRNTLVLLHQMRRISPFLCLKDRRDFRFPQEMVKGSQLQKAHVMSVLHEMLQQIFSLFHTERSSAAWNMTLLDQLHTGLHQQLQHLETCLLQVVGEGESAGAISSPALTLRRYFQGIRVYLKEKKYSDCAWEVVRMEIMKSLFLSTNMQERLRSKDRDLGSS(SEQ ID NO:24)

[0160] In various aspects, 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 IFNω1, 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 IFNω1).

[0161] Human IFNε is identified by UniProt accession number Q86WN2 and has the amino acid sequence listed below, with the signal sequence underlined:

[0162] MIIKHFFGTVLVLLASTTIFS LDLKLIIFQQRQVNQESLKLLNKLQTLSIQQCLPHRK

[0163] NFLLPQKSLSPQQYQKGHTLAILHEMLQQIFSLFRANISLDGWEENHTEKFLIQLH

[0164] QQLEYLEALMGLEAEKLSGTLGSDNLRLQVKMYFRRIHDYLENQDYSTCAWAIVQVEISRCLFFVFSLTEKLSKQGRPLNDMKQELTTEFRSPR(SEQ ID NO:25)

[0165] In various aspects, 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).

[0166] Human IFNκ is identified by UniProt accession number Q9P0W0 and has the amino acid sequence listed below, with the signal sequence underlined:

[0167] MSTKPDMIQKCLWLEILMGIFIAGTLS LDCNLLNVHLRRVTWQNLRHLSSMSNSFP

[0168] VECLRENIAFLPQEFLQYTQPMKRDIKKAFYEMSLQAFNIFSQHTFKYWKERHL

[0169] KQIQIGLDQQAEYLNQCLEEDKNENEDMKEMKENEMKPSEARVPQLSSLELRRYFHRIDNFLKEKKYSDCAWEIVRVEIRRCLYYFYKFTALFRRK(SEQ ID NO:26)

[0170] In various aspects, 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).

[0171] 6.4. IFN masking part

[0172] The present disclosure provides IFN receptor agonists, and its IFN part is shielded by one or more receptor moieties, thereby weakening IFN activity.All human I type interferons are all bound to cell surface receptors (IFNα receptor, IFNAR), and this receptor is the heterodimer (referring to, for example, Novick et al., 1994, Cell77:391) of two transmembrane proteins IFNAR1 and IFNAR2, and these two transmembrane proteins can be used for shielding the IFN part in the IFN receptor agonists of the present disclosure.Therefore, in certain embodiments, the shielding part is IFNAR1 part.In other embodiments, the shielding part is IFNAR2 part.Exemplary IFNAR1 part is disclosed in Section 6.4.1, and exemplary IFNAR2 part is disclosed in Section 6.4.2.

[0173] 6.4.1.IFNAR1

[0174] IFNAR1 is a low-affinity IFN receptor and belongs to the type II helical cytokine receptor. It consists of an extracellular domain composed of four type III fibronectin domains called "subdomains" (SDs), a transmembrane domain, and a 100-amino acid intracellular domain. The four subdomains of IFNAR1 fold into domain 1 (SD1+SD2) and domain 2 (SD3+SD4).

[0175] The sequence of human IFNAR1 has the UniProt identifier P17181. The sequence of human IFNAR1 is reproduced below:

[0176]

[0177] The signal sequence (single underline) corresponds to amino acids 1 to 27, the SD1 domain (bold) corresponds to amino acids 28 to 127, the SD2 domain (double underline) corresponds to amino acids 128 to 227, the SD3 domain (italics) corresponds to amino acids 231 to 329, the SD4 domain (lowercase) corresponds to amino acids 330 to 432, and the extracellular domain corresponds to amino acids 28 to 436 of the full-length human IFNAR1 protein reproduced above.

[0178] The IFNAR1 portion is an amino acid sequence that has at least 70% sequence identity (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity or 100% sequence identity) to the IFN binding portion of a mammalian (e.g., human) IFNAR1. In some embodiments, the IFN binding portion comprises an SD2 domain and an SD3 domain. In various aspects, the IFN binding portion comprises (i) only the SD2 and SD3 domains; (ii) the SD1, SD2, and SD3 domains; (iii) the SD2, SD3, and SD4 domains; (iv) the SD1, SD2, SD3, and SD4 domains; or (v) the entire extracellular domain of IFNAR1.

[0179] 6.4.2.IFNAR2 part

[0180] IFNAR2 is a high-affinity IFN receptor that adopts a two-domain D1 / D2 receptor structure.

[0181] The sequence of human IFNAR2 has the UniProt identifier P48551. The sequence of human IFNAR2 is reproduced below:

[0182]

[0183] The signal sequence (single underline) corresponds to amino acids 1 to 26, the D1 domain (bold) corresponds to amino acids 27 to 136, the D2 domain (double underline) corresponds to amino acids 137 to 232, and the extracellular domain corresponds to amino acids 27 to 243 of the full-length human IFNAR2 protein reproduced above.

[0184] The IFNAR2 portion is an amino acid sequence that has at least 70% sequence identity (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or 100% sequence identity) to the IFN binding portion of mammalian (e.g., human) IFNAR2. In some embodiments, the IFN binding portion comprises a D1 domain. In various aspects, the IFN binding portion comprises (i) only the D1 domain; (ii) the D1 domain and the D2 domain; or (iii) the entire extracellular domain of IFNAR2.

[0185] 6.5. Protease-cleavable linkers

[0186] The IFN receptor agonist optionally includes one or two protease cleavable linkers (PCLs) in at least one half-antibody (or two half-antibodies), while the other linkers are non-cleavable. In some embodiments, the linker adjacent to the IFN portion is a protease cleavable linker. This may result in, for example, the receptor masking agent being released from the IFN portion upon cleavage of the PCL, wherein the IFN portion is retained on the C-terminus of the half-antibody (e.g., in a configuration designated as Fc-IFN-IFNR in Table 1). Alternatively, for other configurations (e.g., a configuration designated as Fc-IFNR-IFN in Table 1), incorporation of a PCL adjacent to the IFN portion will result in the release of IFN upon cleavage, wherein the IFNR portion is retained in the half-antibody. In yet other embodiments, the linker between the Fc domain and the IFNR portion is a PCL that is configured such that cleavage releases the IFNR portion while retaining the IFN portion in the half-antibody (e.g., configurations such as Fc-IFN x Fc-R1, wherein cleavage releases the IFNR portion while retaining the IFN portion in the other half-antibody).

[0187] The scope of protease cleavable linkers can be 8 amino acids to 100 or more amino acids. In various embodiments, the scope of protease cleavable linkers can be 8 amino acids to 15 amino acids, 10 amino acids to 20 amino acids, 20 amino acids to 80, and in some aspects, the scope of non-cleavable peptide linkers can be 20 amino acids to 60 amino acids, 20 amino acids to 40 amino acids, 30 amino acids to 50 amino acids, 20 amino acids to 80 amino acids or 30 amino acids to 70 amino acids in length.

[0188] The protease cleavable linker comprises one or more substrate sequences for one or more proteases (e.g., one or more proteases listed in Section 6.5.1). The one or more substrate sequences (e.g., one or more substrate sequences listed in Section 6.5.2) are typically (but not necessarily) flanked by one or more spacer sequences (e.g., the spacer sequences described in Section 6.5.3). Each protease cleavable linker can comprise one, two, three or more substrate sequences. The spacer sequences can be adjacent, overlapping or separated by spacer sequences. Preferably, the C-terminus and N-terminus of the protease cleavable linker contain spacer sequences.

[0189] In various aspects of the IFN receptor agonist comprising four protease cleavable linkers, the first protease and the third protease cleavable linker can be cleaved by the same protease and / or the second protease and the fourth protease cleavable linker can be cleaved by the same protease. In some embodiments, the protease is a protease listed in Table A.

[0190] In further aspects of the IFN receptor agonist comprising four protease cleavable linkers, the first protease and the third protease cleavable linker comprise the same substrate sequence and / or the second protease and the fourth protease cleavable linker comprise the same substrate sequence. In certain embodiments, the substrate sequences are listed in Table B. In further embodiments, the first protease cleavable linker and the third protease cleavable linker also comprise the same spacer sequence and / or the second protease cleavable linker and the fourth protease cleavable linker also comprise the same spacer sequence. In certain embodiments, the spacer sequence is listed in Table C.

[0191] In further aspects of the IFN receptor agonist comprising four protease cleavable linkers, the first linker and the third linker comprise the same linker sequence and / or the second linker and the fourth linker comprise the same linker sequence. In some embodiments, the linker sequences are listed in Table D.

[0192] In some embodiments of the IFN receptor agonist comprising four protease-cleavable linkers, the first protease-cleavable linker and the third protease-cleavable linker are the same as the second protease-cleavable linker and the fourth protease-cleavable linker.

[0193] In other embodiments, the first protease cleavable linker and the third protease cleavable linker are different from the second protease cleavable linker and the fourth protease cleavable linker.

[0194] In the foregoing aspects and embodiments, different linkers may be cleavable by the same protease, different proteases, or when the linker comprises multiple substrate sequences, different linkers may be cleavable by multiple proteases, where one or more proteases are common and where one or more proteases are different.

[0195] Exemplary protease-cleavable linker sequences are listed in Section 6.5.4.

[0196] 6.5.1. Proteases

[0197] Exemplary proteases whose substrate sequences can be incorporated into protease-cleavable linkers are listed in Table A below.

[0198]

[0199]

[0200] In specific embodiments, the protease is matrix metalloproteinase (MMP)-2, MMP-9, endoasparaginase, thrombin, fibroblast activation protease (FAP), MMP-1, MMP-3, MMP-7, MMP-8, MMP-12, MMP-13, MMP-14, membrane type 1 matrix metalloproteinase (MT1-MMP), plasmin, transmembrane protease, serine (TMPRSS-3 / 4), cathepsin A, cathepsin B, cathepsin D , cathepsin E, cathepsin F, cathepsin H, cathepsin K, cathepsin L, cathepsin L2, cathepsin O, cathepsin S, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, human neutrophil elastase, urine Kinase / urokinase-type plasminogen activator (uPA), a disintegrin and metalloproteinase (ADAM) 10, ADAM12, ADAM17, ADAM with thrombospondin motif (ADAMTS), ADAMTS5, β-secretase (BACE), granzyme A, granzyme B, guanidinobenzoic acid enzyme, serine protease, proteinase, proteinase 2, transmembrane peptidase, neprilysin, prostate-specific membrane antigen (PSMA), tumor necrosis factor-converting enzyme (TAC E), 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, γ-secretase component, antiplasmin cleaving enzyme (APCE), decysteine ​​1, apoptosis-related cysteine ​​peptidase, or N-acetylated α-linked acid dipeptidase-like 1.

[0201] 6.5.2. Substrate

[0202] Exemplary substrate sequences that can be cleaved by tumor proteases and that can incorporate protease-cleavable linkers are listed in Table B below.

[0203]

[0204]

[0205]

[0206]

[0207] 6.5.3. Spacers

[0208] Exemplary spacer sequences that can be incorporated into protease-cleavable linkers are listed below in Table C. In addition to the spacer sequences listed in Table C, any of the non-cleavable linker sequences described in Section 6.6 (e.g., the non-cleavable linker sequences listed in Table E) or portions thereof can be used as spacer sequences. In some embodiments, spacer sequences are completely absent from the protease-cleavable linker.

[0209]

[0210]

[0211] In some embodiments, as used in Table C above, n is an integer from 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0212] 6.5.4. Exemplary Protease-Cleavable Linkers

[0213] Exemplary protease-cleavable linkers comprising one or more substrate sequences and a spacer sequence are listed in Table D below.

[0214]

[0215]

[0216]

[0217]

[0218]

[0219] In certain aspects, the protease-cleavable linker comprises an amino acid sequence having up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitution compared to a sequence listed in Table D. Thus, in some embodiments, the protease-cleavable linker comprises or consists of any amino acid sequence in Table D having 1-5 amino acid substitutions compared to a sequence listed in Table D.

[0220] 6.6. Non-cleavable linkers

[0221] In certain aspects, the present disclosure provides IFN receptor agonists, wherein two or more components of the IFN receptor agonist are connected to each other via a peptide linker. By way of example and not limitation, a linker can be used to connect an Fc domain and a targeting moiety or different domains within a targeting moiety (e.g., VH and VL domains in an scFv), an Fc domain and an IFN or IFNR moiety, or an IFN moiety and an IFNR moiety.

[0222] Preferably, all linkers in the IFN receptor agonist, except for the designated protease-cleavable linker (when present), are non-cleavable linkers (NCLs).

[0223] The non-cleavable linker can range from 2 amino acids to 60 or more amino acids, and in certain aspects the non-cleavable peptide linker can range from 3 amino acids to 50 amino acids, 4 to 30 amino acids, 5 to 25 amino acids, 10 to 25 amino acids, 10 amino acids to 60 amino acids, 12 amino acids to 20 amino acids, 20 amino acids to 50 amino acids, or 25 amino acids to 35 amino acids in length.

[0224] In specific aspects, the non-cleavable linker is at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids in length, and optionally is up to 30 amino acids, up to 40 amino acids, up to 50 amino acids, or up to 60 amino acids in length.

[0225] In some of the foregoing embodiments, the non-cleavable linker ranges from 5 amino acids to 50 amino acids in length, e.g., ranging from 5 to 50 amino acids, 5 to 45 amino acids, 5 to 40 amino acids, 5 to 35 amino acids, 5 to 30 amino acids, 5 to 25 amino acids, or 5 to 20 amino acids in length. In other embodiments of the foregoing, the non-cleavable linker ranges from 6 amino acids to 50 amino acids in length, e.g., ranging from 6 to 50 amino acids, 6 to 45 amino acids, 6 to 40 amino acids, 6 to 35 amino acids, 6 to 30 amino acids, 6 to 25 amino acids, or 6 to 20 amino acids in length. In yet other embodiments, the non-cleavable linker ranges from 7 amino acids to 50 amino acids in length, e.g., ranging from 7 to 50 amino acids, 7 to 45 amino acids, 7 to 40 amino acids, 7 to 35 amino acids, 7 to 30 amino acids, 7 to 25 amino acids, or 7 to 20 amino acids in length.

[0226] Charged (eg, charged hydrophilic linkers) and / or flexible non-cleavable linkers are particularly preferred.

[0227] Examples of flexible non-cleavable linkers that can be used in the IFN receptor agonists of the present disclosure include those disclosed by Chen et al., 2013, Adv Drug Deliv Rev. 65(10):1357-1369 and Klein et al., 2014, Protein Engineering, Design & Selection 27(10):325-330. Particularly useful flexible non-cleavable linkers are or include repeating sequences of glycine and serine, e.g., G nS (SEQ ID NO: 302) or SG n (SEQ ID NO: 303), wherein n is an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the non-cleavable linker is or comprises a monomer or multimer of a G4S (SEQ ID NO: 304) repeating sequence, for example, (GGGGS) n (SEQ ID NO:304).

[0228] Polyglycine non-cleavable linkers can be suitably used in the IFN receptor agonists of the present disclosure. In some embodiments, the peptide non-cleavable linker comprises two consecutive glycines (2Gly), three consecutive glycines (3Gly), four consecutive glycines (4Gly (SEQ ID NO: 305)), five consecutive glycines (5Gly (SEQ ID NO: 306)), six consecutive glycines (6Gly (SEQ ID NO: 307)), seven consecutive glycines (7Gly (SEQ ID NO: 308)), eight consecutive glycines (8Gly (SEQ ID NO: 309)), or nine consecutive glycines (9Gly (SEQ ID NO: 310)).

[0229] Exemplary non-cleavable linker sequences are listed in Table E below.

[0230]

[0231]

[0232]

[0233] In certain aspects, the IFN receptor agonists of the present disclosure may comprise a polypeptide chain comprising a targeting moiety (or targeting moiety chain), a hinge domain, and an Fc domain in an N-terminal to C-terminal orientation. Thus, the hinge domain can be said to constitute a type of linker. Exemplary hinge domains are listed in Section 6.9.3.

[0234] 6.7. Targeting moiety

[0235] Incorporation of targeting moieties into the IFN receptor agonists of the present disclosure allows for the delivery of high concentrations of IFN to the tumor microenvironment while reducing systemic exposure, resulting in fewer side effects than obtained with non-targeted IFN molecules.

[0236] It is expected that any type of target molecule present in a specific lesion or tissue or capable of driving an IFN receptor agonist can be targeted by an IFN receptor agonist of the present invention. In certain embodiments, an IFN receptor agonist is intended to treat cancer, for example, by inducing a local immune response against tumor tissue. Therefore, the targeting molecule can be any local tumor and a related target molecule. The target molecule identified by the targeting moiety of an IFN receptor agonist of the present invention is typically present in, for example, on the surface of activated T cells, on the surface of tumor cells, on the surface of dendritic cells or other antigen presenting cells, on the surface of natural killer (NK) cells, on the surface of virus-infected cells, on the surface of other diseased cells, in free form in serum, in immune cells present in the extracellular matrix (ECM) or in the target site, for example, tumor reactive lymphocytes, dendritic cells or other antigen presenting cells or natural killer cells.

[0237] 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), a checkpoint inhibitor, a tumor associated antigen (TAA), a dendritic cell (DC) or other antigen presenting cell (APC) antigen or a natural killer (NK) cell antigen. Technicians will recognize that the target molecules of the aforementioned categories are not mutually exclusive, and therefore a given target molecule can fall into more than one target molecule of the aforementioned categories. For example, some molecules can be considered as TAA and ECM proteins, while other molecules can be considered as TCA and checkpoint inhibitors.

[0238] Exemplary cancer types that can be targeted include acute lymphocytic leukemia, acute myeloid leukemia, cholangiocarcinoma, B cell leukemia, B cell lymphoma, bile duct cancer, bone cancer, brain cancer, breast cancer, triple negative breast cancer, cervical cancer, Burkitt lymphoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastric cancer, gastrointestinal cancer, glioma, hairy cell leukemia, head and neck cancer, Hodgkin lymphoma, liver cancer, lung cancer, medullary thyroid cancer, melanoma, multiple myeloma, ovarian cancer, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, lung cancer, kidney cancer, sarcoma, skin cancer, testicular cancer, urothelial carcinoma and other bladder cancers. However, those skilled in the art will recognize that TAAs and other target molecules associated with the tumor microenvironment are known for almost any type of cancer.

[0239] Non-limiting examples of ECM antigens include syndecan, heparanase, integrin, osteopontin, connexin, cadherin, laminin, laminin-type EGF, lectin, fibronectin, notch proteins, adhesion proteins (e.g., adhesion protein-4), tenascin, collagen (e.g., type X collagen), and matrix proteins.

[0240] Other target molecules are cell surface molecules of tumor or viral lymphocytes, for example, T cell co-stimulatory proteins such as CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C and B7-H3.

[0241] In certain embodiments, the target molecule is a checkpoint inhibitor, such as CTLA-4, PD1, PDL1, PDL2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2. In specific embodiments, the target molecule is PD1. In other embodiments, the target molecule is LAG3. In yet other embodiments, the target molecule is PDL1.

[0242] In certain embodiments, the target molecule is located on the surface of dendritic cells or other antigen presenting cells, such as XCR1, Clec9a, CD1c, CD11c, CD14, PDL1, macrophage mannose receptor (CD206), and DEC-205.

[0243] In further embodiments, the target molecule is located on the surface of natural killer (NK) cells, such as CD335, CD38, CD2, NKG2D, NKp44, NKp30, CD16, LFA-1, CD27, KIR, NKH1A, and NKp46.

[0244] Antibodies and antigen binding moieties generally bind to specific antigenic determinants and are capable of directing the IFN receptor agonist to a target site, such as to a specific type of tumor cell or tumor stroma carrying the antigenic determinant. In specific 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 (TNC A2), extra domain B of fibronectin (EDB), melanoma-associated chondroitin sulfate proteoglycan (MCSP), MART-1 / Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cycloserine protease B, colorectal-associated antigen (CRC)-C017-1A / GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2 and PSA-3, prostate-specific membrane antigen (PSMA), T cell receptor / CD3-zeta chain, MAGE family tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE ​​family tumor antigens (e.g.,GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin, and γ-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis protein (APC), fodrin, connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products (such as human papillomavirus proteins), Smad tumor antigen family, Imp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (H OM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, c-erbB-2, Her2, EGFR, IGF-1R, CD2 (T cell surface antigen), CD3 (heteromultimer associated with TCR), CD22 (B cell receptor), CD23 (low affinity IgE receptor), CD30 (cytokine receptor), CD33 (myeloid cell surface antigen), CD40 (tumor necrosis factor receptor), IL-6R-( IL6 receptor), CD20, MCSP, PDGFβR (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, prostate enzyme, prostate enzyme-specific antigen (PSA), PAP, LAGA-1a, p53, prostate, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF1)-I, IGF-II, IGF1 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).

[0245] Suitable targeting moiety formats are described in Section 6.8. The targeting moiety is preferably an antigen binding moiety, eg, an antibody or an antigen binding portion of an antibody, eg, a scFv as described in Section 6.8.2 or a Fab as described in Section 6.8.1.

[0246] In some embodiments, the targeting moiety targets the exemplary target molecules listed in Table F below, with reference to exemplary antibodies or antibody sequences on which the targeting moiety can be based.

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263] In some aspects, the targeting moiety competes with the antibodies listed in Table F to bind to the target molecule. In a further aspect, the targeting moiety includes a CDR having the CDR sequence of the antibody listed in Table F. In certain embodiments, the targeting moiety includes all 6 CDR sequences of the antibody listed in Table F. In other embodiments, the targeting moiety includes at least the heavy chain CDR sequences (CDR-H1, CDR-H2, CDR-H3) and the light chain CDR sequences of a universal light chain listed in Table F. In a further aspect, the targeting moiety includes a VH having an amino acid sequence of the VH of the antibody listed in Table F. In certain embodiments, the targeting moiety also includes a VL having an amino acid sequence of the VL of the antibody listed in Table F. In other embodiments, the targeting moiety further includes a universal light chain VL sequence.

[0264] In some embodiments, the target molecule is PDL1. Table F-1 below provides exemplary anti-PDL1 antibodies and / or antibody sequences on which targeting moieties can be based, for example, which can be incorporated into targeting moieties used in the interferon receptor agonists of the present disclosure.

[0265]

[0266]

[0267] In some aspects, the targeting portion competes with the anti-PDL1 antibodies listed in Table F-1 to bind to PDL1. In a further aspect, the targeting portion includes a CDR having a CDR sequence of an anti-PDL1 antibody listed in Table F-1. In some embodiments, the targeting portion includes all 6 CDR sequences of the anti-PDL1 antibodies listed in Table F-1. In other embodiments, the targeting portion includes at least a heavy chain CDR sequence (CDR-H1, CDR-H2, CDR-H3) of the anti-PDL1 antibodies listed in Table F-1 and a light chain CDR sequence of a universal light chain. In a further aspect, the targeting portion includes a VH comprising an amino acid sequence of the VH of the anti-PDL1 antibodies listed in Table F-1. In some embodiments, the targeting portion further includes a VL comprising an amino acid sequence of the VL of the anti-PDL1 antibodies listed in Table F-1. In other embodiments, the targeting portion further includes a universal light chain VL sequence.

[0268] In some embodiments, the target molecule is PD1. Table F-2 below provides exemplary anti-PD1 antibodies and / or antibody sequences on which targeting moieties can be based, for example, which can be incorporated into targeting moieties used in the interferon receptor agonists of the present disclosure.

[0269]

[0270]

[0271]

[0272] In some aspects, the targeting portion competes with the anti-PD1 antibodies listed in Table F-2 to bind to PD1. In a further aspect, the targeting portion includes a CDR having a CDR sequence of an anti-PD1 antibody listed in Table F-2. In some embodiments, the targeting portion includes all 6 CDR sequences of the anti-PD1 antibodies listed in Table F-2. In other embodiments, the targeting portion includes at least the heavy chain CDR sequence (CDR-H1, CDR-H2, CDR-H3) of the anti-PD1 antibodies listed in Table F-2 and the light chain CDR sequence of a universal light chain. In a further aspect, the targeting portion includes a VH comprising the amino acid sequence of the VH of the anti-PD1 antibodies listed in Table F-2. In some embodiments, the targeting portion further includes a VL comprising the amino acid sequence of the VL of the anti-PD1 antibodies listed in Table F-2. In other embodiments, the targeting portion further includes a universal light chain VL sequence.

[0273] In the case where the target molecule is a checkpoint inhibitor, in some embodiments, the checkpoint inhibitor targeting moiety is non-blocking or weakly blocking ligand-receptor binding. Examples of non-blocking or weakly blocking anti-PD1 antibodies include antibodies having VH / VL amino acid sequences of SEQ ID NO: 2 / 10 of PCT Publication No. WO2015 / 112800A1; SEQ ID NO: 16 / 17 of U.S. Patent No. 11,034,765B2; and SEQ ID NO. 164 / 178, 165 / 179, 166 / 180, 167 / 181, 168 / 182, 169 / 183, 170 / 184, 171 / 185, 172 / 186, 173 / 187, 174 / 188, 175 / 189, 176 / 190, and 177 / 190 of U.S. Patent No. 10,294,299B2. Examples of non-blocking or weakly blocking anti-LAG3 antibodies include antibodies having the VH / VL amino acid sequences of SEQ ID NOs 23 / 24, 3 / 4, and 11 / 12 of U.S. Publication No. US2022 / 0056126A1.

[0274] Additional target molecules that can be targeted by IFN receptor agonists are disclosed in Table 1 below and, for example, in Hafeez et al., 2020, Molecules 25:4764, doi: 10.3390 / molecules25204764 (particularly Table 1). The entire contents of Table 1 of Hafeez et al. are incorporated herein by reference.

[0275] 6.8. Targeting Section Format

[0276] In certain aspects, the targeting moiety of the IFN receptor agonist of the present disclosure can be any type of antibody or fragment thereof that retains specific binding to the antigenic determinant. In one embodiment, the targeting 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 but are not limited to 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.

[0277] 6.8.1.Fab

[0278] Fab domains are traditionally produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. Fab domains may comprise constant domain and variable region sequences from any suitable species and may therefore be murine, chimeric, human or humanized.

[0279] The Fab domain typically comprises a CHI domain attached to a VH domain, which is paired with a CL domain attached to a VL domain. In wild-type immunoglobulins, the VH and VL domains pair to form the Fv region, and the CHI domain pairs with the CL domain to further stabilize the binding site. A disulfide bond between the two constant domains can further stabilize the Fab domain.

[0280] For the IFN receptor agonists of the present disclosure, particularly when the light chain of the targeting moiety is not a common or universal light chain, it is advantageous to use a Fab heterodimerization strategy to allow the correct association of Fab domains belonging to the same targeting moiety and minimize abnormal pairing of Fab domains belonging to different targeting moieties. For example, the Fab heterodimerization strategy shown in Table G below can be used:

[0281]

[0282]

[0283] Thus, in certain embodiments, correct association between two polypeptides of a Fab is promoted by exchanging the VL and VH domains of the Fab with each other or exchanging the CH1 and CL domains with each other, for example, as described in WO 2009 / 080251.

[0284] Correct Fab pairing can also be promoted by introducing one or more amino acid modifications in the CH1 domain of the Fab, one or more amino acid modifications in the CL domain of the Fab, and / or one or more amino acid modifications in the VH domain, and one or more amino acid modifications in the VL domain. The modified amino acids are typically part of the VH:VL and CH1:CL interfaces, such that the Fab components preferentially pair with each other rather than with components of other Fabs.

[0285] In one embodiment, one or more amino acid modifications are limited to conserved framework residues of the variable domains (VH, VL) and constant domains (CH1, CL), as indicated by the Kabat numbering of the residues. Almagro, 2008, Frontiers in Bioscience 13: 1619-1633 provides definitions of framework residues based on the Kabat, Chothia, and IMGT numbering schemes.

[0286] In one embodiment, the modifications introduced into the VH and CH1 and / or VL and CL domains are complementary to each other. Complementarity at the heavy and light chain interfaces can be achieved based on steric and hydrophobic contacts, electrostatic / charge interactions, or a combination of various interactions. Complementarity between protein surfaces is widely described in the literature as lock and key fit, knob into hole, protrusion and cavity, donor and acceptor, etc., all of which imply the nature of the structural and chemical matching between the two interacting surfaces.

[0287] In one embodiment, one or more of the introduced modifications introduce new hydrogen bonds at 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 WO 2014 / 150973 and WO 2014 / 082179, the contents of which are incorporated herein by reference.

[0288] In some embodiments, the Fab domain comprises a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain, which introduce a salt bridge between the CH1 and CL domains (see, eg, Golay et al., 2016, J Immunol 196:3199-211).

[0289] In some embodiments, the Fab domain comprises 143Q and 188V substitutions in the CH1 domain and 113T and 176V substitutions in the CL domain, which serve to exchange the hydrophobic and polar regions of contact between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0290] In some embodiments, the Fab domain may comprise modifications in some or all of the VH, CH1, VL, and CL domains to introduce an orthogonal Fab interface that promotes the correct assembly of the Fab domain (Lewis et al., 2014 Nature Biotechnology 32: 191-198). In one embodiment, 39K and 62E modifications are introduced into the VH domain, H172A and F174G modifications are introduced into the CH1 domain, 1R, 38D and (36F) modifications are introduced into the VL domain, and L135Y and S176W modifications are introduced into the CL domain. In another embodiment, 39Y modifications are introduced into the VH domain and 38R modifications are introduced into the VL domain.

[0291] The Fab domain can also be modified to replace the native CH1:CL disulfide bond with an engineered disulfide bond to improve the efficiency of Fab component pairing. For example, an engineered disulfide bond can be introduced by introducing 126C in the CH1 domain and 121C in the CL domain (see, e.g., Mazor et al., 2015, MAbs 7:377-89).

[0292] The Fab domain can also be modified by replacing the CH1 domain and the CL domain with alternative domains that promote correct assembly. For example, Wu et al., 2015, MAbs 7:364-76, describe replacing the CH1 domain with the constant domain of the T cell receptor and replacing the CL domain with the b domain of the T cell receptor, and pairing these domain replacements with additional charge-charge interactions between the VL and VH domains by introducing a 38D modification in the VL domain and a 39K modification in the VH domain.

[0293] Instead of or in addition to using a Fab heterodimerization strategy to promote correct VH-VL pairing, the VL of a common light chain (also referred to as a universal light chain) can be used for each unique ABD in the IFN receptor agonists of the present disclosure. In various embodiments, the use of a common light chain as described herein reduces the number of inappropriate substances in the IFN receptor agonists compared to the use of the original homologous VL. In various embodiments, the VL domain of the ABD is identified from a monospecific antibody comprising a common light chain. In various embodiments, the VH region of the ABS in the IFN receptor agonist comprises human heavy chain variable gene segments that are rearranged in vivo in mouse B cells that have previously been engineered to express a limited human light chain library, or a single human light chain homologous to a human heavy chain, and in response to exposure to an antigen of interest, produces an antibody library containing multiple human VHs that are homologous to one of a possible human VL or two possible human VLs, wherein the antibody library has specificity for the antigen of interest. Common light chains are derived from a rearranged human Vκ1-39Jκ5 sequence or a rearranged human Vκ3-20Jκ1 sequence and include somatically mutated (eg, affinity matured) versions. See, eg, US Patent No. 10,412,940.

[0294] 6.8.2.scFv

[0295] Single-chain Fv or "scFv" antibody fragments comprise the VH and VL domains of an antibody in a single polypeptide chain, are capable of being expressed as a single polypeptide chain, 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 that enables the scFv to form the desired structure for target binding. Examples of linkers suitable for joining the VH and VL chains of an scFv are the non-cleavable linkers identified in Section 6.6.

[0296] Unless otherwise indicated, as used herein, a scFv can have the VL and VH variable regions in either order, e.g., relative to the N-terminus and C-terminus of the polypeptide, a scFv can comprise VL-linker-VH or can comprise VH-linker-VL.

[0297] The scFv may comprise VH and VL sequences from any suitable species, such as murine, human, or humanized VH and VL sequences.

[0298] To produce a nucleic acid encoding an scFv, the VH and VL encoding DNA fragments are operably linked to another fragment encoding a linker, e.g., any of the linkers described in Section 6.6 (typically repeats of a sequence containing the amino acids glycine and serine, such as the amino acid sequence (Gly4-Ser)3 (SEQ ID NO: 182), such that the VH and VL sequences can be expressed as a contiguous single-chain protein in which the VL and VH regions are 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).

[0299] 6.9. Fc Region

[0300] The IFN receptor agonists of the present disclosure generally include a pair of Fc domains that associate to form an Fc region. In native antibodies, the Fc region includes a hinge region at its N-terminus to form a constant domain. Throughout this disclosure, unless otherwise indicated, reference to an Fc domain encompasses an Fc domain having a hinge domain at its N-terminus.

[0301] The Fc domain can be derived from any suitable species that is operably connected to an ABD or its components. In one embodiment, the Fc domain is derived from a human Fc domain. In a preferred embodiment, the targeting moiety or its components are fused to an IgG Fc molecule. The targeting moiety or its components can be fused to the N-terminal or C-terminal or both of the IgG Fc domain.

[0302] The Fc domain can be derived from an antibody of any suitable class, including IgA (including subclass IgA1 and IgA2), IgD, IgE, IgG (including subclass 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. Table Y below provides exemplary sequences of Fc domains from IgG1, IgG2, IgG3 and IgG4.

[0303]

[0304] In some aspects, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 410. Where the Fc domain comprises at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 410 (e.g., between 90% and 99% sequence identity to SEQ ID NO: 410), the Fc domain may further comprise one or more amino acid substitutions described herein, e.g., one or more substitutions that reduce effector function (e.g., as described in Section 6.9.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.9.2).

[0305] In some aspects, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 411. Where the Fc domain comprises at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 411 (e.g., between 90% and 99% sequence identity to SEQ ID NO: 411), the Fc domain can further comprise one or more amino acid substitutions described herein, e.g., one or more substitutions that reduce effector function (e.g., as described in Section 6.9.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.9.2).

[0306] In some aspects, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 412. Where the Fc domain comprises at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 412 (e.g., between 90% and 99% sequence identity to SEQ ID NO: 412), the Fc domain can further comprise one or more amino acid substitutions described herein, e.g., one or more substitutions that reduce effector function (e.g., as described in Section 6.9.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.9.2).

[0307] In some aspects, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 413. Where the Fc domain comprises at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 413 (e.g., between 90% and 99% sequence identity to SEQ ID NO: 413), the Fc domain can further comprise one or more amino acid substitutions described herein, e.g., one or more substitutions that reduce effector function (e.g., as described in Section 6.9.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.9.2).

[0308] The two Fc domains within the Fc region can be identical or different from each other. In native antibodies, the Fc domains are typically identical, but for the purpose of generating multispecific binding molecules, e.g., the IFN receptor agonists of the present disclosure and the MBMs generated by their activation, the Fc domains may advantageously differ to allow heterodimerization, as described in Section 6.9.2 below.

[0309] 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 heavy chain Fc domain of IgE and IgM consists of three heavy chain constant domains (CH2, CH3, and CH4). These dimers form the Fc region.

[0310] In the IFN receptor agonists of the present disclosure, the Fc region and / or the Fc domain therein may comprise heavy chain constant domains from one or more different classes (eg, one, two, or three different classes) of antibodies.

[0311] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG1.

[0312] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG2.

[0313] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG3.

[0314] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG4.

[0315] 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.

[0316] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG and a CH4 domain derived from IgM.

[0317] It should be understood that the heavy chain constant domains of the Fc regions used to generate the IFN receptor agonists of the present disclosure may include variants of the above-described naturally occurring constant domains. Such variants may comprise one or more amino acid variations compared to the wild-type constant domain. In one example, the Fc region of the present disclosure comprises at least one constant domain that differs in sequence from the wild-type constant domain. It should be understood that the variant constant domain may be longer or shorter than the wild-type constant domain. Preferably, the variant constant domain is at least 60% identical or similar to the wild-type constant domain. In another example, the variant constant domain is at least 70% identical or similar. In another example, the variant constant domain is at least 80% identical or similar. In another example, the variant constant domain is at least 90% identical or similar. In another example, the variant constant domain is at least 95% identical or similar.

[0318] IgM and IgA naturally exist in the human body as covalent polymers of the common H2L2 antibody unit. When IgM incorporates a J chain, it appears as a pentamer; or when IgM lacks a J chain, it appears as a hexamer. IgA appears as a monomer and a dimer. The heavy chains of IgM and IgA have 18 amino acids extending to the C-terminal constant domain, called the tail segment. The tail segment contains cysteine ​​residues that form disulfide bonds between the heavy chains of the polymer and are believed to play an important role in polymerization. The tail segment also contains a glycosylation site. In certain embodiments, the IFN receptor agonists of the present disclosure do not contain a tail segment.

[0319] The Fc domain incorporated into the IFN receptor agonists of the present disclosure may include one or more modifications that alter the functional properties of the protein, such as binding to an Fc receptor such as FcRn or a leukocyte receptor, binding to complement, a modified disulfide bond architecture, or an altered glycosylation pattern. Exemplary Fc modifications that alter effector function are described in Section 6.9.1.

[0320] The Fc domain can also be altered to include modifications that improve the manufacturability of asymmetric IFN receptor agonists, for example, by allowing heterodimerization, which is the preferential pairing of non-identical Fc domains relative to identical Fc domains. Heterodimerization allows the production of IFN receptor agonists in which different polypeptide components are linked to each other via Fc regions containing Fc domains that differ in sequence. Examples of heterodimerization strategies are illustrated in Section 6.9.2.

[0321] It will be appreciated that any of the above modifications may be combined in any suitable manner to achieve desired functional properties and / or combined with other modifications to alter the properties of the IFN receptor agonist.

[0322] 6.9.1. Fc Domains with Altered Effector Function

[0323] In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and / or effector function.

[0324] 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 activated Fc receptor. In a specific embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group consisting of complement dependent cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and cytokine secretion. In a specific embodiment, the effector function is ADCC.

[0325] In one embodiment, the Fc domain (e.g., the Fc domain of an IFN receptor agonist half antibody) or Fc region (e.g., one or two Fc domains of an IFN receptor agonist that can associate to form an Fc region) comprises an amino acid substitution at a position selected from the group consisting of E233, L234, L235, N297, P331, and P329 (numbering according to the Kabat EU index). In a more specific embodiment, the Fc domain or Fc region comprises an amino acid substitution at a position selected from the group consisting of L234, L235, and P329 (numbering according to the Kabat EU index). In some embodiments, the Fc domain or Fc region comprises the amino acid substitutions L234A and L235A (numbering according to the Kabat EU index). In one such embodiment, the Fc domain or region is an Igd Fc domain or region, particularly a human Igd Fc domain or region. In one embodiment, the Fc domain or Fc region comprises an amino acid substitution at position P329. In a more specific embodiment, amino acid replacement is P329A or P329G, particularly P329G (according to Kabat EU index numbering). In one embodiment, Fc domains or Fc district comprise amino acid replacement at position P329 and comprise further amino acid replacement at the position selected from E233, L234, L235, N297 and P331 (according to the numbering of Kabat EU index). In a more specific embodiment, further amino acid replacement is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a specific embodiment, Fc domains or Fc district comprise amino acid replacement (according to Kabat EU index numbering) at position P329, L234 and L235. In a more specific embodiment, Fc domains comprise amino acid mutations L234A, L235A and P329G (" P329G LALA ", " PGLALA " or " LALAPG ").

[0326] Typically, the same one or more amino acid substitutions are present in each of the two Fc domains of the Fc region. Thus, in specific embodiments, each Fc domain of the Fc region comprises the amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering), i.e., in each of the first and second Fc domains of the Fc region, the leucine residue at position 234 is substituted with an alanine residue (L234A), the leucine residue at position 235 is substituted with an alanine residue (L235A), and the proline residue at position 329 is substituted with a glycine residue (P329G) (numbering according to the Kabat EU index).

[0327] In one embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In some embodiments, the IgG1 Fc domain is a variant IgG1 comprising D265A, N297A mutations (EU numbering) to reduce effector function.

[0328] In another embodiment, the Fc domain is an IgG4 Fc domain with reduced binding to an Fc receptor. An exemplary IgG4 Fc domain with reduced binding to an Fc receptor can comprise an amino acid sequence selected from the following Table H: In some embodiments, the Fc domain includes only the bold portion of the sequence shown below:

[0329]

[0330]

[0331]

[0332]

[0333] In a specific embodiment, an IgG4 with reduced effector function comprises the bold portion of the amino acid sequence of SEQ ID NO: 31 of WO2014 / 121087, sometimes referred to herein as IgG4 or hIgG4, having the following amino acid sequence: ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 414).

[0334] For heterodimeric Fc regions, combinations of the above-mentioned variant IgG4 Fc sequences can be incorporated, for example, an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 30 of WO2014 / 121087 (or the bold portion thereof) and an Fc domain comprising the amino acid sequence of SEQ ID NO: 37 of WO2014 / 121087 (or the bold 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 bold portion thereof) and an Fc domain comprising the amino acid sequence of SEQ ID NO: 38 of WO2014 / 121087 (or the bold portion thereof).

[0335] 6.9.2. Fc Heterodimerization Variants

[0336] Some IFN receptor agonists require dimerization between two Fc domains, which, unlike natural immunoglobulins, are operably linked to non-identical N-terminal or C-terminal regions. Insufficient heterodimerization of the two Fc domains to form an Fc region may become an obstacle to increasing the yield of the desired heterodimeric molecule and poses a challenge to purification. Various methods available in the art can be used to enhance the dimerization of the Fc domains that may be present in the IFN receptor agonists of the present disclosure, for example, as disclosed in the following documents: EP 1870459A1; U.S. Patent No. 5,582,996; U.S. Patent No. 5,731,168; U.S. Patent No. 5,910,573; U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,441; U.S. Patent No. 7,183,076; U.S. Patent Application Publication No. 2006204493A1; and PCT Publication No. WO 2009 / 089004A1.

[0337] In some embodiments, the present disclosure provides IFN receptor agonists comprising an Fc heterodimer, i.e., an Fc region 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, and preferably the constant region of an antibody of the IgG (IgG1, IgG2, IgG3, and IgG4) class, as described in the previous section.

[0338] Heterodimerization of two different heavy chains at the CH3 domain produces the desired IFN receptor agonist, while homodimerization of the same heavy chain will reduce the production of the desired IFN receptor agonist. Therefore, in a preferred embodiment, the polypeptides that associate to form the IFN receptor agonists of the present disclosure will contain a CH3 domain modified to favor heterodimeric association relative to an unmodified Fc domain.

[0339] In a specific embodiment, the modification that promotes the formation of Fc heterodimers is a so-called "knob-in-hole" or "knob-in-hole" modification, comprising a "knob" modification in one Fc domain and a "hole" modification in another Fc domain. The knob-in-hole technique is described in, for example, U.S. Patent No. 5,731,168; US 7,695,936; Ridgway et al., 1996, Prot Eng 9:617-621, and Carter, 2001, Immunol Meth 248:7-15. Typically, the method includes introducing a protrusion ("knob") at the interface of a first polypeptide and introducing a corresponding cavity ("hole") in the interface of a second polypeptide so that the protrusion can be positioned in the cavity, thereby promoting heterodimer formation and hindering homodimer formation. The protrusion is constructed by replacing the small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of the same or similar size to the protuberance are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (eg, alanine or threonine).

[0340] Therefore, in some embodiments, the amino acid residues in the CH3 domain of the first subunit of the Fc domain are replaced by amino acid residues with larger side chain volumes, thereby generating a protrusion in the CH3 domain of the first subunit, which can be positioned in a cavity in the CH3 domain of the second subunit, and the amino acid residues in the CH3 domain of the second subunit of the Fc domain are replaced by amino acid residues with smaller side chain volumes, thereby generating a cavity in the CH3 domain of the second subunit, in which the protrusion in the CH3 domain of the first subunit can be positioned. Preferably, the amino acid residue with larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W). Preferably, the amino acid residue with smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T) and valine (V). The protrusion and cavity can be prepared by changing the nucleic acid encoding the polypeptide, for example, by site-specific mutagenesis or by peptide synthesis. An exemplary substitution is Y470T.

[0341] In a specific embodiment of this invention, in the first Fc domain, the threonine residue at position 366 is substituted with a tryptophan residue (T366W), and in the Fc domain, the tyrosine residue at position 407 is substituted with a valine residue (Y407V), and optionally, the threonine residue at position 366 is substituted with a serine residue (T366S) and the leucine residue at position 368 is substituted with an alanine residue (L368A) (numbering according to the Kabat EU index). In a further embodiment, in the first Fc domain, the serine residue at position 354 is additionally substituted with a cysteine ​​residue (S354C), or the glutamic acid residue at position 356 is substituted with a cysteine ​​residue (E356C) (particularly, the serine residue at position 354 is substituted with a cysteine ​​residue), and in the second Fc domain, the tyrosine residue at position 349 is additionally substituted with a cysteine ​​residue (Y349C) (numbering according to the Kabat EU index). 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 (numbering according to the Kabat EU index).

[0342] In some embodiments, electrostatic steering (eg, as described in Gunasekaran et al., 2010, J Biol Chem 285(25): 19637-46) can be used to promote binding of the first and second Fc domains of an Fc region.

[0343] As an alternative or in addition to using an Fc domain modified to promote heterodimerization, the Fc domain can be modified to allow a purification strategy that can select for Fc heterodimers. In one such embodiment, a polypeptide comprises a modified Fc domain that eliminates its binding to protein A, thereby enabling a purification method for producing heterodimeric proteins. See, for example, U.S. Patent No. 8,586,713. Thus, an IFN receptor agonist 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 wherein the at least one amino acid difference reduces the binding of the IFN receptor agonist to protein A compared to a corresponding IFN receptor agonist lacking the amino acid difference. In one embodiment, the first CH3 domain binds protein A and the second CH3 domain contains a mutation / modification that reduces or eliminates protein A binding, such as an H95R modification (by IMGT exon numbering; by EU numbering H435R). The second CH3 may further comprise a Y96F modification (by IMGT; by EU Y436F). Such modifications are referred to herein as "star" mutations.

[0344] In some embodiments, the Fc may contain one or more mutations (eg, knob and hole mutations) to promote heterodimerization and a star mutation to facilitate purification.

[0345] 6.9.3. Hinge domain

[0346] The IFN receptor agonist of the present disclosure may include an Fc domain that includes a hinge domain at its N-terminus. The hinge region may be a natural or modified hinge region. The hinge region is typically located at the N-terminus of the Fc region. Unless the context otherwise specifies, the term "hinge domain" refers to a hinge sequence that occurs naturally or non-naturally, which is a monomeric hinge domain in the context of a single or monomeric polypeptide chain and may include two associated hinge sequences on a separate polypeptide chain in the context of a dimeric polypeptide (e.g., a homodimer or heterodimer IFN receptor agonist formed by the association of two Fc domains). Sometimes, two related hinge sequences are referred to as "hinge regions." In certain embodiments of the IFN receptor agonist, additional iterations of the hinge region may be incorporated into the polypeptide sequence.

[0347] The native hinge region is the hinge region usually found between the Fab and Fc domains of naturally occurring antibodies. The modified hinge region is any hinge that is different from the native hinge region in length and / or component. Such hinges can include hinge regions from other species, such as humans, mice, rats, rabbits, sharks, pigs, hamsters, camels, llamas or goat hinge regions. Other modified hinge regions can include complete hinge regions derived from antibodies of different classes or subclasses than the heavy chain Fc domain or Fc region. Alternatively, the modified hinge region can include a portion of a native hinge or a repeat unit, wherein each unit in the repeat is derived from the native hinge region. In another alternative, the native hinge region can be changed by converting one or more cysteines or other residues into neutral residues, such as serine or alanine, or by converting appropriately placed residues into cysteine ​​residues. In this way, the number of cysteine ​​residues in the hinge region can increase or decrease. Other modified hinge regions can be fully synthetic and can be designed to have desired properties, such as length, cysteine ​​composition and flexibility.

[0348] Many modified hinge regions have been described in, for example, US Patent No. 5,677,425, WO 99 / 15549, WO 2005 / 003170, WO 2005 / 003169, WO 2005 / 003170, WO 98 / 25971, and WO 2005 / 003171 and the contents of which are incorporated herein by reference.

[0349] In one embodiment, the IFN receptor agonists of the present disclosure comprise an Fc region, wherein one or both Fc domains have an intact hinge domain at their N-termini.

[0350] In various embodiments, positions 233-236 within the hinge region can be G, G, G, and unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied, unoccupied, and unoccupied; or all unoccupied, where the positions are numbered by EU numbering.

[0351] In some embodiments, the IFN receptor agonists of the present disclosure comprise a modified hinge region that has reduced binding affinity for an Fcγ receptor relative to a wild-type hinge region of the same isotype (eg, human IgG1 or human IgG4).

[0352] In an embodiment, the IFN receptor agonist of the present disclosure comprises an Fc region, wherein each Fc domain has a complete hinge domain at its N-terminus, wherein each Fc domain and hinge domain are derived from IgG4 and each hinge domain includes a modified sequence CPPC (SEQ ID NO: 377). Compared to IgG1 containing the sequence CPPC (SEQ ID NO: 377), the core hinge region of human IgG4 contains the sequence CPSC (SEQ ID NO: 378). The serine residues present in the IgG4 sequence increase the flexibility of this region, so that a portion of the molecules form disulfide bonds (intrachain disulfide bonds) within the same protein chain, rather than bridging to other heavy chains in the IgG molecule to form interchain disulfide bonds. (Angel et al., 1993, Mol Immunol 30 (1): 105-108). Changing the serine residue to proline to obtain the same core sequence as IgG1 can completely form interchain disulfide bonds in the IgG4 hinge region, thereby reducing heterogeneity in the purified product. This altered isotype is called IgG4P.

[0353] 6.9.3.1. Chimeric Hinge Sequences

[0354] The hinge domain may be a chimeric hinge domain.

[0355] For example, a chimeric hinge can 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.

[0356] In specific embodiments, the chimeric hinge region comprises the amino acid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 379) (previously disclosed as SEQ ID NO: 8 of WO2014 / 121087, which is incorporated herein by reference in its entirety) or ESKYGPPCPPCPAPPVA (SEQ ID NO: 380) (previously disclosed as SEQ ID NO: 9 of WO2014 / 121087). Such chimeric hinge sequences can be suitably linked to an IgG4 CH2 region (e.g., by incorporation of an IgG4 Fc domain, e.g., a human or murine Fc domain, which can be further modified in the CH2 and / or CH3 domains to reduce effector function, e.g., as described in Section 6.9.1).

[0357] 6.9.3.2. Hinge sequences with reduced effector function

[0358] In further embodiments, the hinge region can be modified to reduce effector function, for example as described in WO2016161010A2, which is incorporated herein by reference in its entirety. In various embodiments, positions 233-236 of the modified hinge region are G, G, G, and unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied, unoccupied, and unoccupied; or all unoccupied, where positions are numbered by EU numbering (e.g., WO2016161010A2). Figure 1 ). These fragments can be represented as GGG-, GG--, G---, or ----, where "-" indicates an unoccupied position.

[0359] Position 236 is unoccupied in canonical human IgG2, but is occupied in other canonical human IgG isotypes. In all four human isotypes, positions 233-235 are occupied by residues other than G (as in WO2016161010A2). Figure 1 shown).

[0360] Hinge modifications within positions 233-236 can be combined with position 228, which is 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 beneficial for stabilizing IgG4 antibodies and reducing the exchange of heavy and light chain pairs between exogenous and endogenous antibodies. Preferably, positions 226-229 are occupied by C, P, P, and C, respectively.

[0361] Exemplary hinge regions have residues 226-236, sometimes referred to as the middle (or core) and lower hinge, occupied by modified hinge sequences designated GGG-(233-236), GG--(233-236), G---(233-236), and no G(233-236). Optionally, the hinge domain amino acid sequence includes CPPCPAPGGG-GPSVF (SEQ ID NO: 381) (previously disclosed as SEQ ID NO: 1 of WO2016161010A2), CPPCPAPGG--GPSVF (SEQ ID NO: 382) (previously disclosed as SEQ ID NO: 2 of WO2016161010A2), CPPCPAPG---GPSVF (SEQ ID NO: 383) (previously disclosed as SEQ ID NO: 3 of WO2016161010A2), or CPPCPAP----GPSVF (SEQ ID NO: 384) (previously disclosed as SEQ ID NO: 4 of WO2016161010A2).

[0362] The hinge region of the above modifications can be incorporated into a heavy chain constant region, which generally includes CH2 and CH3 domains, and it can have an additional hinge segment (e.g., upper hinge) flanking a specified region. Such other constant region fragments present generally have the same isotype, preferably human isotype, although it can be a heterozygote of different isotypes. The isotype of such other human constant region fragments is preferably human IgG4, but it can also be human IgG1, IgG2 or IgG3 or its domains have a heterozygote of different isotypes. The exemplary sequences of human IgG1, IgG2 and IgG4 are shown in Figures 2 to 4 of WO2016161010A2.

[0363] In specific embodiments, the modified hinge sequence can be linked to the IgG4 CH2 region (e.g., by incorporating an IgG4 Fc domain, e.g., a human or murine Fc domain, which can be further modified in the CH2 and / or CH3 domains to reduce effector function, e.g., as described in Section 6.9.1).

[0364] 6.10. Nucleic Acids and Host Cells

[0365] In another aspect, the present disclosure provides nucleic acids encoding the IFN receptor agonists of the present disclosure. In some embodiments, the IFN receptor agonist is encoded by a single nucleic acid. In other embodiments, the IFN receptor agonist may be encoded by multiple (e.g., two, three, four or more) nucleic acids.

[0366] A single nucleic acid can encode an IFN receptor agonist comprising a single polypeptide chain, an IFN receptor agonist comprising two or more polypeptide chains, or a portion of an IFN receptor agonist comprising two or more polypeptide chains (e.g., a single nucleic acid can encode two polypeptide chains of an IFN receptor agonist comprising three, four or more polypeptide chains, or three polypeptide chains of an IFN receptor agonist comprising four or more polypeptide chains). For separate control of expression, the open reading frames encoding the two or more polypeptide chains can be under the control of separate transcriptional regulatory elements (e.g., promoters and / or enhancers). The open reading frames encoding the two or more polypeptides can also be controlled by the same transcriptional regulatory element and separated by an internal ribosome entry site (IRES) sequence, thereby allowing translation into separate polypeptides.

[0367] In some embodiments, an IFN receptor agonist comprising two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding an IFN receptor agonist may be equal to or less than the number of polypeptide chains in the IFN receptor agonist (e.g., when more than one polypeptide chain is encoded by a single nucleic acid).

[0368] The nucleic acid of the present disclosure can be DNA or RNA (eg, mRNA).

[0369] In another aspect, the present disclosure provides host cells and vectors containing the nucleic acids of the present disclosure. The nucleic acids can be present in a single vector or in separate vectors in the same host cell or in separate host cells, as described in more detail below.

[0370] 6.10.1. Carrier

[0371] The present disclosure provides vectors comprising a nucleotide sequence encoding an IFN receptor agonist or a component thereof as described herein, such as one or two polypeptide chains of a half-antibody of an IFN receptor agonist. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs).

[0372] A variety of vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses, such as, for example, bovine papilloma virus, polyoma virus, 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 flavivirus.

[0373] In addition, can select the cell that DNA is stably integrated into its chromosome by introducing one or more markers that allow to select the host cell of institute's transfection.Mark can for example provide the original plasmonicity to auxotrophic host, biocide resistance (for example, antibiotic) or to the resistance of heavy metal (for example copper) etc. Selectable marker gene can be directly connected to dna sequence dna to be expressed, or introduces in the same cell by cotransformation.The best synthesis of mRNA may also need other element.These elements can comprise splicing signal and transcription promoter, enhancer and termination signal.

[0374] Once the expression vector or DNA sequence containing the construct is prepared for expression, the expression vector can be transfected or introduced into an appropriate host cell. Various techniques can be used to achieve this, such as, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques. Methods and conditions for culturing the resulting transfected cells and for recovering the expressed polypeptide are known to those skilled in the art and can be varied or optimized based on the present specification according to the specific expression vector and mammalian host cell used.

[0375] 6.10.2 Cells

[0376] The disclosure also provides host cells comprising the nucleic acids of the disclosure.

[0377] In one embodiment, the host cell is genetically engineered to contain one or more nucleic acids described herein.

[0378] In one embodiment, host cells are genetically engineered using expression cassettes. The phrase "expression cassette" refers to a nucleotide sequence that is capable of influencing the expression of a gene in a host compatible with such sequence. Such cassettes may include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or helpful to influence expression, such as, for example, inducible promoters, may also be used.

[0379] The present disclosure also provides host cells comprising the vectors described herein.

[0380] Cells can be, but are not limited to, eukaryotic cells, bacterial cells, insect cells, or human cells. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells.

[0381] 6.11. Pharmaceutical Compositions

[0382] The IFN receptor agonists of the present disclosure can be in the form of a composition comprising an IFN receptor agonist and one or more carriers, excipients and / or diluents. The composition can be formulated for specific uses, such as for veterinary use or human pharmaceutical use. The form of the composition used (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents and / or carriers will depend on the intended use of the IFN receptor agonist and, for therapeutic use, also on the mode of administration.

[0383] For therapeutic use, composition can be provided as a part of a sterile pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition can be in any suitable form (depending on the method of its expectation to be applied to the patient). The pharmaceutical composition can be applied to the patient by a variety of routes, such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, local (topically) or local (locally) administration. The most suitable route of administration in any given case will depend on the nature and severity of the specific IFN receptor agonist, subject and disease and the physical condition of the subject. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

[0384] The pharmaceutical composition can be conveniently present in a unit dosage form containing a predetermined amount of an IFN receptor agonist of the present invention per dose. The amount of the IFN receptor agonist contained in the unit dose will depend on the disease being treated and other factors well known in the art. Such unit doses can be in the form of a lyophilized dry powder containing a certain amount of IFN receptor agonist suitable for single administration, or in the form of a liquid. The dry powder unit dosage form can be packaged in a kit together with a syringe, an appropriate amount of diluent and / or other components for administration. The unit dose in the form of a liquid can be conveniently supplied in the form of a syringe pre-filled with a certain amount of IFN receptor agonist suitable for single administration.

[0385] The pharmaceutical composition may also be supplied in bulk containing an amount of IFN receptor agonist suitable for multiple administrations.

[0386] Pharmaceutical compositions can be prepared by mixing an IFN receptor agonist having the desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (all of which are referred to herein as "carriers") commonly used in the art (i.e., buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, antioxidants, and other various additives) for storage as lyophilized formulations or aqueous solutions. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives should be nontoxic to the recipient at the dosages and concentrations employed.

[0387] Buffers help maintain pH in a range close to physiological conditions. They can be present in a variety of concentrations, but are typically present in a concentration range of about 2 mM to about 50 mM. Suitable buffers for use in the present disclosure include 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, In some embodiments, the present invention provides a buffered saline buffer (e.g., a fumaric acid-disodium fumarate mixture, a monosodium fumarate-disodium fumarate mixture, etc.), a gluconate buffer (e.g., a gluconic acid-sodium gluconate mixture, a gluconic acid-sodium hydroxide mixture, a gluconic acid-potassium gluconate mixture, etc.), an oxalate buffer (e.g., an oxalic acid-sodium oxalate mixture, an oxalic acid-sodium hydroxide mixture, an oxalic acid-potassium oxalate mixture, etc.), a lactate buffer (e.g., a lactic acid-sodium lactate mixture, a lactic acid-sodium hydroxide mixture, a lactic acid-potassium lactate mixture, etc.), and an acetate buffer (e.g., an acetic acid-sodium acetate mixture, an acetic acid-sodium hydroxide mixture, etc.). In addition, a phosphate buffer, a histidine buffer, and a trimethylamine salt (such as Tris) can also be used.

[0388] Preservatives can be added to slow down microbial growth and can be added in an amount within the range of about 0.2% to 1% (w / v). Suitable preservatives for the present disclosure include phenol, benzyl alcohol, metacresol, methylparaben, propylparaben, octadecyldimethylbenzyl ammonium chloride, benzalkonium chloride halides (e.g., chloride, bromide and iodide), hexamethylammonium chloride and alkylparabens (such as methylparaben or propylparaben), catechol, resorcinol, cyclohexanol and 3-pentanol. Isotonic agents (sometimes referred to as "stabilizers") can be added to ensure the isotonicity of the liquid composition of the present disclosure, and isotonic agents include polyols, such as trivalent or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol and mannitol. Stabilizers refer to a large class of excipients whose functional range includes from fillers to additives, which can dissolve therapeutic agents or help prevent denaturation or adhesion to the container wall. Typical stabilizers can be polyols (listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, inositol, galactitol, glycerol, etc., including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents such as urea. , glutathione, lipoic acid, sodium thioacetate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides of 10 residues or less); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose, sucrose and trehalose; and trisaccharides such as raffinose; and polysaccharides such as dextran. The stabilizer may be present in an amount of 0.5 to 10% by weight per weight of the IFN receptor agonist.

[0389] Can add nonionic surfactant or detergent (also referred to as " wetting agent ") to help dissolve glycoprotein and protection glycoprotein avoid the aggregation that stirring causes, this also allows preparation to be exposed to shear surface stress and can not cause the denaturation of albumen.Suitable nonionic surfactant comprises polysorbate (20,80 etc.), polyoxysalicylate (184,188 etc.) and pluronic polyol.Nonionic surfactant can exist with the scope of about 0.05mg / mL to about 1.0mg / mL (for example, about 0.07mg / mL to about 0.2mg / mL).

[0390] Other miscellaneous excipients include fillers (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), and co-solvents.

[0391] The IFN receptor agonists of the present disclosure can be formulated into pharmaceutical compositions comprising the IFN receptor agonists, for example, containing one or more pharmaceutically acceptable excipients or carriers. To prepare pharmaceutical or sterile compositions comprising the IFN receptor agonists of the present disclosure, the IFN receptor agonist preparation can be combined with one or more pharmaceutically acceptable excipients or carriers.

[0392] For example, formulations of the IFN receptor agonist can be prepared by mixing the IFN receptor agonist with a physiologically acceptable carrier, excipient, or stabilizer in the form of, for example, 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 Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie, 2000, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

[0393] The effective amount for a particular subject may vary depending on a variety of factors, such as the condition being treated, the subject's overall health, the route and dosage 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).

[0394] Compositions of the present disclosure can also be administered by one or more routes of administration using one or more methods of various methods known in the art. As will be appreciated by those skilled in the art, routes of administration and / or modes will vary according to the desired result. The selected routes of administration of IFN receptor agonists include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other general routes of administration, such as by injection or infusion. General administration can represent a mode of administration other than enteral and topical administration, typically by injection, and includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, compositions of the present disclosure can be administered by non-general routes, such as topical, epidermal or mucosal routes of administration, such as intranasal, oral, vaginal, rectal, sublingual or topical. In one embodiment, IFN receptor agonists are administered by infusion. In another embodiment, IFN receptor agonists of the present disclosure are administered subcutaneously.

[0395] 6.11.1. Pharmaceutical Compositions for Delivery of Nucleic Acids Encoding IFN Receptor Agonists

[0396] The IFN receptor agonists of the present disclosure can be delivered by any method useful for gene therapy, for example, as mRNA or by a viral vector encoding the IFN receptor agonist under the control of a suitable promoter.

[0397] Exemplary viral vectors include recombinant adenovirus and adeno-associated virus vectors (rAAV). The rAAV vector is based on a defective and non-pathogenic parvovirus adeno-associated virus type 2. Most of these vectors are derived from plasmids that retain only the AAV inverted terminal repeats flanking the transgenic expression cassette. A key feature of this vector system is that efficient gene transfer and stable transgene delivery are achieved by integration into the genome of the transduced cells. AAV serotypes can be used to deliver IL27 transgenes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV 8.2, AAV9, and AAV rh10, as well as pseudotyped AAVs, such as AAV2 / 8, AAV2 / 5, and AAV2 / 6.

[0398] AAV can be produced on a clinical scale by a number of different processes. Examples of systems that can be used include (1) plasmid DNA transfection in mammalian cells, (2) Ad infection of stable mammalian cell lines, (3) infection of mammalian cells with recombinant herpes simplex virus (rHSV), and (4) infection of insect cells (Sf9 cells) with recombinant baculovirus (reviewed by Penaud-Budloo et al., 2018, Mol Ther Methods Clin Dev. 8:166-180).

[0399] Replication-deficient recombinant adenoviral vectors (Ad) can be produced in high titers and readily infect many different cell types. Most adenoviral vectors are engineered to replace the Ad Ela, Elb, and / or E3 genes with transgenes; the replication-deficient vectors are then propagated in human 293 cells, which provide the missing transgene function. Ad vectors can transduce a variety of tissue types in vivo, including non-dividing, differentiated cells such as those found in the liver, kidney, and muscle. Conventional Ad vectors have a large carrying capacity.

[0400] Packaging cells are used to form viral particles capable of infecting host cells. Such cells include 293 cells for packaging adenoviruses, and w2 cells or PA317 cells for packaging retroviruses. Viral vectors used in gene therapy are typically produced by producer cell lines that package nucleic acid vectors into viral particles. The vector typically contains the minimal viral sequences required for packaging and subsequent integration into the host (if applicable), with other viral sequences replaced by expression cassettes encoding the protein to be expressed. The missing viral functions are provided in trans by the packaging cell line. For example, AAV vectors used for gene therapy typically only have inverted terminal repeat (ITR) sequences from the AAV genome, which are required for packaging and integration into the host genome. The viral DNA is packaged in a cell line that contains a helper plasmid encoding other AAV genes, namely rep and cap, but lacks ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus promotes the replication of the AAV vector and the expression of AAV genes from the helper plasmid. Due to the lack of ITR sequences, the helper plasmid is not packaged in large quantities. Contamination with adenovirus can be reduced, for example, by heat treatment, which is more sensitive than AAV.

[0401] Nucleic acid molecules (e.g., mRNA) or viruses can be formulated into the only active pharmaceutical ingredient in a pharmaceutical composition, or can be combined with other active agents for the specific condition being treated. Alternatively, other medicaments, pharmaceutical agents, carriers, adjuvants, diluents can be included in the compositions provided herein. For example, any one or more of a wetting agent, emulsifier, and lubricant (such as sodium lauryl sulfate and magnesium stearate) and a colorant, a release agent, a coating agent, a sweetener, a flavoring agent, a preservative, an antioxidant, a chelating agent, and an inert gas may also be present in the composition. Exemplary other agents and excipients that can be included in the composition include, for example, water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol; and metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0402] 6.12. Therapeutic Indications and Usage

[0403] The present disclosure provides methods of use and applications for the IFN receptor agonists of the present disclosure.

[0404] The IFN receptor agonists of the present disclosure can be used to stimulate immune responses in a variety of applications.

[0405] In certain aspects, the present disclosure provides a method for treating cancer comprising administering an IFN receptor agonist or pharmaceutical composition as described herein to a subject in need thereof. In some embodiments, when the IFN receptor agonist comprises one or more protease-cleavable linkers, the activated IFN protein comprising the IFN portion is produced by cleavage of the one or more protease-cleavable linkers in the IFN receptor agonist by one or more proteases expressed by cancerous tissue. Thus, in some embodiments, the IFN receptor agonist is selectively activated in cancerous tissue.

[0406] In some embodiments, the present disclosure provides a method for treating cancer using an IFN protein that is selectively activated in cancer tissue, comprising administering to a subject in need thereof an IFN receptor agonist or pharmaceutical composition as described herein comprising one or more protease-cleavable linkers, each protease-cleavable linker comprising one or more substrates for one or more proteases expressed by the cancer tissue for which the IFN protein is intended. Thus, the activated IFN protein comprising the IFN portion is produced by cleavage of the one or more protease-cleavable linkers in the IFN receptor agonist by one or more proteases in the cancer tissue.

[0407] The present disclosure further provides a method for local delivery of an IFN protein, comprising administering to a subject an IFN receptor agonist or pharmaceutical composition as described herein, wherein the IFN receptor agonist has one or more targeting moieties and / or protease-cleavable linkers, each protease-cleavable linker comprising one or more substrates for one or more proteases expressed by the tissue to which the IFN protein is to be locally delivered. As used herein, the term "local delivery" does not require local administration, but rather means that the active ingredient of the IFN receptor agonist is selectively targeted with a targeting moiety that recognizes a target molecule expressed in the intended site and / or the protein is activated by a protease active at the intended site.

[0408] The present disclosure further provides a method of administering to a subject an IFN therapy with reduced systemic exposure and / or reduced systemic toxicity, comprising administering to the subject an IFN therapy in the form of an IFN receptor agonist or pharmaceutical composition as described herein, wherein the IFN receptor agonist has one or more targeting moieties and / or protease-cleavable linkers that bind to a target molecule expressed by a tissue for which IFN therapy is desired and / or anticipated, each protease-cleavable linker comprising one or more substrates for one or more proteases expressed by a tissue for which IFN therapy is desired and / or anticipated.

[0409] Thus, the aforementioned approach allows for reduction of off-target side effects of IFN therapy due to preferential delivery and / or activation of IFN receptor agonists at the lesions where IFN treatment is intended.

[0410] Thus, the present disclosure provides a method for targeted delivery of an activated IFN protein to a lesion (e.g., cancerous tissue) intended for treatment, comprising administering to a subject an IFN receptor agonist or pharmaceutical composition as described herein, wherein the IFN comprises one or more targeting moieties that recognize a target molecule in the lesion or expressed by the tissue intended for treatment (e.g., cancerous tissue), and the IFN optionally has one or more protease-cleavable linkers, each protease-cleavable linker comprising one or more substrates for one or more proteases expressed by the tissue for which IFN therapy is desired and / or anticipated.

[0411] The present disclosure further provides a method for inducing an immune response locally in a target tissue, comprising administering to a subject an IFN receptor agonist or pharmaceutical composition as described herein, wherein the IFN receptor agonist or pharmaceutical composition has one or more targeting moieties capable of binding to a target molecule expressed in the target tissue and optionally one or more protease-cleavable linkers, each protease-cleavable linker comprising one or more substrates for one or more proteases expressed in the target tissue. When a protease-activated linker is present, an activated IFN protein comprising an IFN portion can then be produced by cleaving one or more protease-cleavable linkers in the IFN receptor agonist 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. In the absence of activation, an IFN protein with weakened activity (e.g., due to masking) can induce an immune response against at least one cell type in the target tissue.

[0412] In some embodiments, administration is not local to a tissue. For example, when the target tissue is cancerous tissue, administration can be systemic or subcutaneous.

[0413] The IFN receptor agonists of the present disclosure can be used to treat any proliferative disorder (e.g., cancer) that expresses a target molecule (on tumor cells or in the tumor microenvironment, e.g., extracellular matrix or tumor lymphocytes). In specific embodiments, the cancer is acute lymphocytic 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 lymphoma, cancer of unknown primary, heart tumor, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasms, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, leukemia ... Tubular carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, 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 lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cancer, liver cancer, lobular primordial tumor carcinoma of the nasal cavity, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck carcinoma with occult primary, midline tract cancer involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin 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, stomach cancer, T-cell lymphoma, teratoma, testicular cancer, pharyngeal cancer, thymoma and thymic cancer, thyroid cancer, urethra cancer, uterine cancer, vaginal cancer, vulvar cancer, or Wilms' tumor.

[0414] Table I below shows exemplary indications for which IFN receptor agonists directed to specific target molecules can be used.

[0415]

[0416]

[0417]

[0418] Other target molecules and corresponding indications are disclosed in, for example, Hafeez et al., 2020, Molecules 25:4764, doi: 10.3390 / molecules25204764, particularly in Table 1. Table 1 is incorporated herein by reference in its entirety.

[0419] In a further embodiment, IFN receptor agonists can be used to enhance the immune response triggered by another agent. Therefore, in some embodiments, the IFN receptor agonists of the present disclosure are administered as an adjuvant therapy of an immunogenic agent. In some embodiments, the immunogenic agent is an adjuvanted or unadjuvanted vaccine. Therefore, IFN receptor agonists can enhance the antigen-specific immune response triggered by the vaccine. In various embodiments, the vaccine is a preventive or therapeutic cancer vaccine or a preventive or therapeutic vaccine against an infectious agent (e.g., a virus, bacteria, or parasite).

[0420] 7. Numbered Examples

[0421] While various specific embodiments have been shown and described, it will be understood that various changes can be made without departing from the spirit and scope of the present disclosure.The present disclosure is illustrated by the numbered embodiments set forth below.

[0422] In the following numbered embodiments, the targeting moiety preferably binds to a mammalian target molecule, the IFN moiety is preferably derived from a mammalian IFN, the Fc domain is preferably derived from a mammalian antibody, and the subject is preferably a mammal. More preferably, the mammal is a human.

[0423] 1. A type I interferon (IFN) receptor agonist comprising

[0424] (a) a first polypeptide chain comprising a first Fc domain;

[0425] (b) a second polypeptide chain comprising a second Fc domain associated with the first Fc domain;

[0426] wherein the first polypeptide chain and / or the second polypeptide chain comprises a type I interferon (IFN) portion that is optionally attenuated by: (i) masking by a type I interferon receptor (IFNR) portion; (ii) one or more mutations in the IFN portion compared to wild-type interferon, e.g., by one or more amino acid substitutions and / or truncation; (iii) use of a native IFN sequence with a lower receptor affinity than IFNα2b and / or IFNβ, or (iv) any combination of two or all three of (i), (ii) and (iii).

[0427] 2. The IFN receptor agonist of embodiment 1, comprising an IFNR portion located on the same polypeptide chain as the IFN portion.

[0428] 3. The IFN receptor agonist of embodiment 1, comprising an IFNR portion located on a different polypeptide chain from the IFN portion.

[0429] 4. The IFN receptor agonist of any one of embodiments 1 to 3, wherein the first polypeptide chain comprises a first IFN portion and the second polypeptide chain comprises a second IFN portion, the first IFN portion and the second IFN portion each being masked by an IFNR portion.

[0430] 5. The IFN receptor agonist of any one of embodiments 1 to 4, which is bivalent with respect to the IFN moiety.

[0431] 6. The IFN receptor agonist of embodiment 5, wherein each IFN moiety is singly masked.

[0432] 7. The IFN receptor agonist of embodiment 5, wherein each IFN moiety is doubly masked.

[0433] 8. The IFN receptor agonist of any one of embodiments 4 to 7, wherein the first IFN moiety is located N-terminally to the first Fc domain, and the second IFN moiety is located N-terminally to the second Fc domain.

[0434] 9. The IFN receptor agonist of any one of embodiments 4 to 7, wherein the first IFN moiety is located at the C-terminus of the first Fc domain, and the second IFN moiety is located at the C-terminus of the second Fc domain.

[0435] 10. The IFN receptor agonist of any one of embodiments 1 to 3, wherein said first polypeptide chain or said second polypeptide chain comprises an IFN moiety masked by an IFNR moiety.

[0436] 11. The IFN receptor agonist of any one of embodiments 1 to 3 and 10, which is monovalent with respect to the IFN moiety.

[0437] 12. The IFN receptor agonist of embodiment 11, wherein the IFN moiety is monomasked.

[0438] 13. The IFN receptor agonist of embodiment 11, wherein the IFN moiety is doubly masked.

[0439] 14. The IFN receptor agonist of any one of embodiments 10 to 13, wherein the IFN moiety is located at the N-terminus of the first Fc domain or the N-terminus of the second Fc domain.

[0440] 15. The IFN receptor agonist of any one of embodiments 10 to 13, wherein the IFN moiety is located at the C-terminus of the first Fc domain or the C-terminus of the second Fc domain.

[0441] 16. An IFN receptor agonist, optionally the IFN receptor agonist according to any one of embodiments 1 to 15, comprising any of the half-antibody pairs designated 1 to 23 listed in Table 2.

[0442] 17. The IFN receptor agonist of any one of embodiments 1 to 16, which does not comprise a protease cleavable linker (PCL).

[0443] 18. The IFN receptor agonist of any one of embodiments 1 to 16, comprising at least one protease cleavable linker (PCL).

[0444] 19. The IFN receptor agonist of embodiment 18, wherein the PCL comprises a substrate sequence cleavable by any protease listed in Table A.

[0445] 20. The IFN receptor agonist of embodiment 18 or embodiment 19, wherein the PCL comprises one or more substrate sequences selected from the substrate sequences listed in Table B.

[0446] 21. The IFN receptor agonist of any one of embodiments 18 to 20, wherein the PCL comprises one or more spacer sequences selected from the substrate sequences listed in Table C.

[0447] 22. The IFN receptor agonist of any one of embodiments 18 to 21, wherein the PCL comprises the amino acid sequence of any one of the PCL sequences listed in Table D or a variant thereof having up to 5 amino acid substitutions, e.g., a variant thereof having 1 amino acid substitution, 2 amino acid substitutions, 3 amino acid substitutions, 4 amino acid substitutions, or 5 amino acid substitutions.

[0448] 23. The IFN receptor agonist of any one of embodiments 18 to 22, which is configured such that cleavage of the protease cleavable linker (PCL) does not mask the IFN moiety.

[0449] 24. The IFN receptor agonist of embodiment 23, wherein the unmasked IFN portion is in the form of an IFN polypeptide comprising an Fc domain.

[0450] 25. The IFN receptor agonist of embodiment 23, wherein the unmasked IFN portion is in the form of an IFN polypeptide lacking an Fc domain.

[0451] 26. The IFN receptor agonist of any one of embodiments 1 to 25, wherein each IFN portion comprises an amino acid sequence having at least about 90% sequence identity to: (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ, or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ with up to one truncation of 15 amino acids at the N-terminus and / or C-terminus.

[0452] 27. The IFN receptor agonist of any one of embodiments 1 to 25, wherein each IFN portion comprises an amino acid sequence having at least about 95% sequence identity to: (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ, or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ with up to one truncation of 15 amino acids at the N-terminus and / or C-terminus.

[0453] 28. The IFN receptor agonist of any one of embodiments 1 to 25, wherein each IFN portion comprises an amino acid sequence having at least about 98% sequence identity to: (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ, or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ with at most one truncation of 15 amino acids at the N-terminus and / or C-terminus.

[0454] 29. The IFN receptor agonist of any one of embodiments 1 to 28, wherein each IFN portion comprises an amino acid sequence having one or more attenuating mutations compared to mature human IFNα1 or IFNα2b.

[0455] 30. The IFN receptor agonist of any one of embodiments 1 to 28, having one or more mutations selected from the group consisting of L26A, F27A, R33A, R33K, L30A, D35E, H57Y, E58N, Q61S, H57S, E58S, H57A, E58A, Q61A, Q90A, E96A, R120A, L135A, R144A, R144S, R144T, R144Y, R144I, R144L, A145D, A145H, A145K, A145M, A145V, A145Y, R149A, R149K, S152A, R162A, and E165D.

[0456] 31. The IFN receptor agonist of any one of embodiments 1 to 29, comprising the amino acid substitution R33A.

[0457] 32. The IFN receptor agonist of any one of embodiments 1 to 29, comprising the amino acid substitution R33K.

[0458] 33. The IFN receptor agonist of any one of embodiments 1 to 29, comprising the amino acid substitution Q90A.

[0459] 34. The IFN receptor agonist of any one of embodiments 1 to 29, comprising the amino acid substitution E96A.

[0460] 35. The IFN receptor agonist of any one of embodiments 1 to 29, comprising the amino acid substitution R120A.

[0461] 36. The IFN receptor agonist of any one of embodiments 1 to 29, comprising the amino acid substitution A145M.

[0462] 37. The IFN receptor agonist of any one of embodiments 1 to 29, comprising the amino acid substitution R149A.

[0463] 38. The IFN receptor agonist of any one of embodiments 1 to 29, comprising the amino acid substitution R149K.

[0464] 39. The IFN receptor agonist of any one of embodiments 1 to 29, comprising the amino acid substitution S152A.

[0465] 40. The IFN receptor agonist of any one of embodiments 1 to 29, comprising amino acid substitutions R33A, H57Y, E58N, and Q61S.

[0466] 41. The IFN receptor agonist of any one of embodiments 1 to 29, comprising amino acid substitutions H57Y, E58N, Q61S, and R144A.

[0467] 42. The IFN receptor agonist of any one of embodiments 1 to 29, comprising amino acid substitutions A145M and R149K.

[0468] 43. The IFN receptor agonist of any one of embodiments 1 to 29, comprising amino acid substitutions Q90A and R120A.

[0469] 44. The IFN receptor agonist of any one of embodiments 1 to 43, wherein the IFNR moiety is an interferon alpha receptor (IFNAR) moiety.

[0470] 45. The IFN receptor agonist of embodiment 44, wherein the IFNAR moiety is an IFNAR1 moiety.

[0471] 46. ​​The IFN receptor agonist of embodiment 45, wherein the IFNAR1 portion comprises an amino acid sequence having at least 90% sequence identity to: (i) the SD2 domain and the SD3 domain of human IFNAR1, (ii) the SD1 domain, SD2 domain, and SD3 domain of human IFNAR1, or (iii) the SD1 domain, SD2 domain, SD3 domain, and SD4 domain of human IFNAR1.

[0472] 47. The IFN receptor agonist of embodiment 46, wherein the IFNAR1 portion comprises an amino acid sequence having at least 90% sequence identity to the SD2 and SD3 domains of human IFNAR1.

[0473] 48. The IFN receptor agonist of embodiment 46, wherein the IFNAR1 portion comprises an amino acid sequence having at least 90% sequence identity to the SD1 domain, SD2 domain, and SD3 domain of human IFNAR1.

[0474] 49. The IFN receptor agonist of embodiment 46, wherein the IFNAR1 portion comprises an amino acid sequence having at least 90% sequence identity to the SD1 domain, SD2 domain, SD3 domain, and SD4 domain of human IFNAR1.

[0475] 50. The IFN receptor agonist of embodiment 45, wherein the IFNAR1 portion comprises an amino acid sequence having at least 95% sequence identity to: (i) the SD2 domain and the SD3 domain of human IFNAR1, (ii) the SD1 domain, SD2 domain, and SD3 domain of human IFNAR1, or (iii) the SD1 domain, SD2 domain, SD3 domain, and SD4 domain of human IFNAR1.

[0476] 51. The IFN receptor agonist of embodiment 50, wherein the IFNAR1 portion comprises an amino acid sequence having at least 95% sequence identity to the SD2 and SD3 domains of human IFNAR1.

[0477] 52. The IFN receptor agonist of embodiment 50, wherein the IFNAR1 portion comprises an amino acid sequence having at least 95% sequence identity to the SD1 domain, SD2 domain, and SD3 domain of human IFNAR1.

[0478] 53. The IFN receptor agonist of embodiment 50, wherein the IFNAR1 portion comprises an amino acid sequence having at least 95% sequence identity to the SD1 domain, SD2 domain, SD3 domain, and SD4 domain of human IFNAR1.

[0479] 54. The IFN receptor agonist of embodiment 45, wherein the IFNAR1 portion comprises an amino acid sequence having at least 98% sequence identity to: (i) the SD2 domain and the SD3 domain of human IFNAR1, (ii) the SD1 domain, SD2 domain, and SD3 domain of human IFNAR1, or (iii) the SD1 domain, SD2 domain, SD3 domain, and SD4 domain of human IFNAR1.

[0480] 55. The IFN receptor agonist of embodiment 54, wherein the IFNARl portion comprises an amino acid sequence having at least 98% sequence identity to the SD2 and SD3 domains of human IFNARl.

[0481] 56. The IFN receptor agonist of embodiment 54, wherein the IFNAR1 portion comprises an amino acid sequence having at least 98% sequence identity to the SD1 domain, SD2 domain, and SD3 domain of human IFNAR1.

[0482] 57. The IFN receptor agonist of embodiment 54, wherein the IFNAR1 portion comprises an amino acid sequence having at least 98% sequence identity to the SD1 domain, SD2 domain, SD3 domain, and SD4 domain of human IFNAR1.

[0483] 58. The IFN receptor agonist of embodiment 44, wherein the IFNAR moiety is an IFNAR2 moiety.

[0484] 59. The IFN receptor agonist of embodiment 58, wherein the IFNAR2 portion comprises an amino acid sequence having at least 90% sequence identity to: (i) the D1 domain of human IFNAR2 or (ii) the D1 domain and D2 domain of human IFNAR2.

[0485] 60. The IFN receptor agonist of embodiment 59, wherein the IFNAR2 portion comprises an amino acid sequence having at least 90% sequence identity to the D1 domain of human IFNAR2.

[0486] 61. The IFN receptor agonist of embodiment 59, wherein the IFNAR2 portion comprises an amino acid sequence having at least 90% sequence identity to the D1 domain and D2 domain of human IFNAR2.

[0487] 62. The IFN receptor agonist of embodiment 58, wherein the IFNAR2 portion comprises an amino acid sequence having at least 95% sequence identity to: (i) the D1 domain of human IFNAR2 or (ii) the D1 domain and D2 domain of human IFNAR2.

[0488] 63. The IFN receptor agonist of embodiment 62, wherein the IFNAR2 portion comprises an amino acid sequence having at least 95% sequence identity to the D1 domain of human IFNAR2.

[0489] 64. The IFN receptor agonist of embodiment 62, wherein the IFNAR2 portion comprises an amino acid sequence having at least 95% sequence identity to the D1 domain and D2 domain of human IFNAR2.

[0490] 65. The IFN receptor agonist of embodiment 58, wherein the IFNAR2 portion comprises an amino acid sequence having at least 98% sequence identity to: (i) the D1 domain of human IFNAR2 or (ii) the D1 domain and D2 domain of human IFNAR2.

[0491] 66. The IFN receptor agonist of embodiment 65, wherein the IFNAR2 portion comprises an amino acid sequence having at least 98% sequence identity to the D1 domain of human IFNAR2.

[0492] 67. The IFN receptor agonist of embodiment 65, wherein the IFNAR2 portion comprises an amino acid sequence having at least 98% sequence identity to the D1 domain and D2 domain of human IFNAR2.

[0493] 68. The IFN receptor agonist of any one of embodiments 1 to 67, wherein the IFN moiety is masked by an IFNAR1 moiety and an IFNAR2 moiety.

[0494] 69. The IFN receptor agonist of claim 68, which is monovalent with respect to the IFN portion, the IFNAR1 portion, and the IFNAR2 portion.

[0495] 70. The IFN receptor agonist of claim 68, which is bivalent with respect to the IFN portion, the IFNAR1 portion, and the IFNAR2 portion.

[0496] 71. The IFN receptor agonist of any one of embodiments 68 to 70, wherein the IFNARl portion comprises an amino acid sequence having at least about 90% sequence identity to the SD2 and SD3 domains of human IFNARl.

[0497] 72. The IFN receptor agonist of any one of embodiments 68 to 70, wherein the IFNARl portion comprises an amino acid sequence having at least about 95% sequence identity to the SD2 and SD3 domains of human IFNARl.

[0498] 73. The IFN receptor agonist of any one of embodiments 68 to 70, wherein the IFNARl portion comprises an amino acid sequence having at least about 98% sequence identity to the SD2 and SD3 domains of human IFNARl.

[0499] 74. The IFN receptor agonist of any one of embodiments 68 to 70, wherein said IFNAR1 portion comprises said amino acid sequence of said SD2 and SD3 domains of human IFNAR1.

[0500] 75. The IFN receptor agonist of any one of embodiments 68 to 70, wherein the IFNARl portion comprises an amino acid sequence having at least about 90% sequence identity to the SDl, SD2, and SD3 domains of human IFNARl.

[0501] 76. The IFN receptor agonist of any one of embodiments 68 to 70, wherein the IFNARl portion comprises an amino acid sequence having at least about 95% sequence identity to the SDl, SD2, and SD3 domains of human IFNARl.

[0502] 77. The IFN receptor agonist of any one of embodiments 68 to 70, wherein the IFNARl portion comprises an amino acid sequence having at least about 98% sequence identity to the SDl, SD2, and SD3 domains of human IFNARl.

[0503] 78. The IFN receptor agonist of any one of embodiments 68 to 70, wherein the IFNAR1 portion comprises the amino acid sequence of the SD1 domain, SD2 domain, and SD3 domain of human IFNAR1.

[0504] 79. The IFN receptor agonist of any one of embodiments 68 to 70, wherein the IFNAR1 portion comprises an amino acid sequence having at least 90% sequence identity to the SD1, SD2, SD3, and SD4 domains of human IFNAR1.

[0505] 80. The IFN receptor agonist of any one of embodiments 68 to 70, wherein the IFNARl portion comprises an amino acid sequence having at least 95% sequence identity to the SDl, SD2, SD3, and SD4 domains of human IFNARl.

[0506] 81. The IFN receptor agonist of any one of embodiments 68 to 70, wherein the IFNARl portion comprises an amino acid sequence having at least 98% sequence identity to the SDl, SD2, SD3, and SD4 domains of human IFNARl.

[0507] 82. The IFN receptor agonist of any one of embodiments 68 to 70, wherein the IFNAR1 portion comprises the amino acid sequence of the SD1, SD2, SD3, and SD4 domains of human IFNAR1.

[0508] 83. The IFN receptor agonist of any one of embodiments 68 to 82, wherein the IFNAR2 portion comprises an amino acid sequence having at least about 90% sequence identity to the D1 domain of human IFNAR2.

[0509] 84. The IFN receptor agonist of any one of embodiments 68 to 82, wherein the IFNAR2 portion comprises an amino acid sequence having at least about 95% sequence identity to the D1 domain of human IFNAR2.

[0510] 85. The IFN receptor agonist of any one of embodiments 68 to 82, wherein the IFNAR2 portion comprises an amino acid sequence having at least about 98% sequence identity to the D1 domain of human IFNAR2.

[0511] 86. The IFN receptor agonist of any one of embodiments 68 to 82, wherein said IFNAR2 portion comprises said amino acid sequence of said D1 domain of human IFNAR2.

[0512] 87. The IFN receptor agonist of any one of embodiments 68 to 82, wherein the IFNAR2 portion comprises an amino acid sequence having at least about 90% sequence identity to the D1 and D2 domains of human IFNAR2.

[0513] 88. The IFN receptor agonist of any one of embodiments 68 to 82, wherein the IFNAR2 portion comprises an amino acid sequence having at least about 95% sequence identity to the D1 and D2 domains of human IFNAR2.

[0514] 89. The IFN receptor agonist of any one of embodiments 68 to 82, wherein the IFNAR2 portion comprises an amino acid sequence having at least about 98% sequence identity to the D1 and D2 domains of human IFNAR2.

[0515] 90. The IFN receptor agonist of any one of embodiments 68 to 82, wherein said IFNAR2 portion comprises said amino acid sequence of said D1 domain and D2 domain of human IFNAR2.

[0516] 91. The IFN receptor agonist of any one of embodiments 68 to 90, wherein the IFN portion has one or more mutations selected from the group consisting of L26A, F27A, R33A, R33K, L30A, D35E, H57Y, E58N, Q61S, H57S, E58S, H57A, E58A, Q61A, Q90A, E96A, R120A, L135A, R144A, R144S, R144T, R144Y, R144I, R144L, A145D, A145H, A145K, A145M, A145V, A145Y, R149A, R149K, S152A, R162A, and E165D, optionally wherein:

[0517] (a) the IFN portion comprises the amino acid substitution R33A;

[0518] (b) the IFN portion comprises the amino acid substitution R33K;

[0519] (c) the IFN portion comprises the amino acid substitution Q90A;

[0520] (d) the IFN portion comprises the amino acid substitution E96A;

[0521] (e) the IFN portion comprises the amino acid substitution R120A;

[0522] (f) the IFN portion comprises the amino acid substitution A145M;

[0523] (g) the IFN portion comprises the amino acid substitution R149A;

[0524] (h) the IFN portion comprises the amino acid substitution R149K;

[0525] (i) the IFN portion comprises the amino acid substitution S152A;

[0526] (j) the IFN portion comprises the amino acid substitutions R33A, H57Y, E58N, and Q61S;

[0527] (k) the IFN portion comprises the amino acid substitutions H57Y, E58N, Q61S, and R144A;

[0528] (1) the IFN portion comprises the amino acid substitutions A145M and R149K; or

[0529] (m) the IFN portion comprises the amino acid substitutions Q90A and R120A.

[0530] 92. The IFN receptor agonist of any one of embodiments 68 to 91, wherein the IFN portion and the IFNAR1 portion are on the same polypeptide chain.

[0531] 93. The IFN receptor agonist of any one of embodiments 68 to 91, wherein the IFN portion and the IFNAR2 portion are located on the same polypeptide chain.

[0532] 94. The IFN receptor agonist of any one of embodiments 68 to 91, wherein the IFN portion and the IFNAR1 portion are on different polypeptide chains.

[0533] 95. The IFN receptor agonist of any one of embodiments 68 to 91, wherein the IFN portion and the IFNAR2 portion are located on different polypeptide chains.

[0534] 96. The IFN receptor agonist of any one of embodiments 68 to 91, wherein said IFN portion, said IFNAR1 portion, and said IFNAR2 portion are located on the same polypeptide chain.

[0535] 97. The IFN receptor agonist of any one of embodiments 68 to 96, wherein the first polypeptide chain comprises the IFN portion.

[0536] 98. The IFN receptor agonist of embodiment 97, wherein the IFN portion is located at the N-terminus of the first Fc domain.

[0537] 99. The IFN receptor agonist of embodiment 97, wherein the IFN portion is located at the C-terminus of the first Fc domain.

[0538] 100. The IFN receptor agonist of any one of embodiments 97 to 99, wherein said first polypeptide chain comprises said IFNAR1 portion.

[0539] 101. The IFN receptor agonist of embodiment 100, wherein the IFNAR1 portion is located at the N-terminus of the IFN portion.

[0540] 102. The IFN receptor agonist of embodiment 100, wherein the IFNAR1 portion is located at the C-terminus of the IFN portion.

[0541] 103. The IFN receptor agonist of any one of embodiments 97 to 102, wherein said first polypeptide chain comprises said IFNAR2 portion.

[0542] 104. The IFN receptor agonist of embodiment 103, wherein the IFNAR2 portion is located at the N-terminus of the IFN portion.

[0543] 105. The IFN receptor agonist of embodiment 103, wherein the IFNAR2 portion is located at the C-terminus of the IFN portion.

[0544] 106. The IFN receptor agonist of any one of embodiments 97 to 105, further comprising one or more linkers connecting two or more of the first Fc domain, the IFN portion, the IFNAR1 portion, and the IFNAR2 portion.

[0545] 107. The IFN receptor agonist of embodiment 97, wherein said first polypeptide comprises said first Fc domain, said IFNAR1 portion, said IFN portion, and said IFNAR2 portion in an N-terminal to C-terminal orientation.

[0546] 108. The IFN receptor agonist of embodiment 107, further comprising a first linker connecting the first Fc domain and the first IFNAR1 portion, a second linker connecting the IFNAR1 portion and the IFN portion, and a third linker connecting the IFN portion and the IFNAR2 portion.

[0547] 109. The IFN receptor agonist of embodiment 97, wherein said first polypeptide comprises, in an N-terminal to C-terminal orientation, said first Fc domain, a first linker, said IFNAR2 portion, a second linker, said IFN portion, a third linker, and said IFNAR1 portion.

[0548] 110. The IFN receptor agonist of embodiment 109, further comprising a first linker connecting the first Fc domain and the first IFNAR2 portion, a second linker connecting the IFNAR2 portion and the IFN portion, and a third linker connecting the IFN portion and the IFNAR1 portion.

[0549] 111. The IFN receptor agonist of embodiment 97, wherein (i) said first polypeptide comprises said first Fc domain, said IFNAR2 portion, and said IFN portion in an N-terminal to C-terminal orientation, and (ii) said second polypeptide comprises said second Fc domain and said IFNAR1 portion in an N-terminal to C-terminal orientation.

[0550] 112. The IFN receptor agonist of embodiment 111, further comprising a first linker connecting the first Fc domain and the IFNAR2 portion, a second linker connecting the IFNAR2 portion and the IFN portion, and a third linker connecting the second Fc domain and the IFNAR1 portion.

[0551] 113. The IFN receptor agonist of embodiment 97, wherein (i) said first polypeptide comprises said first Fc domain, said IFNAR1 portion, and said IFN portion in an N-terminal to C-terminal orientation, and (ii) said second polypeptide comprises said second Fc domain and said IFNAR2 portion in an N-terminal to C-terminal orientation.

[0552] 114. The IFN receptor agonist of embodiment 113, further comprising a first linker connecting the first Fc domain and the IFNAR1 portion, a second linker connecting the IFNAR1 portion and the IFN portion, and a third linker connecting the second Fc domain and the IFNAR2 portion.

[0553] 115. The IFN receptor agonist of any one of embodiments 1 to 114, wherein said first Fc domain and / or said second Fc domain comprises a hinge domain.

[0554] 116. The IFN receptor agonist of any one of embodiments 1 to 115, wherein the Fc region is a homodimer.

[0555] 117. The IFN receptor agonist of any one of embodiments 1 to 115, wherein the Fc region is a heterodimer.

[0556] 118. The IFN receptor agonist of any one of embodiments 1 to 117, comprising any pair of half antibodies depicted in Table 2.

[0557] 119. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 117, comprising Figure 2B The polypeptide chains of the two half antibodies in the configuration shown.

[0558] 120. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 117, comprising Figure 2C The polypeptide chains of the two half antibodies in the configuration shown.

[0559] 121. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 117, comprising Figure 2D The polypeptide chains of the two half antibodies in the configuration shown.

[0560] 122. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 117, comprising Figure 2E The polypeptide chains of the two half antibodies in the configuration shown.

[0561] 123. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 117, comprising Figure 2F The polypeptide chains of the two half antibodies in the configuration shown.

[0562] 124. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 117, comprising Figure 2G The polypeptide chains of the two half antibodies in the configuration shown.

[0563] 125. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 117, comprising Figure 2H The polypeptide chains of the two half antibodies in the configuration shown.

[0564] 126. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 117, comprising Figure 2I The polypeptide chains of the two half antibodies in the configuration shown.

[0565] 127. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 117, comprising Figure 2J The polypeptide chains of the two half antibodies in the configuration shown.

[0566] 128. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 117, comprising Figure 2K The polypeptide chains of the two half antibodies in the configuration shown.

[0567] 129. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 117, comprising Figure 2L The polypeptide chains of the two half antibodies in the configuration shown.

[0568] 130. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 117, comprising Figure 2M The polypeptide chains of the two half antibodies in the configuration shown.

[0569] 131. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 117, comprising Figure 2N The polypeptide chains of the two half antibodies in the configuration shown.

[0570] 132. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 117, comprising Figure 2O The polypeptide chains of the two half antibodies in the configuration shown.

[0571] 133. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 117, comprising Figure 2P The polypeptide chains of the two half antibodies in the configuration shown.

[0572] 134. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 117, comprising Figure 2Q The polypeptide chains of the two half antibodies in the configuration shown.

[0573] 135. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 117, comprising Figure 2R The polypeptide chains of the two half antibodies in the configuration shown.

[0574] 136. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 117, comprising Figure 2S The polypeptide chains of the two half antibodies in the configuration shown.

[0575] 137. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 117, comprising Figure 2T The polypeptide chains of the two half antibodies in the configuration shown.

[0576] 138. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 117, comprising Figure 2U The polypeptide chains of the two half antibodies in the configuration shown.

[0577] 139. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 117, comprising Figure 2V The polypeptide chains of the two half antibodies in the configuration shown.

[0578] 140. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 117, comprising Figure 2W The polypeptide chains of the two half antibodies in the configuration shown.

[0579] 141. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 117, comprising Figure 2X The polypeptide chains of the two half antibodies in the configuration shown.

[0580] 142. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 1 to 141, comprising any of the half-antibody pairs designated 1 to 23 listed in Table 2.

[0581] 143. The IFN receptor agonist of any one of embodiments 1 to 142, further comprising one or more targeting moieties that bind to one or more target molecules.

[0582] 144. The IFN receptor agonist of embodiment 143, comprising a first targeting moiety that binds to a first target molecule and optionally a second targeting moiety that binds to a second target molecule.

[0583] 145. The IFN receptor agonist of embodiment 144, wherein the first targeting moiety and optional second targeting moiety are antibodies or antigen-binding fragments thereof.

[0584] 146. An IFN receptor agonist according to embodiment 145, wherein the first targeting moiety and optional second targeting moiety are Fab or scFv.

[0585] 147. An IFN receptor agonist according to any one of embodiments 144 to 146, wherein the first targeting moiety and / or the optional 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) or other antigen presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0586] 148. An IFN receptor agonist according to any one of embodiments 144 to 147, wherein the first targeting moiety and / or optional second targeting moiety is capable of binding to any target molecule identified in Section 6.7.

[0587] 149. An IFN receptor agonist according to any one of embodiments 144 to 148, wherein the first targeting moiety and / or optional second targeting moiety (a) comprises (i) CDRs or (ii) VH sequences and VL sequences of an antibody listed in Table F or (b) competes with the antibody listed in Table F for binding to the target molecule.

[0588] 150. An IFN receptor agonist according to any one of embodiments 144 to 148, wherein the first targeting moiety and / or optional second targeting moiety is capable of binding to an ECM antigen, and the ECM antigen is optionally selected from syndecan, heparanase, integrin, osteopontin, connexin, cadherin, laminin, laminin-type EGF, lectin, fibronectin, notch protein, adhesion protein (e.g., adhesion protein-4), tenascin, collagen (e.g., type X collagen) and matrix protein.

[0589] 151. The IFN receptor agonist of embodiment 150, wherein the first targeting moiety and / or the optional second targeting moiety is capable of binding to an adhesion protein, eg, adhesion protein 4.

[0590] 152. The IFN receptor agonist of embodiment 150, wherein the first targeting moiety and / or the optional second targeting moiety is capable of binding to collagen, eg, collagen X.

[0591] 153. An IFN receptor agonist according to any one of embodiments 144 to 148, wherein the first targeting moiety and / or optional second targeting moiety is capable of binding to a cell surface molecule of a tumor or viral lymphocyte.

[0592] 154. The IFN receptor agonist of embodiment 153, wherein the antigen is a T cell co-stimulatory protein.

[0593] 155. The IFN receptor agonist of embodiment 154, wherein the T cell co-stimulatory 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.

[0594] 156. The IFN receptor agonist of embodiment 155, wherein the T cell co-stimulatory protein is B7-H3.

[0595] 157. The IFN receptor agonist of any one of embodiments 144 to 148, wherein the first targeting moiety and / or optional second targeting moiety is capable of binding to a checkpoint inhibitor.

[0596] 158. An IFN receptor agonist according to embodiment 157, 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.

[0597] 159. The IFN receptor agonist of embodiment 158, wherein the checkpoint inhibitor is PDL1.

[0598] 160. The IFN receptor agonist of embodiment 158, wherein the checkpoint inhibitor is PD1.

[0599] 161. The IFN receptor agonist of embodiment 158, wherein the checkpoint inhibitor is LAG3.

[0600] 162. An IFN receptor agonist according to any one of embodiments 144 to 148, wherein the first targeting moiety and / or optional second targeting moiety is capable of binding to a tumor associated antigen (TAA).

[0601] 163. The IFN receptor agonist of embodiment 162, wherein the first targeting moiety and / or the optional second targeting moiety is capable of binding to AFP, ALK, BAGE protein, BIRC5 (survivin), BIRC7, beta-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, Her 2, 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, O X40, 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.

[0602] 164. The IFN receptor agonist of embodiment 163, wherein the TAA is EGFR.

[0603] 165. The IFN receptor agonist of embodiment 163, wherein the TAA is HER2.

[0604] 166. The IFN receptor agonist of embodiment 163, wherein the TAA is EPCAM.

[0605] 167. The IFN receptor agonist of embodiment 163, wherein the TAA is CEACAM5.

[0606] 168. The IFN receptor agonist of embodiment 163, wherein the TAA is CD20.

[0607] 169. An IFN receptor agonist according to any one of embodiments 144 to 148, wherein the first targeting moiety and / or the optional second targeting moiety is capable of binding to a dendritic cell (DC) or other antigen presenting cell (APC) antigen, and the APC antigen is optionally selected from XCR1, Clec9a, CD1c, CD11c, CD14, PDL1, macrophage mannose receptor (CD206) and DEC-205.

[0608] 170. The IFN receptor agonist of embodiment 169, wherein the dendritic cell antigen is XCR1.

[0609] 171. The IFN receptor agonist according to embodiment 169, wherein the dendritic cell antigen is Clec9a.

[0610] 172. The IFN receptor agonist of embodiment 169, wherein the dendritic cell antigen is DEC-205.

[0611] 173. The IFN receptor agonist of any one of embodiments 144 to 148, wherein the first targeting moiety and / or optional second targeting moiety is capable of binding to a natural killer (NK) cell antigen.

[0612] 174. The IFN receptor agonist of any one of embodiments 1 to 142, further comprising one or more targeting moieties, each targeting moiety comprising means for binding to a target molecule.

[0613] 175. The IFN receptor agonist of embodiment 174, comprising a first targeting moiety comprising means for binding to a first target molecule and optionally a second targeting moiety comprising means for binding to a second target molecule.

[0614] 176. An IFN receptor agonist according to embodiment 175, wherein the first targeting moiety and optional second targeting moiety are antibodies or antigen-binding fragments thereof.

[0615] 177. The IFN receptor agonist of embodiment 176, wherein the first antibody or antigen-binding fragment thereof and the optional second antibody or antigen-binding fragment thereof are Fab or scFv.

[0616] 178. An IFN receptor agonist according to any one of embodiments 175 to 177, wherein the first targeting moiety and / or optional second targeting moiety comprises means for binding to extracellular matrix (ECM) antigens, tumor-reactive lymphocyte antigens, cell surface molecules of tumor or viral lymphocytes, T cell antigens (TCAs), checkpoint inhibitors, tumor-associated antigens (TAAs), dendritic cells (DCs) or other antigen presenting cell (APC) antigens or natural killer (NK) cell antigens.

[0617] 179. An IFN receptor agonist according to any one of embodiments 175 to 178, wherein the first targeting moiety and / or optional second targeting moiety comprises means for binding to any target molecule identified in Section 6.7.

[0618] 180. An IFN receptor agonist according to any one of embodiments 175 to 179, wherein the first targeting moiety and / or optional second targeting moiety comprises means for binding to an ECM antigen, wherein the ECM antigen is optionally selected from syndecan, heparanase, integrin, osteopontin, connexin, cadherin, laminin, laminin-type EGF, lectin, fibronectin, notch protein, adhesion protein (e.g., adhesion protein-4), tenascin, collagen (e.g., type X collagen) and matrix protein.

[0619] 181. The IFN receptor agonist of embodiment 180, wherein the first targeting moiety and / or the optional second targeting moiety comprises means for binding to a zonulin, eg, laminin 4.

[0620] 182. The IFN receptor agonist of embodiment 180, wherein the first targeting moiety and / or optional second targeting moiety comprises means for binding to collagen, eg, collagen X.

[0621] 183. An IFN receptor agonist according to any one of embodiments 175 to 177, wherein the first targeting moiety and / or optional second targeting moiety comprises means for binding to a cell surface molecule of a tumor or viral lymphocyte.

[0622] 184. The IFN receptor agonist of embodiment 183, wherein the cell surface molecule is a T cell co-stimulatory protein.

[0623] 185. An IFN receptor agonist according to embodiment 184, wherein the T cell co-stimulatory 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.

[0624] 186. The IFN receptor agonist of embodiment 185, wherein the T cell co-stimulatory protein is B7-H3.

[0625] 187. An IFN receptor agonist according to any one of embodiments 175 to 177, wherein the first targeting moiety and / or optional second targeting moiety comprises means for binding to a checkpoint inhibitor.

[0626] 188. An IFN receptor agonist according to embodiment 187, 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.

[0627] 189. The IFN receptor agonist of embodiment 188, wherein the checkpoint inhibitor is PDL1.

[0628] 190. The IFN receptor agonist of embodiment 188, wherein the checkpoint inhibitor is PD1.

[0629] 191. The IFN receptor agonist of embodiment 188, wherein the checkpoint inhibitor is LAG3.

[0630] 192. An IFN receptor agonist according to any one of embodiments 175 to 177, wherein the first targeting moiety and / or optional second targeting moiety comprises means for binding to a tumor associated antigen (TAA).

[0631] 193. The IFN receptor agonist of embodiment 192, wherein the TAA is AFP, ALK, BAGE protein, BIRC5 (survivin), BIRC7, beta-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-ra f, 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, p 15, 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.

[0632] 194. The IFN receptor agonist of embodiment 193, wherein the TAA is EGFR.

[0633] 195. The IFN receptor agonist of embodiment 193, wherein the TAA is HER2.

[0634] 196. The IFN receptor agonist of embodiment 193, wherein the TAA is EPCAM.

[0635] 197. The IFN receptor agonist of embodiment 193, wherein the TAA is CEACAM5.

[0636] 198. The IFN receptor agonist of embodiment 193, wherein the TAA is CD20.

[0637] 199. An IFN receptor agonist according to any one of embodiments 175 to 177, wherein the first targeting moiety and / or optional second targeting moiety comprises means for binding to a dendritic cell (DC) or other antigen presenting cell (APC) antigen, the APC antigen being optionally selected from XCR1, Clec9a, CD1c, CD11c, CD14, PDL1, macrophage mannose receptor (CD206) and DEC-205.

[0638] 200. The IFN receptor agonist of embodiment 199, wherein the dendritic cell antigen is XCR1.

[0639] 201. The IFN receptor agonist according to embodiment 199, wherein the dendritic cell antigen is Clec9a.

[0640] 202. The IFN receptor agonist according to embodiment 199, wherein the dendritic cell antigen is DEC-205.

[0641] 203. An IFN receptor agonist according to any one of embodiments 175 to 177, wherein the first targeting moiety and / or optional second targeting moiety comprises means for binding to a natural killer (NK) cell antigen.

[0642] 204. The IFN receptor agonist of any one of embodiments 143 to 203, comprising any pair of half antibodies depicted in Table 2.

[0643] 205. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 143 to 203, comprising Figure 3B The polypeptide chains of the two half antibodies in the configuration shown.

[0644] 206. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 143 to 203, comprising Figure 3C The polypeptide chains of the two half antibodies in the configuration shown.

[0645] 207. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 143 to 203, comprising Figure 3D The polypeptide chains of the two half antibodies in the configuration shown.

[0646] 208. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 143 to 203, comprising Figure 3E The polypeptide chains of the two half antibodies in the configuration shown.

[0647] 209. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 143 to 203, comprising Figure 3F The polypeptide chains of the two half antibodies in the configuration shown.

[0648] 210. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 143 to 203, comprising Figure 3G The polypeptide chains of the two half antibodies in the configuration shown.

[0649] 211. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 143 to 203, comprising Figure 3H The polypeptide chains of the two half antibodies in the configuration shown.

[0650] 212. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 143 to 203, comprising Figure 3I The polypeptide chains of the two half antibodies in the configuration shown.

[0651] 213. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 143 to 203, comprising Figure 3J The polypeptide chains of the two half antibodies in the configuration shown.

[0652] 214. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 143 to 203, comprising Figure 3K The polypeptide chains of the two half antibodies in the configuration shown.

[0653] 215. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 143 to 203, comprising Figure 3L The polypeptide chains of the two half antibodies in the configuration shown.

[0654] 216. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 143 to 203, comprising Figure 3M The polypeptide chains of the two half antibodies in the configuration shown.

[0655] 217. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 143 to 203, comprising Figure 3N The polypeptide chains of the two half antibodies in the configuration shown.

[0656] 218. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 143 to 203, comprising Figure 3O The polypeptide chains of the two half antibodies in the configuration shown.

[0657] 219. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 143 to 203, comprising Figure 3P The polypeptide chains of the two half antibodies in the configuration shown.

[0658] 220. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 143 to 203, comprising Figure 3Q The polypeptide chains of the two half antibodies in the configuration shown.

[0659] 221. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 143 to 203, comprising Figure 3R The polypeptide chains of the two half antibodies in the configuration shown.

[0660] 222. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 143 to 203, comprising Figure 3S The polypeptide chains of the two half antibodies in the configuration shown.

[0661] 223. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 143 to 203, comprising Figure 3T The polypeptide chains of the two half antibodies in the configuration shown.

[0662] 224. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 143 to 203, comprising Figure 3U The polypeptide chains of the two half antibodies in the configuration shown.

[0663] 225. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 143 to 203, comprising Figure 3V The polypeptide chains of the two half antibodies in the configuration shown.

[0664] 226. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 143 to 203, comprising Figure 3W The polypeptide chains of the two half antibodies in the configuration shown.

[0665] 227. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 143 to 203, comprising Figure 3X The polypeptide chains of the two half antibodies in the configuration shown.

[0666] 228. A nucleic acid or nucleic acids encoding the IFN receptor agonist of any one of embodiments 1 to 227.

[0667] 229. A host cell engineered to express the IFN receptor agonist of any one of embodiments 1 to 227 or the nucleic acid of embodiment 228.

[0668] 230. A method of producing the IFN receptor agonist of any one of embodiments 1 to 227, comprising culturing the host cell of embodiment 229 and recovering the IFN receptor agonist expressed thereby.

[0669] 231. A pharmaceutical composition comprising the IFN receptor agonist of any one of embodiments 1 to 227 and an excipient.

[0670] 232. A method of treating cancer comprising administering to a subject in need thereof the IFN receptor agonist of any one of embodiments 1 to 227 or the pharmaceutical composition of embodiment 231.

[0671] 233. The method of embodiment 232, wherein the IFN receptor agonist comprises at least one targeting portion capable of binding to a target molecule.

[0672] 234. The method of embodiment 232, wherein the IFN receptor agonist comprises at least one targeting moiety comprising means for binding to a target molecule.

[0673] 235. The method of embodiment 233 or 234, wherein the cancer is associated with expression of the target molecule, e.g., TAA and related cancers as listed in Table 1.

[0674] 236. The method of any one of embodiments 232 to 235, wherein the activated IFN protein comprising the IFN portion is produced by cleavage of one or more protease-cleavable linkers in the IFN receptor agonist by one or more proteases expressed by the cancer tissue.

[0675] 237. The method of embodiment 236, wherein the IFN protein is selectively activated in the cancer tissue.

[0676] 238. A method for local delivery of an IFN protein, comprising administering to a subject an IFN receptor agonist according to any one of embodiments 1 to 227 (or a pharmaceutical composition comprising the IFN receptor agonist and an excipient), the IFN receptor agonist having one or more protease-cleavable linkers, each protease-cleavable linker comprising one or more substrates for one or more proteases expressed by the tissue to which the IFN protein is to be locally delivered.

[0677] 239. The method of embodiment 238, wherein the IFN receptor agonist comprises one or more targeting moieties that recognize a target molecule expressed by the tissue.

[0678] 240. The method of embodiment 239, wherein the IFN receptor agonist comprises two targeting moieties, each targeting moiety recognizing a target molecule expressed by the tissue.

[0679] 241. The method of embodiment 238, wherein the IFN receptor agonist comprises one or more targeting moieties, each targeting moiety comprising means for binding to a target molecule expressed by the tissue.

[0680] 242. The method of embodiment 241, wherein the IFN receptor agonist comprises two targeting moieties, each targeting moiety comprising means for binding to a target molecule expressed by the tissue.

[0681] 243. The method of any one of embodiments 238 to 242, wherein the tissue is cancerous tissue.

[0682] 244. A method according to embodiment 243, wherein the target molecule expressed by the tissue is an extracellular matrix (ECM) antigen, a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or viral lymphocyte, a T cell antigen (TCA), a checkpoint inhibitor, a tumor-associated antigen (TAA), a dendritic cell (DC) or other antigen presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0683] 245. The method of any one of embodiments 238 to 244, wherein the activated IFN protein comprising said IFN portion is produced by cleavage of one or more protease-cleavable linkers in said IFN receptor agonist by one or more proteases in said tissue.

[0684] 246. 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 receptor agonist according to any one of embodiments 1 to 227 (or a pharmaceutical composition comprising the IFN receptor agonist and an excipient), wherein the IFN receptor agonist has one or more protease-cleavable linkers, each protease-cleavable linker comprising one or more substrates for one or more proteases expressed by cancer tissue, e.g., cancer tissue targeted by the IFN protein.

[0685] 247. A method according to embodiment 246, wherein the IFN receptor agonist comprises one or more targeting moieties that recognize a target molecule expressed by the cancer tissue or associated immune cells.

[0686] 248. A method according to embodiment 247, wherein the IFN receptor agonist comprises two targeting moieties, each targeting moiety recognizing a target molecule expressed by the cancer tissue or associated immune cells.

[0687] 249. A method according to embodiment 246, wherein the IFN receptor agonist comprises one or more targeting moieties, each targeting moiety comprising means for binding to a target molecule expressed by the cancer tissue or associated immune cells.

[0688] 250. The method of embodiment 249, wherein the IFN receptor agonist comprises two targeting moieties, each targeting moiety comprising means for binding to a target molecule expressed by the cancer tissue or associated immune cells.

[0689] 251. The method of any one of embodiments 246 to 250, 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) or other antigen presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0690] 252. The method of any one of embodiments 246 to 251, wherein the activated IFN protein comprising the IFN portion is produced by cleavage of one or more protease-cleavable linkers in the IFN receptor agonist by one or more proteases in the cancer tissue.

[0691] 253. A method of administering to a subject an IFN therapy with reduced systemic exposure and / or reduced systemic toxicity, comprising administering to the subject the IFN therapy in the form of an IFN receptor agonist (or a pharmaceutical composition comprising the IFN receptor agonist and an excipient) according to any one of embodiments 1 to 227, wherein the IFN receptor agonist has one or more protease-cleavable linkers, each protease-cleavable linker comprising one or more substrates for one or more proteases expressed by tissue for which IFN therapy is desired and / or anticipated.

[0692] 254. The method of embodiment 253, wherein the IFN receptor agonist comprises one or more targeting moieties that recognize a target molecule expressed by the tissue.

[0693] 255. The method of embodiment 254, wherein the IFN receptor agonist comprises two targeting moieties, each targeting moiety recognizing a target molecule expressed by the tissue.

[0694] 256. The method of embodiment 253, wherein the IFN receptor agonist comprises one or more targeting moieties comprising means for binding to a target molecule expressed by the tissue.

[0695] 257. The method of embodiment 256, wherein the IFN receptor agonist comprises two targeting moieties, the targeting moieties comprising means for binding to a target molecule expressed by the tissue.

[0696] 258. The method of any one of embodiments 253 to 257, wherein the tissue is cancerous tissue or associated immune cells.

[0697] 259. A method according to embodiment 258, wherein the target molecule expressed by the tissue is an extracellular matrix (ECM) antigen, a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or viral lymphocyte, a T cell antigen (TCA), a checkpoint inhibitor, a tumor-associated antigen (TAA), a dendritic cell (DC) or other antigen presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0698] 260. The method of any one of embodiments 253 to 259, wherein the activated IFN protein comprising said IFN portion is produced by cleavage of one or more protease-cleavable linkers in said IFN receptor agonist by one or more proteases in said tissue.

[0699] 261. 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 receptor agonist according to any one of embodiments 1 to 227 (or a pharmaceutical composition comprising the IFN receptor agonist and an excipient), wherein the IFN receptor agonist has one or more protease-cleavable linkers, each protease-cleavable linker comprising one or more substrates of one or more proteases expressed by the cancer tissue.

[0700] 262. A method according to embodiment 261, wherein the IFN receptor agonist comprises one or more targeting moieties that recognize a target molecule expressed by the cancer tissue or associated immune cells.

[0701] 263. A method according to embodiment 262, wherein the IFN receptor agonist comprises two targeting portions, each of which recognizes a target molecule expressed by the cancer tissue or associated immune cells.

[0702] 264. A method according to embodiment 261, wherein the IFN receptor agonist comprises one or more targeting moieties, each targeting moiety comprising means for binding to a target molecule expressed by the cancer tissue or associated immune cells.

[0703] 265. A method according to embodiment 264, wherein the IFN receptor agonist comprises two targeting moieties, each targeting moiety comprising means for binding to a target molecule expressed by the cancer tissue or associated immune cells.

[0704] 266. A method according to any one of embodiments 261 to 265, 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) or other antigen presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0705] 267. The method of any one of embodiments 261 to 266, wherein the activated IFN protein comprising the IFN portion is produced by cleavage of one or more protease-cleavable linkers in the IFN receptor agonist by one or more proteases in the cancer tissue.

[0706] 268. A method for targeted delivery of activated IFN protein to cancer tissue, comprising administering to a subject the IFN receptor agonist of any one of embodiments 1 to 227 (or a pharmaceutical composition comprising the IFN receptor agonist and an excipient), wherein the IFN receptor agonist:

[0707] (a) comprising (i) one or more targeting moieties that recognize a target molecule expressed by said cancer tissue or associated immune cells, or (ii) one or more targeting moieties, each of which comprises means for binding to a target molecule expressed by said cancer tissue or associated immune cells; and

[0708] (b) has one or more protease-cleavable linkers, each protease-cleavable linker comprising one or more substrates for one or more proteases expressed in the tissue for which IFN therapy is desired and / or anticipated.

[0709] 269. A method according to embodiment 268, wherein the IFN receptor agonist comprises (i) two targeting portions each recognizing a target molecule expressed by the cancer tissue or associated immune cells, or (ii) two targeting portions each comprising a means for binding to a target molecule expressed by the cancer tissue or associated immune cells.

[0710] 270. The method of embodiment 268 or embodiment 269, 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) or other antigen presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0711] 271. The method of any one of embodiments 268 to 270, wherein the activated IFN protein comprising the IFN portion is produced by cleavage of one or more protease-cleavable linkers in the IFN receptor agonist by one or more proteases in the cancer tissue.

[0712] 272. A method for inducing an immune response locally in a target tissue, comprising administering to a subject an IFN receptor agonist according to any one of embodiments 1 to 227 (or a pharmaceutical composition comprising the IFN receptor agonist and an excipient), the IFN receptor agonist having (i) one or more targeting moieties capable of binding to a target molecule expressed in the target tissue, or (ii) one or more targeting moieties comprising means for binding to a target molecule expressed in the target tissue, and one or more protease-cleavable linkers, each protease-cleavable linker comprising one or more substrates for one or more proteases expressed in the target tissue.

[0713] 273. A method according to embodiment 272, wherein the IFN receptor agonist comprises (i) two targeting portions each recognizing a target molecule expressed in the target tissue or associated immune cell, or (ii) two targeting portions each comprising a means for binding to a target molecule expressed in the target tissue or associated immune cell.

[0714] 274. The method of embodiment 272 or embodiment 273, wherein the target tissue is cancerous tissue.

[0715] 275. The method of any one of embodiments 272 to 274, 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) or other antigen presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0716] 276. The method of any one of embodiments 272 to 275, wherein the activated IFN protein comprising the IFN portion is produced by cleavage of one or more protease-cleavable linkers in the IFN receptor agonist by one or more proteases in the target tissue.

[0717] 277. The method of embodiment 276, wherein said IFN protein induces said immune response against at least one cell type in said target tissue.

[0718] 278. A method of enhancing an immune response to an antigen, comprising administering to a subject an immunogenic agent that elicits an immune response to the antigen together with an IFN receptor agonist according to any one of claims 1 to 227 (or a pharmaceutical composition comprising the IFN receptor agonist and an excipient) or a nucleic acid encoding such an IFN receptor agonist, e.g., as described in Section 6.11.1).

[0719] 279. The method of embodiment 278, wherein the administration of the immunogenic agent and the IFN receptor agonist is concurrent, separate but simultaneous, or sequential.

[0720] 280. The method of embodiment 278 or embodiment 279, wherein the immunogenic agent is a vaccine, optionally wherein the vaccine is a cancer vaccine or a vaccine against an infectious agent.

[0721] 281. The method of any one of embodiments 232 to 280, wherein said administration is non-local.

[0722] 282. The method of embodiment 281, wherein said administration is systemic.

[0723] 283. The method of embodiment 281, wherein said administration is subcutaneous.

[0724] 284. A type I interferon (IFN) receptor agonist comprising:

[0725] (a) a first polypeptide chain comprising a first Fc domain and a type I interferon (IFN) portion that is attenuated by being masked by an interferon alpha receptor 1 (IFNAR) portion and an interferon alpha receptor 2 (IFNAR2) portion; and

[0726] (b) a second polypeptide chain comprising a second Fc domain associated with the first Fc domain.

[0727] 285. The IFN receptor agonist of embodiment 284, wherein the IFN portion is located at the N-terminus of the first Fc domain.

[0728] 286. The IFN receptor agonist of embodiment 284, wherein the IFN portion is located at the C-terminus of the first Fc domain.

[0729] 287. The IFN receptor agonist of any one of embodiments 284 to 286, wherein said first polypeptide chain comprises said IFNAR1 portion.

[0730] 288. The IFN receptor agonist of embodiment 287, wherein the IFNAR1 portion is located at the N-terminus of the IFN portion.

[0731] 289. The IFN receptor agonist of embodiment 287, wherein the IFNAR1 portion is located at the C-terminus of the IFN portion.

[0732] 290. The IFN receptor agonist of any one of embodiments 284 to 289, wherein said first polypeptide chain comprises said IFNAR2 portion.

[0733] 291. The IFN receptor agonist of embodiment 290, wherein the IFNAR2 portion is located at the N-terminus of the IFN portion.

[0734] 292. The IFN receptor agonist of embodiment 290, wherein the IFNAR2 portion is located at the C-terminus of the IFN portion.

[0735] 293. The IFN receptor agonist of any one of embodiments 284 to 292, further comprising one or more linkers connecting two or more of said first Fc domain, said IFN portion, said IFNAR1 portion, and said IFNAR2 portion.

[0736] 294. The IFN receptor agonist of embodiment 284, wherein said first polypeptide comprises said first Fc domain, said IFNAR1 portion, said IFN portion, and said IFNAR2 portion in an N-terminal to C-terminal orientation.

[0737] 295. The IFN receptor agonist of embodiment 294, further comprising a first linker connecting the first Fc domain and the first IFNAR1 portion, a second linker connecting the IFNAR1 portion and the IFN portion, and a third linker connecting the IFN portion and the IFNAR2 portion.

[0738] 296. The IFN receptor agonist of embodiment 284, wherein said first polypeptide comprises, in an N-terminal to C-terminal orientation, said first Fc domain, a first linker, said IFNAR2 portion, a second linker, said IFN portion, a third linker, and said IFNAR1 portion.

[0739] 297. The IFN receptor agonist of embodiment 296, further comprising a first linker connecting said first Fc domain and said first IFNAR2 portion, a second linker connecting said IFNAR2 portion and said IFN portion, and a third linker connecting said IFN portion and said IFNAR1 portion.

[0740] 298. An IFN receptor agonist according to embodiment 284, wherein (i) said first polypeptide comprises said first Fc domain, said IFNAR2 portion and said IFN portion in an N-terminal to C-terminal orientation, and (ii) said second polypeptide comprises said second Fc domain and said IFNAR1 portion in an N-terminal to C-terminal orientation.

[0741] 299. The IFN receptor agonist of embodiment 298, further comprising a first linker connecting the first Fc domain and the IFNAR2 portion, a second linker connecting the IFNAR2 portion and the IFN portion, and a third linker connecting the second Fc domain and the IFNAR1 portion.

[0742] 300. The IFN receptor agonist of embodiment 284, wherein (i) said first polypeptide comprises said first Fc domain, said IFNAR1 portion, and said IFN portion in an N-terminal to C-terminal orientation, and (ii) said second polypeptide comprises said second Fc domain and said IFNAR2 portion in an N-terminal to C-terminal orientation.

[0743] 301. The IFN receptor agonist of embodiment 300, further comprising a first linker connecting the first Fc domain and the IFNAR1 portion, a second linker connecting the IFNAR1 portion and the IFN portion, and a third linker connecting the second Fc domain and the IFNAR2 portion.

[0744] 302. The IFN receptor agonist of any one of embodiments 295, 297, 299, or 301, wherein one or more of said first linker, said second linker, and said third linker is a protease cleavable linker (PCL).

[0745] 303. The IFN receptor agonist of embodiment 302, wherein the second linker is PCL.

[0746] 304. The IFN receptor agonist of embodiment 302 or 303, wherein the PCL comprises a substrate sequence cleavable by any protease listed in Table A.

[0747] 305. The IFN receptor agonist of any one of embodiments 302 to 304, wherein said PCL comprises one or more substrate sequences selected from said substrate sequences listed in Table B.

[0748] 306. The IFN receptor agonist of any one of embodiments 302 to 305, wherein said PCL comprises one or more spacer sequences selected from said spacer sequences listed in Table C.

[0749] 307. The IFN receptor agonist of any one of embodiments 302 to 306, wherein said PCL comprises the amino acid sequence of any one of said PCL sequences listed in Table D or a variant thereof having up to 5 amino acid substitutions.

[0750] 308. The IFN receptor agonist of any one of embodiments 302 to 307, which is configured such that cleavage of the protease cleavable linker (PCL) does not mask the IFN portion.

[0751] 309. The IFN receptor agonist of any one of embodiments 284 to 308, wherein the second polypeptide chain comprises an additional IFN moiety masked by an additional IFNAR1 moiety and an additional IFNAR2 moiety.

[0752] 310. An IFN receptor agonist according to any one of embodiments 284 to 309, wherein the IFN portion comprises an amino acid sequence having at least about 90%, at least about 95% or at least about 98% sequence identity to: (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ, or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ having at most one truncation of 15 amino acids at the N-terminus and / or C-terminus.

[0753] 311. The IFN receptor agonist of any one of embodiments 284 to 310, wherein the IFN portion comprises an amino acid sequence having one or more attenuating mutations compared to mature human IFNα1 or IFNα2b.

[0754] 312. The IFN receptor agonist of any one of embodiments 284 to 311, wherein the IFN portion has one or more mutations selected from the group consisting of L26A, F27A, R33A, R33K, L30A, D35E, H57Y, E58N, Q61S, H57S, E58S, H57A, E58A, Q61A, Q90A, E96A, R120A, L135A, R144A, R144S, R144T, R144Y, R144I, R144L, A145D, A145H, A145K, A145M, A145V, A145Y, R149A, R149K, S152A, R162A, and E165D.

[0755] 313. An IFN receptor agonist according to any one of embodiments 284 to 312, wherein the IFNAR1 portion comprises an amino acid sequence having at least 90%, at least 95% or at least 98% sequence identity to: (i) the SD2 domain and SD3 domain of human IFNAR1, (ii) the SD1 domain, SD2 domain and SD3 domain of human IFNAR1 or (iii) the SD1 domain, SD2 domain, SD3 domain and SD4 domain of human IFNAR1.

[0756] 314. The IFN receptor agonist of any one of embodiments 284 to 313, wherein the IFNAR2 portion comprises an amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity to: (i) the D1 domain of human IFNAR2 or (ii) the D1 domain and D2 domain of human IFNAR2.

[0757] 315. The IFN receptor agonist of any one of embodiments 284 to 314, wherein said first Fc domain and / or said second Fc domain comprises a hinge domain.

[0758] 316. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 284 to 315, comprising Figure 2N The polypeptide chains of the two half antibodies in the configuration shown.

[0759] 317. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 284 to 315, comprising Figure 2L The polypeptide chains of the two half antibodies in the configuration shown.

[0760] 318. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 284 to 315, comprising Figure 2M The polypeptide chains of the two half antibodies in the configuration shown.

[0761] 319. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 284 to 315, comprising Figure 2O The polypeptide chains of the two half antibodies in the configuration shown.

[0762] 320. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 284 to 315, comprising Figure 2P The polypeptide chains of the two half antibodies in the configuration shown.

[0763] 321. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 284 to 315, comprising Figure 2Q The polypeptide chains of the two half antibodies in the configuration shown.

[0764] 322. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 284 to 315, comprising Figure 2U The polypeptide chains of the two half antibodies in the configuration shown.

[0765] 323. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 284 to 315, comprising Figure 2V The polypeptide chains of the two half antibodies in the configuration shown.

[0766] 324. The IFN receptor agonist of any one of embodiments 284 to 323, comprising a first targeting moiety that binds to a first target molecule and optionally a second targeting moiety that binds to a second target molecule.

[0767] 325. An IFN receptor agonist according to embodiment 324, wherein the first targeting moiety and optional second targeting moiety are antibodies or antigen-binding fragments thereof.

[0768] 326. An IFN receptor agonist according to embodiment 324 or 325, wherein the first targeting moiety and / or the optional 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) or other antigen presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

[0769] 327. An IFN receptor agonist according to any one of embodiments 324 to 326, wherein the first targeting moiety and / or optional second targeting moiety (a) comprises (i) CDRs or (ii) VH sequences and VL sequences of an antibody listed in Table F or (b) competes with the antibody listed in Table F for binding to the target molecule.

[0770] 328. An IFN receptor agonist according to any one of embodiments 324 to 326, wherein the first targeting moiety and / or optional second targeting moiety is capable of binding to an ECM antigen, and the ECM antigen is optionally selected from syndecan, heparanase, integrin, osteopontin, connexin, cadherin, laminin, laminin-type EGF, lectin, fibronectin, notch protein, adhesion protein (e.g., adhesion protein-4), tenascin, collagen (e.g., type X collagen) and matrix protein.

[0771] 329. An IFN receptor agonist according to any one of embodiments 324 to 326, wherein the first targeting moiety and / or optional second targeting moiety is capable of binding to a cell surface molecule of a tumor or viral lymphocyte.

[0772] 330. The IFN receptor agonist of embodiment 329, wherein the cell surface molecule is a T cell co-stimulatory 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.

[0773] 331. The IFN receptor agonist of any one of embodiments 324 to 326, wherein the first targeting moiety and / or optional second targeting moiety is capable of binding to a checkpoint inhibitor.

[0774] 332. The IFN receptor agonist of embodiment 331, wherein the checkpoint inhibitor is PDL1.

[0775] 333. The IFN receptor agonist of embodiment 331, wherein the checkpoint inhibitor is PD1.

[0776] 334. The IFN receptor agonist of any one of embodiments 324 to 326, wherein the first targeting moiety and / or the optional second targeting moiety is capable of binding to a tumor associated antigen (TAA), the TAA being optionally selected from AFP, ALK, BAGE protein, BIRC5 (survivin), BIRC7, beta-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, and uroplaquelin-3.

[0777] 335. An IFN receptor agonist according to any one of embodiments 324 to 326, wherein the first targeting moiety and / or optional second targeting moiety is capable of binding to a dendritic cell (DC) or other antigen presenting cell (APC) antigen, and the APC antigen is optionally selected from XCR1, Clec9a, CD1c, CD11c, CD14, macrophage mannose receptor (CD206) and DEC-205.

[0778] 336. The IFN receptor agonist of any one of embodiments 324 to 326, wherein the first targeting moiety and / or optional second targeting moiety is capable of binding to a natural killer (NK) cell antigen.

[0779] 337. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 324 to 336, comprising Figure 3N The polypeptide chains of the two half antibodies in the configuration shown.

[0780] 338. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 324 to 336, comprising Figure 3L The polypeptide chains of the two half antibodies in the configuration shown.

[0781] 339. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 324 to 336, comprising Figure 3M The polypeptide chains of the two half antibodies in the configuration shown.

[0782] 340. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 324 to 336, comprising Figure 3O The polypeptide chains of the two half antibodies in the configuration shown.

[0783] 341. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 324 to 336, comprising Figure 3P The polypeptide chains of the two half antibodies in the configuration shown.

[0784] 342. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 324 to 336, comprising Figure 3Q The polypeptide chains of the two half antibodies in the configuration shown.

[0785] 343. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 324 to 336, comprising Figure 3U The polypeptide chains of the two half antibodies in the configuration shown.

[0786] 344. An IFN receptor agonist, optionally an IFN receptor agonist according to any one of embodiments 324 to 336, comprising Figure 3V The polypeptide chains of the two half antibodies in the configuration shown.

[0787] 345. A nucleic acid or nucleic acids encoding the IFN receptor agonist of any one of embodiments 284 to 344.

[0788] 346. A host cell engineered to express the IFN receptor agonist of any one of embodiments 284 to 344 or the nucleic acid of embodiment 345.

[0789] 347. A method of producing the IFN receptor agonist of any one of embodiments 284 to 344, comprising culturing the host cell of embodiment 346 and recovering the IFN receptor agonist expressed thereby.

[0790] 348. A pharmaceutical composition comprising the IFN receptor agonist of any one of embodiments 284 to 344 and an excipient.

[0791] 349. A method of treating cancer comprising administering to a subject in need thereof an IFN receptor agonist according to any one of embodiments 284 to 344 or a pharmaceutical composition according to embodiment 348.

[0792] 350. A method of local delivery of an IFN protein, comprising administering to a subject an IFN receptor agonist according to any one of embodiments 284 to 344 (or a pharmaceutical composition comprising the IFN receptor agonist and an excipient), the IFN receptor agonist having one or more protease-cleavable linkers, each protease-cleavable linker comprising one or more substrates for one or more proteases expressed by the tissue to which the IFN protein is to be locally delivered.

[0793] 351. 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 receptor agonist according to any one of embodiments 284 to 344 (or a pharmaceutical composition comprising the IFN receptor agonist and an excipient), wherein the IFN receptor agonist has one or more protease-cleavable linkers, each protease-cleavable linker comprising one or more substrates of one or more proteases expressed by cancer tissue.

[0794] 352. A method of administering to a subject an IFN therapy with reduced systemic exposure and / or reduced systemic toxicity, comprising administering to the subject the IFN therapy in the form of an IFN receptor agonist (or a pharmaceutical composition comprising the IFN receptor agonist and an excipient) according to any one of embodiments 284 to 344, wherein the IFN receptor agonist has one or more protease-cleavable linkers, each protease-cleavable linker comprising one or more substrates for one or more proteases expressed by a tissue for which IFN therapy is desired and / or anticipated.

[0795] 353. A method for targeted delivery of activated IFN protein to cancer tissue, comprising administering to a subject an IFN receptor agonist according to any one of embodiments 284 to 344 (or a pharmaceutical composition comprising the IFN receptor agonist and an excipient), wherein the IFN receptor agonist:

[0796] (a) comprising one or more targeting moieties that recognize target molecules expressed by the cancer tissue or associated immune cells; and

[0797] (b) has one or more protease-cleavable linkers, each protease-cleavable linker comprising one or more substrates for one or more proteases expressed in the tissue for which IFN therapy is desired and / or anticipated.

[0798] 354. A method for locally inducing an immune response in a target tissue, comprising administering to a subject an IFN receptor agonist according to any one of embodiments 284 to 344 (or a pharmaceutical composition comprising the IFN receptor agonist and an excipient), the IFN receptor agonist having one or more targeting moieties capable of binding to a target molecule expressed in the target tissue, and one or more protease-cleavable linkers, each protease-cleavable linker comprising one or more substrates for one or more proteases expressed in the target tissue.

[0799] 355. A method of enhancing an immune response to an antigen, comprising administering to a subject an immunogenic agent that elicits an immune response to the antigen together with an IFN receptor agonist according to any one of embodiments 284 to 344 (or a pharmaceutical composition comprising the IFN receptor agonist and an excipient) or a nucleic acid encoding such an IFN receptor agonist.

[0800] 356. The method of any one of embodiments 349 to 355, wherein said administration is non-local.

[0801] 8. Examples

[0802] 8.1. IFN Receptor Agonist Construct Sequence

[0803] Table 6 below provides the sequences of the IFN receptor agonists and control constructs used in the studies described herein. Targeting moieties may be included in all of these as described above.

[0804]

[0805]

[0806]

[0807]

[0808]

[0809]

[0810]

[0811]

[0812]

[0813]

[0814]

[0815]

[0816]

[0817]

[0818]

[0819]

[0820]

[0821]

[0822] Materials and methods

[0823] 8.2.1. Generation of IFN Receptor Agonist Constructs

[0824] The construct encoding IFN receptor agonist is produced in a standard mammalian protein expression DNA vector (pcDNA3.4 or similar vector), which is suitable for high-yield protein production and contains standard elements, such as promoter sequences, polyA sequences, regulatory elements, and resistance genes. Where applicable, the sequence is codon optimized. 29 amino acid signal sequences from mouse inactivated tyrosine protein kinase transmembrane receptor ROR1 (mROR1) are added to the N-terminus of the construct as a signal for secretion. All IFN receptor agonists are expressed as preproteins containing signal sequences, which are cut to produce mature proteins by intracellular processing. Constructs were expressed in Expi293F™ cells by transient transfection (Thermo Fisher Scientific). Proteins in Expi293F supernatants were purified using ProteinMaker systems (Protein BioSolutions, Gaithersburg, Maryland) and HiTrap™ Protein G HP or MabSelect SuRe pcc columns (Cytiva). After a single elution step, the protein was neutralized, dialyzed into a final buffer of phosphate buffered saline (PBS) containing 5% glycerol, aliquoted, and stored at -80° C. The samples were further analyzed by SE-UPLC to determine the presence of high or low molecular weight species relative to the species of interest.

[0825] 8.2.2. Engineering of reporter gene KG-1a cells

[0826] The promyelocytic macrophage cell line KG-1a was transduced with an interferon-stimulated response element (ISRE)-driven luciferase reporter gene construct and maintained in Iscove's modified Dulbecco's medium supplemented with 2mM L-glutamine / penicillin / streptomycin+20% FBS+1μg / mL puromycin. A single cell clone with high responsiveness to IFNα2b was isolated. PDL1 expression in this clone was knocked out using CRISPR-Cas9 technology, and the resulting cell line KG-1a / ISRE-Luc / PDL1 KO (also referred to as PDL1 KO KG-1a cells) was verified by flow cytometry. KG-1a / ISRE-Luc cells were engineered to overexpress PDL1 (amino acids M1-T290 of accession number NP_054862.1), followed by flow cytometric sorting of high PDL1-expressing cells to generate the cell line KG-1a / ISRE-Luc / hPDL1 (also referred to as PDL1 OE KG-1a cells).

[0827] 8.2.3. Luciferase assay setup

[0828] RPMI1640 medium supplemented with 2 mM L-glutamine / penicillin / streptomycin + 10% FBS was used as assay medium to prepare cell suspensions and fusion protein dilutions.

[0829] On the day of the assay, cells were centrifuged and assayed at 5 x 10 5 Resuspend in assay medium at a density of 100 μg / mL. Recombinant IFNα2b (sometimes referred to as "recombinant IFN" or simply "IFN"), IFNα1, IFNβ, or IFN fusion protein is diluted 1:5 over an 11-point dilution range (100 nM to 10.2 fM range or 500 nM to 51.2 fM range), with the 12th point containing no recombinant protein. 2.5 x 10 4 Reporter cells were added to 96-well white flat-bottom plates and incubated with serially diluted recombinant IFN or IFN fusion proteins. The plates were incubated at 37°C and 5% CO2 for 5 hours before adding 100 μL ONE-Glo TM Cells were lysed with luciferase (Promega) reagent and luciferase activity was measured. Emission was captured in relative light units (RLU) on a multi-label plate reader, Envision (PerkinElmer). All serial dilutions were tested in duplicate.

[0830] 8.2.4. Spleen cell culture

[0831] Mice expressing human IFNAR1 and IFNAR2 receptors were generated in-house. Spleens were removed and homogenized. Cell suspensions were lysed with RBC lysis buffer for 5 minutes and then washed in RPMI1640 supplemented with 10% FBS. Cells were plated at 2.5 x 10 5 Cells were plated at a density of 10 cells / well in 96-well U-bottom plates.

[0832] 8.2.5. PBMC Culture

[0833] Human PBMCs were thawed and recovered overnight in RPMI1640 supplemented with 10% FBS. On the day of stimulation, cells were collected and plated at 7.5 x 10 4 Cells were plated at a density of 100 μg / mL in 96-well U-bottom plates.

[0834] 8.2.6. Detection of pSTAT1

[0835] On the day of stimulation, the molecules were diluted to an 11-point dilution range (100 nM to 10.2 fM) and added to the plated splenocytes and PBMCs. The plates were incubated at 37°C and 5% CO2 for 20 minutes. After stimulation, the cells were washed and fixed and washed with BD Cytofix buffer (Catalog 554655), and the fixed cells were incubated at 37°C and 5% CO2 for 12 minutes. The cells were spun and permeabilized on ice with BD Perm Buffer III (Catalog 558050) for 10 minutes. The cells were washed twice and stained with cell surface and intracellular antibodies (BD: CD4, B220, CD11b, CD44, CD3, CD8a, NK1.1, pSTAT1) prepared in BD Horizon Brilliant buffer (Catalog 566349) containing 2% mouse serum for 60 minutes at room temperature. The cells were washed twice and collected on a BD Fortessa flow cytometer.

[0836] 8.3. Example 1: SE-UPLC profile of Fc-linked interferon molecules

[0837] SE-UPLC was performed to evaluate the IFN molecules that could be incorporated into the IFN agonists of the present disclosure. Figure 5A )、Fc-IFNα2b( Figure 5B )、IFNα2b-Fc( Figure 5C ) and Fc-IFN x Fc( Figure 5D The four exemplary constructs analyzed showed varying levels of discrete main peaks of high molecular weight species. The main peak area percentage for Fc-IFNα1 was calculated to be 37.43, while for Fc-IFNα2b and IFNα2b-Fc, these percentages were larger, calculated to be 57.66 and 56.4, respectively. The maximum main peak area percentage value observed for Fc-IFNxFc was 85.

[0838] 8.4. Example 2: Activity of interferon molecules

[0839] An interferon-stimulated response element (ISRE)-driven luciferase reporter gene was incorporated into the promyelocytic macrophage cell line KG-1a as described in Section 8.2.2 and used to evaluate the ability of IFN molecules to drive ISRE-dependent transcriptional responses in KG-1a cells as described in Section 8.2.3.

[0840] The results shown in Figure 6 show that the Fc fusions of recombinant proteins and IFN variants exhibited varying degrees of attenuation in the in vitro luciferase assay for interferon-stimulated response elements (ISREs). Figure 6AAll three IFN molecular structures shown in the Figure 2 exhibit weak interferon signaling ( Figure 6B However, the levels of attenuation were more similar for Fc-IFNα2b and IFNα2b-Fc, both being associated with slightly higher attenuation than Fc-IFNα2b x Fc. Next, the in vitro activities of the two Fc-IFN constructs, Fc-IFNα2b and Fc-IFNα1, were compared with the activities of the three IFN variants, IFNα2b, IFNα1, and IFNβ ( Figure 6C Among IFN variants, the highest activity levels were observed with IFNβ and IFNα2b, while IFNα1 activity was relatively weak. Recombinant proteins with Fc fusions exhibited attenuated activity compared to IFN variants. Overall, Fc fusions lead to attenuated interferon signaling relative to free interferon.

[0841] 8.5. Example 3: SE-UPLC profiles of mutant IFN constructs

[0842] SE-UPLC was performed to evaluate mutant IFN molecules linked to the Fc domain at the C-terminus. Figure 7A )、Fc-IFNα2bR149A( Figure 7B )、Fc-IFNα2bR120A( Figure 7C ) and Fc-IFNα2bS152A ( Figure 7D ) showed discrete main peaks of high molecular weight species at varying levels.

[0843] 8.6. Example 4: Activity of mutant IFN constructs

[0844] An ISRE-driven luciferase reporter gene was incorporated into the promyelocytic macrophage cell line KG-1a as described in Section 8.2.2 and used to evaluate the ability of mutant IFN constructs to drive an ISRE-dependent transcriptional response in KG-1a cells as described in Section 8.2.3.

[0845] The activity of IFN variants is related to their affinity for IFNAR. Therefore, mutations that affect IFN-IFNAR binding can affect the activity of Fc-IFN constructs. A series of mutations were introduced at the IFNAR1 or IFNAR2 interface of IFNα2b ( Figure 8A and 8BMost mutations that interfered with IFNAR1 or IFNAR2 binding of Fc-IFNα2b attenuated ISRE-luciferase activity relative to wild-type Fc-IFNα2b. Moreover, the extent of this attenuation varied; while some mutations caused only a slight attenuation of activity, others resulted in very high levels of attenuation ( Figure 8B ).

[0846] 8.7. Example 5: SEC Profiles of Exemplary Interferon Receptor Agonist Constructs

[0847] As described in Section 8.2.1, SE-UPLC was performed to assess the presence of high or low molecular weight species in samples of exemplary IFN receptor agonist constructs. FIG9 depicts the profiles of the six exemplary IFN receptor agonist constructs described in FIG4: Fc-IFNAR1(SD1-3)-IFNα2b( Figure 9A ), Fc-R1(SD1-3)-IFNα2b x Fc( Figure 9B )、Fc-IFNα2b-IFNAR2(D1)( Figure 9C )、Fc-IFNAR2(D1)-IFNα2b( Figure 9D )、Fc-IFNα2b x Fc-R2(D1-2)( Figure 9E ) and Fc-IFNα2b-R2(D1-2)x Fc( Figure 9F In general, all six receptor agonist constructs showed a discrete main peak with smaller peaks at varying levels corresponding to high molecular weight species.

[0848] 8.8. Example 6: Activity of Exemplary IFN Receptor Agonist Constructs

[0849] An ISRE-driven luciferase reporter gene was incorporated into the promyelocytic macrophage cell line KG-1a as described in Section 8.2.2 and used as described in Section 8.2.3 to evaluate the ability of IFN receptor agonist constructs to drive ISRE-dependent transcriptional responses in KG-1a cells.

[0850] Figure 10 The results shown indicate that IFNα2b is significantly different from recombinant IFNα2b. Figure 10 IFN receptor agonists exhibit varying degrees of attenuation of ISRE luciferase activity. Receptor masking attenuates wild-type interferon activity to varying degrees, depending on the receptor masking agent used.

[0851] 8.9. Example 7: Effect of Masking on Interferon Receptor Agonist Activity

[0852] An interferon-stimulated response element (ISRE)-driven luciferase reporter gene was incorporated into the promyelocytic macrophage cell line KG-1a as described in Section 8.2.2 and used to evaluate the effect of masking on the ability of IFN fusion proteins to drive ISRE-dependent transcriptional responses in KG-1a cells as described in Section 8.2.3.

[0853] Figure 11A The results shown indicate that masked Fc-IFN constructs attenuate reporter gene activity, but with varying potency. Figure 11A and 11B Compared to the reporter gene responses obtained with the "recombinant IFN" construct ("recombinant IFN"), both N-terminal and C-terminal Fc fusions of IFN attenuated reporter gene activity. Similar potency was achieved regardless of the N-terminal or C-terminal position of the Fc fusion. The Fc-IFNAR1(SD1-3)-IFN and Fc-IFNAR1(SD2-3)-IFN masking constructs showed relatively higher attenuation of reporter gene responses than Fc-IFN and IFN-Fc, which is consistent with the results of the present study. Figure 10 Similar results were observed in the . Fc-IFNAR2(D1)-IFN further attenuated the reporter gene response than Fc-IFNAR1(SD2-3)-IFN; however, Fc-IFN-IFNAR2(D1) was the most effective construct in attenuating the reporter gene response, with a potency approximately 4.7-fold lower than that of Fc-IFNAR2(D1)-IFN.

[0854] Next, the ability of the heterodimeric IFN fusion protein constructs to drive an ISRE-dependent transcriptional response in KG-1a cells was evaluated. Figure 11B The results shown indicate that the KiH monomeric IFN fusion construct differs from the dimeric Fc-IFN in attenuating the reporter gene response. Figure 11A As can be seen, IFN-Fc and Fc-IFN attenuated the reporter gene response with nearly identical potency. Fc-IFN x Fc was associated with less attenuation than Fc-IFN, as the former was nearly 2-fold more potent than the latter construct in inducing the reporter gene response. Fc-IFN-R2(D1-2) x Fc and Fc-IFN x Fc-R2(D1-2) showed similar potency, suggesting that placing the IFNAR2 masking agent on the same pair of different Fc chains had no detectable effect. In contrast, Fc-R1(SD1-3)-IFN x Fc was more effective than Fc-IFN x Fc-R1(SD1-3) in attenuating the reporter gene response, suggesting that placing the IFNAR1(SD1-3) masking agent on the same Fc chain is associated with greater attenuation.

[0855] 8.10. Example 8: Activity of IFN Receptor Agonists in Mouse Cells Expressing hIFNAR

[0856] To assess the activity of exemplary IFN receptor agonists, mouse splenocytes were isolated as described in Section 8.2.4. As described in Section 8.2.6, different cell types were evaluated for the presence of pSTAT1, which was reported as the percentage of pSTAT1-positive cells.

[0857] hIFNAR CD8 + In T cells, both Fc-IFN and IFN-Fc were associated with attenuation of the % pSTAT1 response relative to that obtained with increasing concentrations of non-fused IFN (IFNα2b). These attenuations showed similar potency regardless of the N-terminal or C-terminal position of the Fc fusion ( Figure 12A Fc-IFNAR2(D1)-IFN and Fc-IFN-IFNAR2(D1) further attenuated the response compared to Fc-IFN and IFN-Fc; however, Fc-IFNAR1(SD1-3)-IFN was the most effective construct in attenuating the response ( Figure 12A Evaluation of KiH fusion constructs revealed that Fc-IFN-R2(D1-2) x Fc and Fc-IFN x Fc-R2(D1-2) exhibited similar potency, indicating that placing the IFNAR2 masking agent on the same pair of different Fc chains had no detectable effect ( Figure 12B In contrast, Fc-R1(SD1-3)-IFN x Fc was more effective in attenuating the pSTAT1 response than Fc-IFN x Fc-R1(SD1-3), suggesting that placing the IFNAR1(SD1-3) masking agent on the same Fc chain is associated with better attenuation ( Figure 12B ).

[0858] hIFNAR CD11b + In cells, responses obtained with both Fc-IFN and IFN-Fc were associated with attenuation of the % pSTAT1 response relative to that obtained with increasing concentrations of non-fusion IFN (IFNα2b), with the attenuation associated with IFN-Fc being more pronounced ( Figure 13A Fc-IFNAR2(D1)-IFN, Fc-IFN-IFNAR2(D1), and Fc-IFNAR1(SD1-3)-IFN further attenuated the response ( Figure 13AEvaluation of heterodimeric KiH fusion constructs revealed that Fc-IFN-R2(D1-2)xFc was associated with slightly less pronounced attenuation than Fc-IFNxFc-R2(D1-2), suggesting that placing the IFNAR2 masking agent on a different Fc chain may be associated with better attenuation ( Figure 13B In contrast, Fc-R1(SD1-3)-IFN x Fc was more effective in attenuating the pSTAT1 response than Fc-IFN x Fc-R1(SD1-3), suggesting that placing the IFNAR1(SD1-3) masking agent on the same Fc chain is associated with better attenuation ( Figure 13B ).

[0859] hIFNAR CD4 + In T cells, both Fc-IFN and IFN-Fc were associated with attenuation of the % pSTAT1 response relative to that obtained with increasing concentrations of non-fused IFN (IFNα2b). These attenuations showed similar potency regardless of the N-terminal or C-terminal position of the Fc fusion ( Figure 14A Fc-IFNAR2(D1)-IFN and Fc-IFN-IFNAR2(D1) further attenuated the response compared to Fc-IFN and IFN-Fc; however, Fc-IFNAR1(SD1-3)-IFN was the most effective construct in attenuating the response ( Figure 14A Evaluation of heterodimeric KiH fusion constructs revealed that Fc-IFN-R2(D1-2)xFc was associated with slightly less pronounced attenuation than Fc-IFNxFc-R2(D1-2), suggesting that placing the IFNAR2 masking agent on a different Fc chain may be associated with better attenuation ( Figure 14B In contrast, Fc-R1(SD1-3)-IFN x Fc was more effective in attenuating the pSTAT1 response than Fc-IFN x Fc-R1(SD1-3), suggesting that placing the IFNAR1(SD1-3) masking agent on the same Fc chain is associated with better attenuation ( Figure 14B ).

[0860] Finally, in hIFNAR NK cells, both Fc-IFN and IFN-Fc were associated with attenuation of the % pSTAT1 response relative to that obtained with increasing concentrations of non-fused IFN (IFNα2b). These attenuations showed similar potency regardless of the N-terminal or C-terminal position of the Fc fusion ( Figure 15AFc-IFNAR2(D1)-IFN and Fc-IFN-IFNAR2(D1) further attenuated the response compared to Fc-IFN and IFN-Fc; however, Fc-IFNAR1(SD1-3)-IFN was the most effective construct in attenuating the response ( Figure 15A Evaluation of heterodimeric KiH fusion constructs revealed that Fc-IFN-R2(D1-2) x Fc was associated with less pronounced attenuation than Fc-IFN x Fc-R2(D1-2), suggesting that placing the IFNAR2 masking agent on different Fc chains is associated with better attenuation ( Figure 14B In contrast, Fc-R1(SD1-3)-IFN x Fc was more effective in attenuating the pSTAT1 response than Fc-IFN x Fc-R1(SD1-3), suggesting that placing the IFNAR1(SD1-3) masking agent on the same Fc chain is associated with better attenuation ( Figure 15B ).

[0861] 8.11. Example 9: Activity of IFN Receptor Agonists in Human Peripheral Blood Mononuclear Cells

[0862] To assess the activity of exemplary IFN receptor agonists, human PBMCs were isolated as described in Section 8.2.5. As described in Section 8.2.6, different cell types were evaluated for the presence of pSTAT1, which was reported as the percentage of pSTAT1-positive cells.

[0863] Both Fc-IFN and Fc-IFN x Fc were associated with similar levels of attenuation of the % pSTAT1 response relative to that of PBMC CD8 + T cells ( Figure 16A ) and PBMC NK cells ( Figure 16B ) in terms of the % pSTAT1 response obtained with increasing concentrations of non-fused IFN. Responses associated with Fc-IFNAR1(SD1-3)-IFN and Fc-R1(SD1-3)-IFN x Fc were even more severely attenuated in both cell types ( Figure 16A and 16B ).

[0864] 8.12. Example 10: Activity of Exemplary Monovalent and Bivalent Masked IFN Receptor Agonists

[0865] As described in Section 8.2.2, an ISRE-driven luciferase reporter gene was incorporated into the promyelocytic macrophage cell line KG-1a and used to evaluate the ability of IFN receptor agonists to drive ISRE-dependent transcriptional responses in KG-1a cells as described in Section 8.2.3. The single-masked or dual-masked monovalent and bivalent IFN receptor agonists used in this evaluation are listed in Table 7 below.

[0866]

[0867] Figure 17 The results shown indicate that monovalent IFN receptor agonists exhibit varying degrees of attenuation of ISRE activation relative to IFNα2b, depending on the type and orientation of the receptor masking agent. The dual masking construct Fc-R1(SD1-3)-IFNα2b-R2(D1-2)xFc exhibited the greatest signal attenuation.

[0868] Similarly, bivalent IFN receptor agonists also showed varying degrees of attenuation of ISRE activation ( Figure 18 In this evaluation, attenuation of ISRE activation was assessed using a dual-masked bivalent and three single-masked bivalent IFN receptor agonists (Table 7), with the dual-masked constructs showing increased attenuation relative to the single-masked constructs ( Figure 18 ).

[0869] 8.13. Example 11: Effect of PDL1 Targeting on the Efficacy of IFN Receptor Agonists

[0870] As described in Section 8.2.2, an ISRE-driven luciferase reporter gene assay was incorporated into the promyelocytic macrophage cell line KG-1a and used to evaluate the ability of PDL1-targeted IFN receptor agonists to drive ISRE-dependent transcriptional responses in KG-1a cells as described in Section 8.2.3. The isotypes ("Iso") or PDL1-targeted monovalent and bivalent IFN receptor agonists used in this evaluation are listed in Table 8 below.

[0871]

[0872] In PDL1 OE KG-1a cells, PDL1-targeted monovalent IFN receptor agonist constructs exhibited enhanced potency relative to their non-targeted (isotype) counterparts ( Figure 19A The difference in potency between PDL1-targeted and non-targeted constructs was absent in PDL1 KO KG-1a cells ( Figure 19B Similar results were observed with the PDL1-targeted bivalent IFN receptor agonist construct relative to its isotype counterpart ( Figure 19C and 19D ).

[0873] 8.14. Example 12: Effect of Linker Length on the Potency of IFN Receptor Agonists

[0874] An ISRE-driven luciferase reporter assay was incorporated into the promyelocytic macrophage cell line KG-1a as described in Section 8.2.2 and used as described in Section 8.2.3 to evaluate the effect of linker length on the ability of PDL1-targeted IFN receptor agonists to drive an ISRE-dependent transcriptional response in KG-1a cells.

[0875] Figures 20A to 20D The structures of the IFN receptor agonist constructs evaluated in this assessment are shown, wherein the linker between the masking moiety of IFNα2b and α2b in the dual-masked monovalent PDL1-targeted or isotype constructs varied from 5 to 20 amino acids. PDL1-targeted constructs with various linker lengths resulted in similar levels of ISRE-luciferase activity in PDL1 OE KG-1a cells ( Figure 20E ), indicating that the potency of the IFN receptor-masked constructs was not affected by increasing linker length.

[0876] 9. Citations

[0877] All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document were individually indicated to be incorporated by reference herein for all purposes. In the event of any inconsistency between the teachings of one or more of the references incorporated herein and the present disclosure, the teachings of the present specification are intended to control.

Claims

1. An interferon type I (IFN) receptor agonist, comprising: (a) A first polypeptide chain comprising a first Fc domain and an interferon type I (IFN) portion attenuated by masking with an interferon alpha receptor 1 (IFNAR1) portion and an interferon alpha receptor 2 (IFNAR2) portion; and (b) A second polypeptide chain comprising a second Fc domain associated with the first Fc domain.

2. The IFN receptor agonist according to claim 1, wherein the IFN portion is located at the N-terminus of the first Fc domain.

3. The IFN receptor agonist according to claim 1, wherein the IFN portion is located at the C-terminus of the first Fc domain.

4. The IFN receptor agonist according to any one of claims 1 to 3, wherein the first polypeptide chain comprises the IFNAR1 portion.

5. The IFN receptor agonist according to claim 4, wherein the IFNAR1 portion is located at the N-terminus of the IFN portion.

6. The IFN receptor agonist according to claim 4, wherein the IFNAR1 portion is located at the C-terminus of the IFN portion.

7. The IFN receptor agonist according to any one of claims 1 to 6, wherein the first polypeptide chain comprises the IFNAR2 portion.

8. The IFN receptor agonist according to claim 7, wherein the IFNAR2 portion is located at the N-terminus of the IFN portion.

9. The IFN receptor agonist according to claim 7, wherein the IFNAR2 portion is located at the C-terminus of the IFN portion.

10. The IFN receptor agonist according to any one of claims 1 to 6, further comprising one or more linkers connecting two or more of the first Fc domain, the IFN portion, the IFNAR1 portion, and the IFNAR2 portion.

11. The IFN receptor agonist according to claim 1, wherein the first polypeptide comprises the first Fc domain, the IFNAR1 portion, the IFN portion, and the IFNAR2 portion in an N-terminus to C-terminus orientation.

12. The IFN receptor agonist according to claim 11, further comprising a first linker connecting the first Fc domain and the first IFNAR1 portion, a second linker connecting the IFNAR1 portion and the IFN portion, and a third linker connecting the IFN portion and the IFNAR2 portion.

13. The IFN receptor agonist according to claim 1, wherein the first polypeptide comprises the first Fc domain, a first linker, the IFNAR2 portion, a second linker, the IFN portion, a third linker, and the IFNAR1 portion in an N-terminus to C-terminus orientation.

14. The IFN receptor agonist according to claim 13, further comprising a first linker connecting the first Fc domain and the first IFNAR2 moiety, a second linker connecting the IFNAR2 moiety and the IFN moiety, and a third linker connecting the IFN moiety and the IFNAR1 moiety.

15. The IFN receptor agonist according to claim 1, wherein (i) the first polypeptide comprises the first Fc domain, the IFNAR2 moiety, and the IFN moiety in an N-terminal to C-terminal orientation, and (ii) the second polypeptide comprises the second Fc domain and the IFNAR1 moiety in an N-terminal to C-terminal orientation.

16. The IFN receptor agonist according to claim 15, further comprising a first linker connecting the first Fc domain and the IFNAR2 moiety, a second linker connecting the IFNAR2 moiety and the IFN moiety, and a third linker connecting the second Fc domain and the IFNAR1 moiety.

17. The IFN receptor agonist according to claim 1, wherein (i) the first polypeptide comprises the first Fc domain, the IFNAR1 moiety, and the IFN moiety in an N-terminal to C-terminal orientation, and (ii) the second polypeptide comprises the second Fc domain and the IFNAR2 moiety in an N-terminal to C-terminal orientation.

18. The IFN receptor agonist according to claim 17, further comprising a first linker connecting the first Fc domain and the IFNAR1 moiety, a second linker connecting the IFNAR1 moiety and the IFN moiety, and a third linker connecting the second Fc domain and the IFNAR2 moiety.

19. The IFN receptor agonist according to any one of claims 12, 14, 16, or 18, wherein one or more of the first linker, the second linker, and the third linker is a protease-cleavable linker (PCL).

20. The IFN receptor agonist according to claim 19, wherein the second linker is a PCL.

21. The IFN receptor agonist according to any one of claims 19 or 20, wherein the PCL comprises a substrate sequence cleavable by any protease listed in Table A.

22. The IFN receptor agonist according to any one of claims 19 to 21, wherein the PCL comprises one or more substrate sequences selected from the substrate sequences listed in Table B.

23. The IFN receptor agonist according to any one of claims 19 to 22, wherein the PCL comprises one or more spacer sequences selected from the spacer sequences listed in Table C.

24. The IFN receptor agonist according to any one of claims 19 to 23, wherein the PCL comprises the amino acid sequence of any one of the PCL sequences listed in Table D or a variant thereof having at most 5 amino acid substitutions.

25. The IFN receptor agonist according to any one of claims 19 to 24, which is constructed such that cleavage of the protease-cleavable linker (PCL) does not unmask the IFN moiety.

26. The IFN receptor agonist according to any one of claims 1 to 25, wherein the second polypeptide chain comprises an additional IFN moiety masked by an additional IFNAR1 moiety and an additional IFNAR2 moiety.

27. The IFN receptor agonist according to any one of claims 1 to 26, wherein the IFN moiety comprises an amino acid sequence having at least about 90%, at least about 95% or at least about 98% sequence identity to: (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ, or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ, which has at most one 15-amino acid truncation at the N-terminus and / or C-terminus.

28. The IFN receptor agonist according to any one of claims 1 to 27, wherein the IFN moiety comprises an amino acid sequence having one or more attenuation mutations compared to mature human IFNα1 or IFNα2b.

29. The IFN receptor agonist according to any one of claims 1 to 28, wherein the IFN moiety has 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.

30. The IFN receptor agonist according to any one of claims 1 to 29, wherein the IFNAR1 moiety comprises an amino acid sequence having at least 90%, at least 95% or at least 98% sequence identity to: (i) the SD2 domain and SD3 domain of human IFNAR1, (ii) the SD1 domain, SD2 domain and SD3 domain of human IFNAR1 or (iii) the SD1 domain, SD2 domain, SD3 domain and SD4 domain of human IFNAR1.

31. The IFN receptor agonist according to any one of claims 1 to 30, wherein the IFNAR2 moiety comprises an amino acid sequence having at least 90%, at least 95% or at least 98% sequence identity to: (i) the D1 domain of human IFNAR2 or (ii) the D1 domain and D2 domain of human IFNAR2.

32. The IFN receptor agonist according to any one of claims 1 to 31, wherein the first Fc domain and / or the second Fc domain comprises a hinge domain.

33. An IFN receptor agonist, optionally the IFN receptor agonist according to any one of claims 1 to 32, comprising a polypeptide chain having the configuration of two half-antibodies shown in Figure 2N.

34. An IFN receptor agonist, optionally the IFN receptor agonist according to any one of claims 1 to 32, comprising a polypeptide chain having the configuration of two half-antibodies shown in Figure 2L.

35. An IFN receptor agonist, optionally the IFN receptor agonist according to any one of claims 1 to 32, comprising a polypeptide chain having the configuration of two half-antibodies shown in Figure 2M.

36. An IFN receptor agonist, optionally the IFN receptor agonist according to any one of claims 1 to 32, comprising a polypeptide chain having the configuration of two half-antibodies shown in Figure 2O.

37. An IFN receptor agonist, optionally the IFN receptor agonist according to any one of claims 1 to 32, comprising a polypeptide chain having the configuration of two half-antibodies shown in Figure 2P.

38. An IFN receptor agonist, optionally the IFN receptor agonist according to any one of claims 1 to 32, comprising a polypeptide chain having the configuration of two half-antibodies shown in Figure 2Q.

39. An IFN receptor agonist, optionally the IFN receptor agonist according to any one of claims 1 to 32, comprising a polypeptide chain having the configuration of two half-antibodies shown in Figure 2U.

40. An IFN receptor agonist, optionally the IFN receptor agonist according to any one of claims 1 to 32, comprising a polypeptide chain having the configuration of two half-antibodies shown in Figure 2V.

41. The IFN receptor agonist according to any one of claims 1 to 40, comprising a first targeting moiety that binds to a first target molecule and optionally a second targeting moiety that binds to a second target molecule.

42. The IFN receptor agonist according to claim 41, wherein the first targeting moiety and optionally the second targeting moiety are an antibody or an antigen-binding fragment thereof.

43. The IFN receptor agonist according to claim 41 or 42, wherein the first targeting moiety and / or optionally 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 virus lymphocyte, a T cell antigen (TCA), a checkpoint inhibitor, a tumor-associated antigen (TAA), a dendritic cell (DC) or other antigen-presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

44. The IFN receptor agonist according to any one of claims 41 to 43, wherein the first targeting moiety and / or optionally the second targeting moiety (a) comprises (i) a CDR or (ii) a VH sequence and a VL sequence of an antibody listed in Table F or (b) competes with the antibody listed in Table F for binding to the target molecule.

45. The IFN receptor agonist according to any one of claims 41 to 44, wherein the first targeting moiety and / or optionally the second targeting moiety is capable of binding to an ECM antigen, which is optionally selected from syndecan, heparanase, integrin, osteopontin, nectin, cadherin, laminin, laminin-type EGF, lectin, fibronectin, notch protein, tenascin (e.g., tenascin-4), tenascin, collagen (e.g., type X collagen), and matrix protein.

46. The IFN receptor agonist according to any one of claims 41 to 44, wherein the first targeting moiety and / or optionally the second targeting moiety is capable of binding to a cell surface molecule of a tumor or a virus lymphocyte.

47. The IFN receptor agonist according to claim 46, wherein the cell surface molecule is a T cell co-stimulatory protein, which is optionally selected from CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

48. The IFN receptor agonist according to any one of claims 41 to 44, wherein the first targeting moiety and / or optionally the second targeting moiety is capable of binding to a checkpoint inhibitor.

49. The IFN receptor agonist according to claim 48, wherein the checkpoint inhibitor is PDL1.

50. The IFN receptor agonist according to claim 48, wherein the checkpoint inhibitor is PD1.

51. The IFN receptor agonist according to any one of claims 41 to 44, wherein the first targeting moiety and / or optionally the second targeting moiety is capable of binding to a tumor-associated antigen (TAA), which TAA is optionally selected from AFP, ALK, BAGE protein, BIRC5 (survivin), BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CEACAM5 (also known as carcinoembryonic antigen or CEA), CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE protein (e.g., GAGE-1 or -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, LMP2, MAGE protein (e.g., MAGE-1, -2, -3, -4, -6 and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase, and uroplakin-3.

52. The IFN receptor agonist according to any one of claims 41 to 44, wherein the first targeting moiety and / or optionally the second targeting moiety is capable of binding to a dendritic cell (DC) or other antigen-presenting cell (APC) antigen, which APC antigen is optionally selected from XCR1, Clec9a, CD1c, CD11c, CD14, macrophage mannose receptor (CD206), and DEC-205.

53. The IFN receptor agonist according to any one of claims 41 to 44, wherein the first targeting moiety and / or optionally the second targeting moiety is capable of binding to a natural killer (NK) cell antigen.

54. An IFN receptor agonist, which is optionally an IFN receptor agonist according to any one of claims 41 to 53, and which comprises a polypeptide chain having the configuration of two half-antibodies shown in Figure 3N.

55. An IFN receptor agonist, which is optionally an IFN receptor agonist according to any one of claims 41 to 53, and which comprises a polypeptide chain having the configuration of two half-antibodies shown in Figure 3L.

56. An IFN receptor agonist, which is optionally an IFN receptor agonist according to any one of claims 41 to 53, and which comprises a polypeptide chain having the configuration of two half-antibodies shown in Figure 3M.

57. An IFN receptor agonist, which is optionally an IFN receptor agonist according to any one of claims 41 to 53, and which comprises a polypeptide chain having the configuration of two half-antibodies shown in Figure 3O.

58. An IFN receptor agonist, which is optionally an IFN receptor agonist according to any one of claims 41 to 53, and which comprises a polypeptide chain having the configuration of two half-antibodies shown in Figure 3P.

59. An IFN receptor agonist, which is optionally an IFN receptor agonist according to any one of claims 41 to 53, and which comprises a polypeptide chain having the configuration of two half-antibodies shown in Figure 3Q.

60. An IFN receptor agonist, which is optionally an IFN receptor agonist according to any one of claims 41 to 53, and which comprises a polypeptide chain having the configuration of two half-antibodies shown in Figure 3U.

61. An IFN receptor agonist, which is optionally an IFN receptor agonist according to any one of claims 41 to 53, and which comprises a polypeptide chain having the configuration of two half-antibodies shown in Figure 3V.

62. A nucleic acid or nucleic acids, which encode an IFN receptor agonist according to any one of claims 1 to 61.

63. A host cell, which is engineered to express an IFN receptor agonist according to any one of claims 1 to 61 or a nucleic acid according to claim 62.

64. A method for producing an IFN receptor agonist according to any one of claims 1 to 61, which comprises culturing a host cell according to claim 63 and recovering the IFN receptor agonist expressed thereby.

65. A pharmaceutical composition, which comprises an IFN receptor agonist according to any one of claims 1 to 61 and an excipient.

66. A method for treating cancer, which comprises administering to a subject in need thereof an IFN receptor agonist according to any one of claims 1 to 61 or a pharmaceutical composition according to claim 65.

67. A method for local delivery of an IFN protein, comprising administering to a subject an IFN receptor agonist (or a pharmaceutical composition comprising the IFN receptor agonist and an excipient) according to any one of claims 1 to 61, wherein the IFN receptor agonist has one or more protease-cleavable linkers, and each protease-cleavable linker comprises one or more substrates for one or more proteases expressed by the tissue to which the IFN protein is to be locally delivered.

68. A method for treating cancer with an IFN protein selectively activated in cancer tissue, comprising administering to a subject in need thereof an IFN receptor agonist (or a pharmaceutical composition comprising the IFN receptor agonist and an excipient) according to any one of claims 1 to 61, wherein the IFN receptor agonist has one or more protease-cleavable linkers, and each protease-cleavable linker comprises one or more substrates for one or more proteases expressed by the cancer tissue.

69. A method for administering an 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 receptor agonist (or a pharmaceutical composition comprising the IFN receptor agonist and an excipient) according to any one of claims 1 to 61, wherein the IFN receptor agonist has one or more protease-cleavable linkers, and each protease-cleavable linker comprises one or more substrates for one or more proteases expressed by the tissue in which IFN therapy is desired and / or anticipated.

70. A method for targeted delivery of an activated IFN protein to cancer tissue, comprising administering to a subject an IFN receptor agonist (or a pharmaceutical composition comprising the IFN receptor agonist and an excipient) according to any one of claims 1 to 61, wherein the IFN receptor agonist: (a) comprises one or more targeting moieties that recognize a target molecule expressed by the cancer tissue or associated immune cells; and (b) has one or more protease-cleavable linkers, and each protease-cleavable linker comprises one or more substrates for one or more proteases expressed by the tissue in which IFN therapy is desired and / or anticipated.

71. A method for locally inducing an immune response in a target tissue, comprising administering to a subject an IFN receptor agonist (or a pharmaceutical composition comprising the IFN receptor agonist and an excipient) according to any one of claims 1 to 61, wherein the IFN receptor agonist has one or more targeting moieties capable of binding to a target molecule expressed in the target tissue and one or more protease-cleavable linkers, and each protease-cleavable linker comprises one or more substrates for one or more proteases expressed in the target tissue.

72. A method of enhancing an immune response against an antigen, comprising administering to a subject an immunogenic agent that elicits an immune response against the antigen together with an IFN receptor agonist according to any one of claims 1 to 61 (or a pharmaceutical composition comprising the IFN receptor agonist and an excipient) or a nucleic acid encoding such an IFN receptor agonist.

73. The method according to any one of claims 66 to 72, wherein the administration is non-local.

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