Interferon receptor antagonists and uses thereof

Abstract

The present disclosure provides interferon (IFN) receptor antagonists. The IFN receptor antagonists disclosed herein comprise an anchoring moiety, an IFN masking moiety, an IFN moiety, and a separating moiety, such as a targeting moiety that recognizes an antigen associated with a cell expressing a type 1 interferon receptor and anchors the IFN receptor antagonist to such cell. The disclosure further provides pharmaceutical compositions comprising the IFN receptor antagonists, and methods of using the IFN receptor antagonists in inhibition of IFN signaling, including methods of treatment. Also disclosed are nucleic acids encoding the IFN receptor antagonists, recombinant cells expressing the IFN receptor antagonists, and methods of producing the IFN receptor antagonists.

Description

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 501,840, filed May 12, 2023, and U.S. Provisional Application No. 63 / 597,502, filed November 9, 2023, the contents of each of which are incorporated by reference in their entirety.

[0003] 2. SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted electronically in XML format and which is hereby incorporated by reference in its entirety. The Sequence Listing, created on May 6, 2024, is named RGN-035WO_SL.xml and is 146,865 bytes in size. BACKGROUND

[0005] Type I interferons (IFNs) are a family of cytokines that act as immune modulators of both the innate and adaptive immune responses. Most cell types produce low levels of Type I IFNs continuously; however, infection and other triggers can stimulate the production of these molecules, which then bind to and signal through the IFNAR1 / IFNAR2 receptor complex.

[0006] Multiple lines of evidence strongly implicate elevated IFNs and increased IFN signaling in the pathogenesis of various autoimmune inflammatory diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSc), and Sjogren’s syndrome (SS). Inhibitors of IFN signaling are currently being tested in multiple clinical trials (Chasset et al., 2021, Front. Pharmacol. 12:633821. doi: 10.3389 / fphar.2021.633821).

[0007] Furthermore, Type I IFN is also upregulated as a response to oncolytic virus (OV) therapy. OVs are biologic therapeutics that are effective against solid tumors. Unfortunately, upregulation of Type I IFN enhances the patient’s autoimmune side effects, increases tumor cell resistance to oncolytic viruses, and decreases the therapeutic efficacy of this approach (El-Sayes et al., 2022, Oncolytics 25: 16-30). Preclinical studies have shown that the combination of OV therapy with inhibitors of IFN signaling is associated with an improvement in treatment outcomes (El-Sayes et al., 2022, Oncolytics 25: 16-30; Ebrahimi et al., 2017, J Cell Biochem. 118(8): 1994-9; Selman et al., 2018, Sci Transl Med. 10(425): eaal613).

[0008] Nonetheless, given that the IFNAR1 / IFNAR2 receptor complex is expressed systemically, and that systemic inhibition of Type I IFN signaling can impair the body’s immune response, such as the ability to fight infection, therapeutics that indiscriminately inhibit Type I IFN signaling can do more harm than good.

[0009] Accordingly, there is a need in the art for novel therapeutics that specifically target and modulate incorrect or prolonged Type I IFN signaling in particular cells. SUMMARY

[0010] The present disclosure relates to novel molecular constructs (referred to herein as “IFN receptor antagonists”) that comprise an IFN moiety masked by an IFNAR1 moiety and are capable of potent IFN signal inhibition. Surprisingly, despite comprising an IFN molecule, the IFN receptor antagonists described herein do not activate IFN signaling in cells, but rather exclusively function as IFN signal blockers, competing with exogenous IFN for binding and preventing or significantly reducing IFN signaling in cells through the Type I interferon receptor.

[0011] In addition to the IFN portion and the IFNAR1 portion, the IFN receptor antagonist generally further comprises an anchor portion (e.g., a targeting portion), a spacer portion (e.g., an Fc domain), and, optionally, a linker connecting the IFNAR1 portion to the IFN portion. The IFN receptor antagonist can further comprise one or more linkers connecting one or more components (e.g., connecting the spacer portion to the IFNAR1 or IFN portion). The anchor portion can bind to a target molecule present on the surface of a cell expressing the type I interferon receptor. For example, the anchor portion can be a targeting portion comprising an antigen binding domain that can bind such a target molecule. The spacer portion is capable of simultaneously allowing the anchor portion to bind to the target molecule and the IFN portion to bind to the type I interferon receptor on the cell. For example, the spacer portion can be an Fc domain.

[0012] Exemplary IFN portions useful in the IFN receptor antagonists of the present disclosure are described in Section 6.3.

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

[0014] Linkers useful in the IFN receptor antagonists of the present disclosure are described in Section 6.5.

[0015] Anchor portions useful in the IFN receptor antagonists of the present disclosure are described in Section 6.6. Targeting portions are described in Section 6.6.1, and targeting portion formats are described in Section 6.6.1.1.

[0016] Spacer portions that can be incorporated into the IFN receptor antagonists of the present disclosure are described in Section 6.7. Fc domains are described in Section 6.7.1.

[0017] Exemplary IFN receptor antagonists of the present disclosure are described in Section 6.2 and numbered embodiments 1-90.

[0018] The present disclosure further provides nucleic acids encoding the IFN receptor antagonists of the present disclosure. The nucleic acid encoding the IFN receptor antagonist can be a single nucleic acid (e.g., a vector encoding all of the polypeptide chains of the IFN receptor antagonist) or multiple nucleic acids (e.g., two or more vectors encoding different polypeptide chains of the IFN receptor antagonist). The present disclosure further provides host cells and cell lines engineered to express the nucleic acids and IFN receptor antagonists of the present disclosure. The present disclosure further provides methods of producing the IFN receptor antagonists of the present disclosure. Exemplary nucleic acids, host cells and cell lines, and methods of producing the IFN receptor antagonists are described in Section 6.8 and numbered embodiments 91-93.

[0019] This disclosure further provides pharmaceutical compositions comprising the IFN receptor antagonists of this disclosure. Exemplary pharmaceutical compositions are described in Section 6.9 and in Examples 94 and 95.

[0020] This document further provides methods for using the IFN receptor antagonists and pharmaceutical compositions of this disclosure, for example, to treat cancer or autoimmune diseases. Exemplary methods are described in Section 6.10 and in Examples 96 through 112. Attached Figure Description

[0021] Figures 1A-1F It is a cartoon representing an IFN receptor antagonist. Figure 1A The components used to produce a masked IFN receptor antagonist are shown (TM = targeting portion; IFN = interferon; IFNAR1 = interferon α / β receptor 1). Figures 1B-1F The structure of the construct targeting the IFN receptor antagonist is shown. Figures 1A-1F As used herein, "IFN" generally refers to any IFN portion (e.g., IFNα2b), and "IFNAR1" generally refers to any IFNAR1 portion. Although Figures 1A-1F The target portion is shown as Fab, but other target portions (e.g., scFv) can be used instead of Fab.

[0022] Figures 2A-2B This paper presents an overview of the staining and bioassay activities of the non-targeted construct containing the IFNAR1 (R1) masking portion. Figure 2A This is a graph showing the coloring profile of the bivalent masking construct. Figure 2B This is a graph showing the in vitro activity of the divalent masking construct.

[0023] Figures 3A-3I This illustrates the in vitro activity of an exemplary IFN receptor-targeting antagonist in the presence or absence of a constant amount of IFNα2b. In PDL1-overexpressing (OE) KG1a / ISRE-Luc cells ( Figures 3A-3C and Figures 3G-3I ) or PDL1 knockout (KO) KG1a / ISRE-Luc cells ( Figures 3D-3F In (in the absence of () Figures 3A-3C ) or the presence of 200 pMIFNα2b ( Figures 3D-3I (titration under the condition of ) to evaluate PDL1-targeted and allotype control constructs ( Figure 3A , Figure 3D and Figure 3G ), masking unit price construct ( Figure 3B , Figure 3E and Figure 3H ) and masking binary constructs ( Figure 3C , Figure 3F andFigure 3I The in vitro activity of ).

[0024] Figures 4A-4L This illustrates the in vitro activity of exemplary IFN receptor-targeting antagonists and anti-IFNAR1 or anti-IFNAR2 antibodies in the presence of constant amounts of IFNα2b or IFNβ. In PDL1 KO ( Figures 4A-4C and Figures 4G-4I ) or PDL1OE ( Figures 4D-4F and Figures 4J-4L KG1a / ISRE-Luc cells (in the presence of 45pM IFNα2b) Figures 4A-4F ) or 95pM IFNβ ( Figures 4G-4L The in vitro activity of PDL1-targeting and isotype control constructs, masked monovalent constructs, masked bivalent constructs, and anti-IFNAR1 or anti-IFNAR2 antibodies was evaluated by titration under the condition of ) .

[0025] Figures 5A-5D An exemplary unmasked control construct is shown ( Figure 5A ), targeted and homologous monovalent constructs ( Figure 5B ) and targeted and isotype bivalent constructs ( Figure 5C ) and anti-IFNAR1 antibodies and anti-IFNAR2 antibodies ( Figure 5D In vitro cell protection activity in PDL1OE KG1a / ISRE-Luc cells.

[0026] Figures 6A-6D An exemplary unmasked control construct is shown ( Figure 6A ), targeted and homologous monovalent constructs ( Figure 6B ) and targeted and isotype bivalent constructs ( Figure 6C ) and anti-IFNAR1 antibodies and anti-IFNAR2 antibodies ( Figure 6D In vitro cell protection activity in PDL1KO KG1a / ISRE-Luc cells.

[0027] Figures 7A-7D An exemplary unmasked control construct is shown ( Figure 7A ), targeted and homologous monovalent constructs ( Figure 7B ) and targeted and isotype bivalent constructs ( Figure 7C ) and anti-IFNAR1 antibodies and anti-IFNAR2 antibodies ( Figure 7D In vitro cell protection activity in PDL1OE KG1a / ISRE-Luc cells.

[0028] Figures 8A-8D An exemplary unmasked control construct is shown ( Figure 8A ), targeted and homologous monovalent constructs ( Figure 8B) and targeted and isotype bivalent constructs ( Figure 8C ) and anti-IFNAR1 antibodies and anti-IFNAR2 antibodies ( Figure 8D In vitro cell protection activity in PDL1KO KG1a / ISRE-Luc cells.

[0029] Figures 9A-9L This shows the presence or absence of a constant IFNα2b ( Figures 9A-9F ) or IFNβ ( Figures 9G-9L Exemplary in vitro activity of targeting IFN receptor antagonists in the case of PDL1 knockout (KO) KG1a / ISRE-Luc cells ( Figures 9A-9C and Figures 9G-9I ) or PDL1 overexpression (OE) KG1a / ISRE-Luc cells ( Figures 9D-9F and Figures 9J-9L In the evaluation of PDL1-targeted and allotype control constructs ( Figure 9A , Figure 9D , Figure 9G and Figure 9J ), masking unit price construct ( Figure 9B , Figure 9E , Figure 9H and Figure 9K ) and masking binary constructs ( Figure 9C , Figure 9F , Figure 9I and Figure 9L The in vitro activity of ).

[0030] Figures 10A-10B This illustrates exemplary targeting and isotype IFN receptor antagonist constructs for IFNα2b-mediated IP10 release from monocyte-derived DCs (MoDCs). Figure 10A ) and PDL1 expression in MoDC ( Figure 10B The impact of ).

[0031] Figures 11A-11F The in vitro activity of exemplary IFN receptor antagonist constructs is shown in the presence or absence of constant amounts of hIFNα2b or hIFNβ. In PDL1 knockout (KO) KG1a / ISRE-Luc cells ( Figures 11A-11C ) and PDL1 overexpression (OE) KG1a / ISRE-Luc cells ( Figures 11D-11F In this study, the in vitro activity of the PDL1-targeting masked bivalent construct and the control construct was evaluated.

[0032] Figures 12A-12FThis illustrates the in vitro activity of an exemplary IFN receptor antagonist construct comprising a universal type I interferon (uIFN) moiety in the presence or absence of a constant amount of hIFNα2b or hIFNβ. In PDL1 knockout (KO) KG1a / ISRE-Luc cells (… Figure 12A , Figure 12C and Figure 12E ) and PDL1 overexpression (OE) KG1a / ISRE-Luc cells ( Figure 12B , Figure 12D and Figure 12F In this study, the in vitro activity of masked bivalent constructs targeting the same type or PDL1 was evaluated.

[0033] Figures 13A-13F This illustrates the in vitro activity of an exemplary IFN receptor antagonist construct comprising a universal type I interferon (uIFN) moiety in the presence or absence of a constant amount of hIFNα2b or hIFNβ. In KG1a / ISRE-Luc cells that do not express EGFR (“EGFR-free”); Figure 13A , Figure 13C and Figure 13E ) and EGFR-overexpressing (OE) KG1a / ISRE-Luc cells ( Figure 13B , Figure 13D and Figure 13F In this study, the in vitro activity of masked bivalent constructs targeting the same type or EGFR was evaluated. Detailed Implementation

[0034] 6.1. Definition

[0035] The following terms, as used in this document, have the following meanings:

[0036] ABD strand, targeting moiety strandThe target moiety and the antigen-binding site (ABD) therein may exist as a single polypeptide chain (e.g., in the case of scFv or scFab) or be formed by the association of more than one polypeptide chain (e.g., in the case of Fab or Fv). As used herein, the terms “ABD chain” and “target moiety chain” refer to all or part of the ABD or target moiety present on a single polypeptide chain. The use of the terms “ABD chain” or “target moiety chain” is for convenience and descriptive purposes only and does not imply a particular configuration or method of production. Furthermore, references to the ABD or target moiety when describing IFN receptor agonists cover the ABD chain or target moiety chain unless the context otherwise requires. Thus, when describing IFN receptor antagonists in which the Fc domain is operatively linked to the target moiety, the Fc domain may be directly or indirectly covalently linked via peptide bonds (e.g., via a linker) to, for example, (1) a first ABD or target moiety chain of Fab or Fv (where other components of Fab or Fv are on a second associated ABD or target moiety chain) or (2) an ABD or target moiety chain containing scFv or scFab.

[0037] about, approximately Throughout the specification, the terms "about," "approximately," etc., are used before numbers to indicate that the number is not necessarily accurate (e.g., considering variations in fractions, measurement accuracy and / or precision, timing, etc.). It should be understood that disclosures of "about X" or "approximately X," where X is a number, are also disclosures of "X." Thus, for example, a disclosure of an embodiment in which one sequence has "about X% sequence identity" with another sequence is also a disclosure of an embodiment in which that sequence has "X% sequence identity" with another sequence.

[0038] anchoring moiety As used herein, the term "anchoring moiety" refers to any molecule or portion thereof that can bind to a cell. Anchoring moieties considered herein include, but are not limited to, cell surface protein-binding molecules (e.g., ligands and other protein-binding couplers, such as those described in Section 6.6) and targeting moieties (e.g., antibody- and antigen-binding fragments, such as those described in Section 6.6.1). As used herein, an anchoring moiety "for a particular cell" means that the anchoring moiety is capable of binding to a particular cell. Binding need not be selective or specific.

[0039] and, or Unless otherwise stated, the conjunction “or” should be used in its proper sense as a Boolean logical operator, encompassing the selection of features in alternatives (A or B, where the selection of A and B are mutually exclusive) and the selection of joint features (A or B, where both A and B are selected). In some places in the text, the terms “and / or” are used for the same purpose, which should not be interpreted as implying that “or” is used to refer to mutually exclusive alternatives.

[0040] antagonistic As used herein, the term "antagonistic" or "antagonist" in relation to IFN receptor antagonists refers to the ability to reduce signaling, activation, or activity of type I interferon receptors in the presence of IFNα2b. In some embodiments, an IFN receptor antagonist is a molecule that reduces interferon signaling by at least 10% as measured by an activity assay in the presence of IFNα2b as described in Section 9.1.4.

[0041] antibodyAs used herein, the term "antibody" refers to a polypeptide (or group of polypeptides) of the immunoglobulin family that can bind nonvalently, reversibly, and specifically to an antigen. For example, a naturally occurring IgG-type "antibody" is a tetramer consisting of at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain contains a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region contains one domain (abbreviated 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 consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of an antibody can mediate the binding of immunoglobulins to host tissues or factors, encompassing various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelified antibodies, chimeric antibodies, bispecific or multispecific antibodies, and anti-idiotype (anti-id) antibodies. Antibodies can be any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Both the light and heavy chains are divided into 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. Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental migration, Fc receptor binding, and complement binding. By convention, the numbering of constant domains increases as they move further away from the antibody's antigen-binding domain or N-terminus. The N-terminus is the variable region, and the C-terminus is the constant region; the CH3 and CL domains represent the C-termini of the heavy and light chains of the native antibody, respectively. For convenience, and unless the context otherwise requires, references to antibodies refer to antibody fragments and engineered antibodies that include and / or have antigen-binding domains with non-natural structures.

[0042] antigen binding domainAs used herein, the term "antigen-binding domain" or "ABD" refers to a portion of an antibody or antibody fragment (e.g., a targeting portion) that has the ability to bind non-covalently, reversibly, and specifically to an antigen. Examples of antibody fragments that may contain an ABD include, but are not limited to: single-chain Fv (scFv); Fab fragments, i.e., monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments, i.e., bivalent fragments containing 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 the antibody; dAb fragments (Ward et al., 1989, Nature 341:544-546), consisting of a VH domain; and separated complementarity-determining regions (CDRs). Thus, the term "antibody fragment" encompasses both proteolytic fragments of antibodies (e.g., Fab and F(ab)2 fragments) and engineered proteins containing one or more portions of an antibody (e.g., scFv). Antibody fragments can also be incorporated into single-domain antibodies, giant antibodies, micro antibodies, intracellular antibodies, bisomal antibodies, trisomal antibodies, tetrasomal antibodies, v-NARs, and bi-scFvs (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology 23: 1126-1136).

[0043] associate In the context of IFN receptor antagonists, the term "association" refers to a functional relationship between two or more polypeptide chains. Specifically, the term "association" means that two or more polypeptides (e.g., non-covalently through molecular interactions or covalently through one or more disulfide bridges or chemical crosslinks) associate with each other to produce a functional IFN receptor antagonist. Examples of association that may exist in the IFN receptor antagonists of this disclosure include, but are not limited to, association between Fc domains to form the Fc region (such as homodimers or heterodimers as described in Section 6.7), association between VH and VL regions in Fab or Fv, and association between CH1 and CL in Fab.

[0044] cancerThe term "cancer" refers to a disease characterized by the uncontrolled (and often rapid) growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. This article describes examples of various cancers, including but not limited to breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, adrenal 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 and digestive system cancers, urinary tract cancer, vaginal cancer, vulvar cancer, lymphoma, leukemia, lung cancer, etc., for example, any TAA-positive cancer of any type in the aforementioned categories.

[0045] complementarity determining regionAs used herein, the term “complementarity-determining region” or “CDR” refers to the sequence of amino acids within the variable region of an antibody that confers 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 those described 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; Lefranc et al., 2003, Dev.Comp.Immunol.27:55-77 (“IMGT” numbering scheme). For example, in 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-H1), and 50-65 (CDR-H1). In Chothia, the CDR amino acid residues in VH are numbered 24-34 (CDR-H1), 52-56 (CDR-H2), and 95-102 (CDR-H3); and the amino acid residues in VL are numbered 26-32 (CDR-H1), 50-52 (CDR-L2), and 91-96 (CDR-L3). By combining the CDR definitions of Kabat and Chothia, the CDR consists 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-H3) in human VL. It consists of (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3).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 the antibody can be determined using the IMGT / DomainGapAlign procedure.

[0046] constant domain The term "constant domain" refers to the CH1, CH2, CH3, or CL domains of an immunoglobulin.

[0047] The term "CH1 domain" refers to a heavy-chain constant region that connects a variable domain to a hinge in a heavy-chain constant domain. In some embodiments, the term "CH1 domain" refers to a region of an immunoglobulin molecule spanning amino acids 118 to 215 (EU number). The term "CH1 domain" encompasses the wild-type CH1 domain and its variants (e.g., non-naturally occurring CH1 domains or modified CH1 domains). For example, the term "CH1 domain" includes wild-type IgG1, IgG2, IgG3, and IgG4 CH1 domains and variants having 1, 2, 3, 4, 5, 1 to 3, 1 to 5, 3 to 5, and / or up to 5, 4, 3, 2, or 1 mutation (e.g., substitution, deletion, and / or addition). Exemplary CH1 domains include CH1 domains with mutations that alter the biological activity of an antibody, such as ADCC, CDC, or half-life.

[0048] The term "CH2 domain" refers to a heavy chain constant region in which a hinge connects to a CH3 domain within a heavy chain constant domain. In some embodiments, the term "CH2 domain" refers to a region of an immunoglobulin molecule spanning amino acids 238 to 340 (EU number). The term "CH2 domain" encompasses the wild-type CH2 domain and its variants (e.g., non-naturally occurring CH2 domains or modified CH2 domains). For example, the term "CH2 domain" includes wild-type IgG1, IgG2, IgG3, and IgG4 CH2 domains and variants having 1, 2, 3, 4, 5, 1 to 3, 1 to 5, 3 to 5, and / or up to 5, 4, 3, 2, or 1 mutation (e.g., substitution, deletion, and / or addition). Exemplary CH2 domains include CH2 domains with mutations that alter the biological activity of an antibody, such as ADCC, CDC, purification, dimerization, and half-life.

[0049] The term "CH3 domain" refers to the heavy chain constant region located at the C-terminus of the CH2 domain within the heavy chain constant domain. In some embodiments, the term "CH3 domain" refers to a region of an immunoglobulin molecule spanning amino acids 341 to 447 (EU number). The term "CH3 domain" encompasses the wild-type CH3 domain and its variants (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 having 1, 2, 3, 4, 5, 1 to 3, 1 to 5, 3 to 5, and / or up to 5, 4, 3, 2, or 1 mutation (e.g., substitution, deletion, and / or addition). Exemplary CH3 domains include CH3 domains with mutations that alter the biological activity of an antibody (such as ADCC, CDC, purification, dimerization, and half-life).

[0050] The term "CL domain" refers to a constant region of the immunoglobulin light chain. The term "CL domain" encompasses wild-type CL domains (e.g., the constant region of the κ or λ light chain) and their variants (e.g., non-naturally occurring CL domains or modified CL domains). For example, the term "CL domain" includes wild-type κ and λ constant domains and their variants having 1, 2, 3, 4, 5, 1 to 3, 1 to 5, 3 to 5, and / or up to 5, 4, 3, 2, or 1 mutation (e.g., substitution, deletion, and / or addition).

[0051] effector functionThe term "effective function" refers to the activity of an antibody molecule mediated by the binding of its domains other than the antigen-binding domain (typically mediated by the binding of effector molecules). Effector functions include complement-mediated effectsor functions, which are mediated by, for example, the binding of the C1 component of complement to the antibody. Complement activation is important in the opsonization and lysis of cellular pathogens. Complement activation also stimulates inflammatory responses and may be involved in autoimmune hypersensitivity reactions. Effector functions also include Fc receptor (FcR)-mediated effectsor functions, which can be triggered by the binding of the constant domain of an antibody to the Fc receptor (FcR). Binding an antibody to an Fc receptor on the cell surface triggers 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 (known as antibody-dependent cell-mediated cytotoxicity or ADCC), release of inflammatory mediators, control of placental transfer, and immunoglobulin production. The effector function of an antibody can be altered (e.g., enhanced or reduced) by changing the antibody's affinity for effector molecules such as Fc receptors or complement components. Binding affinity is typically altered by modifying the binding site of the effector molecule, and in this case, it is appropriate to target the site of interest and modify at least a portion of that site in a suitable manner. It is also envisioned that changes in the binding site on an antibody against an effector molecule do not need to significantly alter the overall binding affinity, but may alter the geometry of the interaction, rendering the effector mechanism ineffective as in nonproductive binding. Furthermore, it is envisioned that effector function can also be altered by modifying sites that do not directly participate in the binding of the effector molecule but otherwise participate in the execution of the effector function.

[0052] epitope An epitope, or antigenic determinant, is a portion of an antigen recognized by an antibody or other antigen-binding moiety as described herein. Epitopes can be linear or conformational.

[0053] FabThe term "Fab" refers to a pair of polypeptide chains, the first polypeptide chain containing a variable heavy chain (VH) domain of the antibody operably linked (typically at the N-terminus) to a first constant domain (referred to herein as C1), and the second polypeptide chain containing a variable light chain (VL) domain at the N-terminus of the antibody operably linked (typically at the N-terminus) to a second constant domain capable of pairing with the first constant domain (referred to herein as C2). In native 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 this disclosure may be arranged according to the natural orientation or include substitutions or exchanges of domains that facilitate proper VH and VL pairing. For example, the CH1 and CL domain pairs in Fab may be replaced with CH3 domain pairs to facilitate proper modified Fab chain pairing in heterodimer molecules. CH1 and CL may also be reversed, such that CH1 is attached to VL and CL is attached to VH; this configuration is commonly referred to as a Crossmab. The term "Fab" also encompasses single-chain Fab.

[0054] Fc domain and Fc region The term "Fc domain" refers to a portion of a heavy chain that pairs with a corresponding portion of another heavy chain. In some embodiments, the Fc domain comprises a CH2 domain followed by a CH3 domain, which may or may not have a hinge region at the N-terminus of the CH2 domain. The term "Fc region" refers to a region formed by the association of two heavy chain Fc domains. The two Fc domains within an Fc region may be identical or different from each other. In natural antibodies, the Fc domains are typically identical, but one or both Fc domains may be modified to allow heterodimerization, for example, via mortise and tenon interactions.

[0055] 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 consists of a dimer composed of a heavy chain variable domain and a light chain variable domain, which are tightly non-covalently associated (VH-VL dimer). It is in this conformation 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 the antibody target binding specificity. However, in some cases, even a single variable domain (or half of an Fv containing only the three target-specific CDRs) can have the ability to recognize and bind to a target. The mention of the VH-VL dimer in this document does not imply the expression of any particular conformation. When present on a single polypeptide chain (e.g., scFv), the VH is located at the N-terminus or C-terminus of the VL.

[0056] half-antibodyThe term "half-antibody" refers to a molecule that contains at least one Fc domain and can associate with another molecule containing an Fc domain through, for example, disulfide bridges or molecular interactions. A half-antibody can consist of one or more polypeptide chains (e.g., the two polypeptide chains of Fab). An example of a half-antibody is a molecule containing both the heavy and light chains of an antibody (e.g., an IgG antibody). Another example of a half-antibody is a molecule containing a first polypeptide containing VL and CL domains and a second polypeptide containing VH, CH1, hinge, CH2, and CH3 domains, wherein the VL and VH domains form an ABD. Yet another example of a half-antibody is a polypeptide containing scFv, CH2, and CH3 domains.

[0057] The IFN receptor antagonists disclosed herein typically comprise two halves, each half containing an IFN moiety masked by one or two receptor moieties (e.g., IFN masking moieties). The one or two masking moieties may be located in the same half as the IFN moiety or in another half derived from the IFN moiety, such as... Figure 1B As illustrated in Figure 1G and in the examples listed in Table 2, exemplary locations of the linker (e.g., an uncleavable linker) in the hapten are shown in Table 1. One or both haptens of the IFN receptor antagonist may further include a targeting moiety, such as scFv or Fab. Exemplary IFN receptor antagonists containing a targeting moiety are shown in Table 1. Figure 1B See Figure 1G and Table 2.

[0058] The term "half-antibody" is used for descriptive purposes only and does not imply a specific configuration or method of production. Describing half-antibodies as "first" half-antibody, "second" half-antibody, "left" half-antibody, "right" half-antibody, etc., is merely for convenience and descriptive purposes.

[0059] host cell or recombinant host cellThe term "host cell" or "recombinant host cell" refers to a cell that has been genetically engineered, for example, by introducing a heterologous nucleic acid. It should be understood that such terms refer not only to a specific subject cell but also to the progeny of such cells. Because certain modifications may occur in offspring due to mutations or environmental influences, these progeny may actually differ from the parent cells but are still included within the scope of the term "host cell" as used herein. Host cells can, for example, transiently carry heterologous nucleic acids on an extrachromosomal heterologous expression vector, or stably carry heterologous nucleic acids, for example, by integrating them into the host cell genome. For the purpose of expressing the IFN receptor antagonist of this disclosure, the host cell is preferably a mammalian-derived or mammalian-like cell line, such as monkey kidney cells (COS, e.g., COS-1, COS-7, HEK293), juvenile 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 glycan-modified derivatives and / or site-specific integration site derivatives.

[0060] interferon As used herein, the term "interferon" refers to full-length interferon or modified interferon, such as truncated and / or mutant interferon. In some embodiments, the modified interferon is attenuated compared to the corresponding wild-type interferon (e.g., retaining 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% of activity in an in vitro luciferase reporter assay, as described in Section 9.1.5). In some embodiments, the modified interferon is attenuated beyond the range defined by any two of the foregoing values, e.g., 0.05% to 50%, 0.1% to 20%, 0.1% to 10%, 0.05% to 5%, 1% to 20%, etc. In other embodiments, the modified interferon substantially retains the biological activity of the corresponding wild-type interferon (e.g., retaining at least 50% of the activity in an in vitro luciferase reporter assay, as described in Section 9.1.5). Interferons include type I interferons (e.g., interferon-α and interferon-β) and type II interferons (e.g., interferon-γ). The term "interferon" also covers synthetic or engineered proteins that have the biological activity of wild-type interferons (e.g., having at least 50% activity in an in vitro luciferase reporter assay as described in Section 9.1.5), such as the universal type I interferon as described in Section 6.3.1.

[0061] linkerAs used herein, the term "linker" refers to a connecting peptide between two parts. For example, a linker can connect an IFN part and an IFN masking part.

[0062] non-cleavable linker As used herein, a non-cleavable linker is a peptide whose amino acid sequence lacks the substrate sequence of a protease. Examples of non-cleavable linkers are illustrated in Section 6.5.

[0063] oncolytic virus The term "oncolytic virus" refers to a virus that replicates in tumor cells. These viruses include those that naturally preferentially replicate and accumulate in tumor cells (such as poxviruses) and those modified to replicate and accumulate in tumor cells. Some oncolytic viruses can kill tumor cells after infecting them. For example, oncolytic viruses can cause tumor cell death by lysing tumor cells or inducing cell death in tumor cells. Exemplary oncolytic viruses include, but are not limited to, poxviruses, herpesviruses, adenoviruses, adeno-associated viruses (AAVs), lentiviruses, retroviruses, rhabdoviruses, papillomaviruses, vesicular stomatitis viruses (VSVs), measles viruses, Newcastle disease viruses, piconemaviruses, Sindbisviruses, papillomaviruses, parvoviruses, reoviruses, and Coxsackieviruses. In some respects, the oncolytic virus of this disclosure is a VSV. Oncolytic viruses and their use in the treatment of cancer are further described, for example, in Chiocca and Rabkin Cancer Immunol Res (2014) 2(4): 295-300.

[0064] operably linked The term "operably linked" refers to a functional relationship between two or more peptide or polypeptide domains or nucleic acid (e.g., DNA) segments. In the context of fusion proteins or other polypeptides, the term "operably linked" means that two or more amino acid fragments are linked to produce a functional polypeptide. For example, in the context of the IFN receptor antagonist of this disclosure, individual components (e.g., the Fc domain and the IFN moiety) can be operably linked directly or via peptide linker sequences. In the context of nucleic acids encoding fusion proteins (such as haptens of the IFN receptor antagonist of this disclosure), "operably linked" means that two nucleic acids are linked such that the amino acid sequences encoded by the two nucleic acids remain within the frame. In the context of transcriptional regulation, the term refers to a functional relationship between a transcriptional regulatory sequence and a transcriptional sequence. For example, if a promoter or enhancer sequence stimulates or regulates transcription of a coding sequence in a suitable host cell or other expression system, then the promoter or enhancer sequence is operably linked to the coding sequence.

[0065] polypeptide, peptide, and protein The terms “polypeptide”, “peptide”, and “protein” are used interchangeably in this document and refer to polymers of amino acid residues.

[0066] recognize As used herein, the term “recognition” refers to an antibody or antibody fragment (e.g., a targeting portion) that discovers and interacts with (e.g., binds to) an epitope.

[0067] single chain Fab or scFab As used herein, the term "single-chain Fab" or "scFab" refers to an ABD comprising a VH domain, a CH1 domain, a VL domain, a CL domain, and a linker. In some embodiments, the aforementioned domains and linker are arranged in one of the following orders in 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 preferably a non-cleavable linker of at least 30 amino acids, preferably between 32 and 50 amino acids. Single-chain Fab fragments are generally stabilized via a native disulfide bond between the CL domain and the CH1 domain. Furthermore, these single-chain Fab molecules can be further stabilized by forming interchain disulfide bonds via the insertion of cysteine ​​residues (e.g., at position 44 in the VH domain and position 100 in the VL domain according to Kabat numbering).

[0068] single chain Fv or scFv As used herein, the term "single-chain Fv" or "scFv" refers to an ABD containing 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, which 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, eds. Rosenburg and Moore (1994), Springer-Verlag, New York, pp. 269–315. VH and VL may be arranged in N-terminus to C-terminus (i.e., VH-VL or VL-VH), typically separated by linkers, such as those listed in Table E.

[0069] separating moiety: As used herein, the term "separator" refers to the following amino acid sequence that, as a component of an IFN receptor antagonist comprising an anchoring moiety and an IFN moiety, provides sufficient spatial separation between the anchoring moiety and the IFN moiety to allow them to bind simultaneously to the same cell, such as the cell described in Section 9.1.3. In some embodiments, the separator is a polypeptide of at least about 100 amino acids in length. In specific embodiments, the separator of this disclosure comprises an Fc domain or a fragment thereof.

[0070] specific (or selective) binding The term "specific (or selective) binding" to an antigen or epitope refers to a binding reaction that identifies the presence of a homologous antigen or epitope in a heterogeneous population of proteins and other molecules. The binding reaction may, but does not necessarily, be mediated by an antibody or antibody fragment. The term "specific binding" does not exclude cross-species reactivity. For example, an antigen-binding domain "specifically binding" to an antigen from one species (e.g., an antigen-binding fragment of an antibody) may also "specifically bind" to that antigen in one or more other species. Therefore, such cross-species reactivity does not, in itself, alter the classification of an antigen-binding domain as a "specific" binder. In some embodiments, the antigen-binding domains of this disclosure that specifically bind to human antigens exhibit cross-species reactivity with one or more non-human mammal 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).

[0071] subject The term "subject" includes both humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. In a preferred embodiment, the subject is a human.

[0072] target molecule As used herein, the term “target molecule” refers to any biomolecule (e.g., protein, carbohydrate, lipid, or combination thereof) expressed on the cell surface or in the extracellular matrix that can be specifically bound by the targeting portion of the IFN receptor antagonist of this disclosure.

[0073] targeting moiety As used herein, the term "targeting moiety" refers to any molecule or its binding portion (e.g., an immunoglobulin or antigen-binding fragment) capable of binding to a cell surface molecule on which the IFN receptor antagonist of this disclosure is to be targeted (e.g., on cells expressing type I interferon receptors, such as lymphocytes involved in autoimmune diseases)). In addition to targeting the IFN receptor antagonist to a specific site, the targeting moiety may also have functional activity. For example, a targeting moiety binding to a checkpoint inhibitor such as PDL1 may also exhibit antitumor activity, for example, by inhibiting PD1 / PDL1 signaling.

[0074] T cell antigen, TCAThe term "T-cell antigen" or "TCA" refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) expressed on the surface of T lymphocytes and can be used to preferentially target a drug at a specific site. In some embodiments, this site is cancerous tissue and / or the T-cell antigen is a tumor-reactive lymphocyte antigen, a cell surface molecule of tumor or viral lymphocytes, or a checkpoint inhibitor expressed on T lymphocytes.

[0075] tumor The term “tumor” is used interchangeably with the term “cancer” in this document, as both terms cover solid and liquid tumors, such as diffuse or circulating tumors. As used herein, the terms “cancer” or “tumor” include precancerous as well as malignant cancers and tumors.

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

[0077] treat / treatment / treatingAs used herein, the term "treat / treatment / treating" refers to a reduction or improvement in the progression, severity, and / or duration of a disease (e.g., an inflammatory disease, an autoimmune disease, or a proliferative disease), or an improvement in one or more symptoms (preferably one or more identifiable symptoms) of a disease caused by the administration of one or more IFN receptor antagonists of this disclosure. In some embodiments, the disease is an inflammatory, autoimmune, or proliferative disease, and the term "treat / treatment / treating" refers to an improvement in at least one measurable physical parameter of an inflammatory, autoimmune, or proliferative disease that is not necessarily identifiable by the patient. In other embodiments, the term "treatment" refers to suppressing the progression of a disease physically by, for example, stabilizing identifiable symptoms, or physiologically by, for example, stabilizing physical parameters, or both. In other embodiments, the term "treat / treatment / treating" refers to a reduction or stabilization of tumor size or cancer cell count in a proliferative disease.

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

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

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

[0081] 6.2. IFN receptor antagonists

[0082] This disclosure relates to IFN receptor antagonists comprising an anchoring portion, a septum portion, a type I interferon (IFN) portion, and a type I interferon α receptor 1 (IFNAR1) portion.

[0083] The IFN receptor antagonists of this disclosure typically comprise one or more anchoring portions that bind to cells expressing type I interferon receptors. Depending on the desired indication or use, the anchoring portions may be designed to specifically bind to a particular cell type expressing a type I interferon receptor. Thus, in some embodiments, the IFN receptor antagonists of this disclosure comprise one or more targeting portions (e.g., antigen-binding domains of antibodies) that target the IFN receptor antagonist to cells expressing type I interferon receptors, such as cancer cells or lymphocytes involved in autoimmune diseases.

[0084] The IFN receptor antagonists of this disclosure typically comprise one or more septa that separate the anchoring portion from the IFN portion and the IFNAR1 portion. The septa can be any molecule or peptide capable of enabling the anchoring portion and the IFN portion to bind simultaneously to the same cell. In some cases, the septa are polymerizing portions that allow two or more individual components to polymerize (e.g., dimerize). Thus, in embodiments where the IFN receptor antagonist is a dimer, the IFN receptor antagonist typically consists of two halves comprising a pair of polymerizing domains, such as Fc domains that associate to form an Fc region (typically containing a hinge sequence). In the IFN receptor antagonists of this disclosure, the two halves together comprise at least one IFN portion, but may comprise two or more IFN portions. The IFN portions in the IFN receptor antagonist may each be masked by an interferon α receptor 1 (IFNAR1) portion.

[0085] Exemplary IFN receptor antagonists are shown in Figure 1B See Figure 1G.

[0086] Table 1 below describes exemplary haptens that can be incorporated into the IFN receptor antagonists of this disclosure. As can be clearly seen from Table 1, each hapten may contain one or more polypeptide chains. For convenience, each hapten described in Table 1 is often referred to herein as an “exemplary monomer” when describing combinations of haptens in the IFN receptor antagonists of this disclosure.

[0087]

[0088] In some embodiments, all linkers in the IFN receptor antagonist are non-cleavable. Exemplary linkers are described in Section 6.5. The Fc domains in the polypeptide chains described in Table 1 preferably include hinge domains as listed in Section 6.7.1.3.

[0089] In some embodiments, the anchoring portion is a targeting portion that binds to a cell surface protein (e.g., as described in Section 6.6.1). In other embodiments, the anchoring portion is a cell surface protein-binding molecule. In some embodiments, the separating portion is an Fc domain (e.g., as described in Section 6.7.1). Not intended to be theoretically construed, the inventors believe that in this configuration, the anchoring portion anchors the IFN receptor antagonist to the cell, thereby enabling the IFNAR1-masked IFN portion to bind to type I interferon receptors on the cell and inhibit IFN signaling.

[0090] Table 2 below illustrates additional exemplary monomer pairings that can be used in the IFN receptor antagonists of this disclosure. The IFN receptor antagonists identified in Table 2 comprise two targeting moieties.

[0091]

[0092] The order and length of the hinge and linker sequences can vary, as can the order of the IFN portion (containing full-length or N-terminal and / or C-terminal truncated IFN sequences and amino acid substitutions). Exemplary IFN portions are described in Section 6.3 and include IFNα- and IFNβ-based portions as described in Sections 6.3.1 and 6.3.2 below, and other type I IFN-based portions as described in Section 6.3.1. Exemplary IFNAR1 portions are disclosed in Section 6.4. Exemplary linker and hinge sequences are disclosed in Sections 6.5 and 6.7.1.3, respectively. Exemplary targeting portions are disclosed in Section 6.6. Exemplary Fc domains (including Fc domains suitable for heterodimerization when the two half-antibodies of an IFN receptor antagonist are not identical) are described in Section 6.7.

[0093] 6.3. IFN section

[0094] There are two main classes of IFNs: type I (IFN-α subtype, IFN-β, etc.) and type II (IFN-γ). Additional IFNs (IFN-like cytokines; IFN-λ subtype) have also been identified.

[0095] The IFN moiety disclosed herein may include any wild-type or modified (e.g., truncated and / or mutant) IFN or IFN-like cytokine sequence, but is preferably a type I IFN moiety. Type I IFNs bind to the heterodimeric plasma membrane receptor IFNAR, composed of IFNAR1 and IFNAR2, which is widely expressed in all nucleated cells. Ligand binding is initiated by the high-affinity receptor subunit IFNAR2 (Piehler et al., 2012, Immunological Reviews, doi.org / 10.1111 / imr.12001). Thus, type I IFNs are capable of acting on almost all cells in the body. Sixteen type I interferon isoforms have been identified, each exhibiting intrinsic variability in affinity and activity for IFNAR2.

[0096] In some embodiments, the type I IFN portion is the interferon-α (IFN α) portion. In other embodiments, the type I IFN portion is the interferon-β (IFN β) portion.

[0097] In other embodiments, the type I IFN portion is interferon-ω (IFNω), interferon-ε (IFNε), or interferon-κ (IFNκ) portion.

[0098] The type I IFN portion may include a sequence that differs from the wild-type IFN sequence due to one or more mutations (e.g., substitution, deletion, or insertion). Substitutions that weaken IFN activity by reducing receptor binding may be appropriately used. Amino acids with N-terminal or C-terminal deletions (or truncations) may also be used, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids truncated from the N-terminus and / or C-terminus of mature type I IFN.

[0099] Further details of the exemplary Type I IFN portion are provided below.

[0100] 6.3.1. Interferon-α portion

[0101] The IFNα gene is a member of the α-interferon gene cluster on chromosome 9. The encoded cytokine is a member of the type I interferon family, produced in response to viral infection, and is a key component of the innate immune response, possessing potent antiviral, antiproliferative, and immunomodulatory properties. IFNα is a protein family with at least 15 known human IFNα subtypes. The major identified subtypes are IFNα1, IFNα2, IFNα8, IFNα10, IFNα14, and IFNα21.

[0102] The IFNα1 gene has two allelic variants: IFNα1a and IFNα1b. The amino acid sequence of human IFNα1a is assigned UniProtKB accession number P01562 and is reproduced below, with the signal peptide underlined:

[0103] MASPFALLMV LVVLSCKSSC SLG CDLPETH SLDNRRTLML LAQMSRISPS SCLMDRHDFGFPQEEFDGNQ FQKAPAISVL HELIQQIFNL FTTKDSSAAW DEDLLDKFCT ELYQQLNDLE ACVMQEERVGETPLMNADSI LAVKKYFRRI TLYLTEKKYS PCAWEVVRAE IMRSLSLSTN LQERLRRKE (SEQ ID NO:1)

[0104] The difference between the human IFNα1b gene and the IFNα1a allele variant lies in 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).

[0105] The IFNα2 allele has three allelic variants: IFNα2a, IFNα2b, and IFNα2c. Allele IFNα2b is the dominant allele, while allele IFNα2a is the subdominant allele, and IFNα2c is only a minor allelic variant. The amino acid sequence of human IFNα2 has been assigned UniProtKB accession number P01563. The sequence of the IFNα2b allele is reproduced below, with the signal peptide underlined:

[0106] MALTFALLVA LLVLSCKSSC SVG CDLPQTH SLGSRRTLML LAQMRRISLF SCLKDRHDFGFPQEEFGNQF QKAETIPVLH EMIQQIFNLF STKDSSAAWD ETLLDKFYTE LYQQLNDLEA CVIQGVGVTETPLMKEDSIL AVRKYFQRIT LYLKEKKYSP CAWEVVRAEI MRSFSLSTNL QESLRSKE (SEQ ID NO:2)

[0107] IFNα2b has arginine (R) at position 23 of the mature protein, while IFNα2a has lysine (K). Therefore, in some embodiments, the IFNα2 moiety has arginine at position 23 corresponding to the mature protein. In other embodiments, the IFNα2 moiety has lysine at position 23 corresponding to the mature protein.

[0108] In all respects, 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 with a fragment having at least 15 amino acids truncated at the N-terminus and / or C-terminus of mature IFNα1a, IFNα1b and / or IFNα2b, IFNα2a or IFNα2c or a truncated segment having at most 15 amino acids truncated at the N-terminus and / or C-terminus of mature IFNα1a, IFNα1b and / or IFNα2b, IFNα2a or IFNα2c.

[0109] In some embodiments, the IFNα portion has one or more amino acid substitutions, for example, substitutions that alter the binding and / or agonistic activity of IFNAR. Exemplary substitutions are found in WO 2013 / 107791, U.S. Patent No. 8,258,263, WO2007 / 000769A2, WO2008 / 124086, WO2010 / 030671, WO2018 / 144999A1, and WO2015 / 007520, WO 2013 / 059885, WO2020156467A1, and WO2021 / 126929A1. In some embodiments, the IFNα portion comprises:

[0110] a) One or more substitutions selected from the following: 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

[0111] b) One or more substitutions at amino acids 57 to 89 and 159 to 165 as described in WO2007000769A2; and / or

[0112] c) Substitution of one or more amino acids of alanine, glycine or threonine at positions 9, 17, 47, 65, 66, 117, 123, 128, 147 and 157 as described in WO2021126929A1.

[0113] The positions of the aforementioned substituted amino acids are given with reference to mature IFNα2b.

[0114] In a further embodiment, the IFNα portion includes one or more amino acid substitutions listed in Table 3. Table 3 lists the IFNα substitutions identified with reference to the amino acid positions within the IFNα2 sequence.

[0115]

[0116] In some embodiments, the IFNα portion includes an amino acid sequence comprising amino acid substitutions for R33A or R33K, Q90A, E96A, R120A, R120E, A145M, R149A or R149K, S152A, or any combination of two or more of the foregoing, such as Q90A + R120A or A145M + R149K.

[0117] Sequences of exemplary IFNα moieties that can be used in the IFN receptor antagonists of this disclosure are listed in Table 4 below:

[0118]

[0119] 6.3.2. Interferon-β fraction

[0120] Interferon-β (IFNβ) is a cytokine naturally produced 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 known as fibroblast interferon. IFNβ binds to the IFNAR receptor, which is composed of the IFNAR1 and IFNAR2 dimers, to induce signal transduction via the JAK / STAT pathway and other pathways. IFNβ can also function by binding to IFNAR1 alone and signaling independently of the Jak-STAT pathway (Ivashkiv and Donlin, 2014, Nat Rev Immunol.14(1):36-49).

[0121] IFNβ contains five α-helices designated as A (ynllgflqrssnfqcqkll (SEQ ID NO:101)), B (kedaaltiyemlqnifaif (SEQ ID NO:102)), C (etivenllanvyhqinhlktvleekl (SEQ ID NO:103)), D (sslhlkryygrilhylka (SEQ ID NO:104)), and E (hcawtivrveilrnfyfinrlt (SEQ ID NO:105)). These five α-helices are interconnected by loops of 2 to 28 residues designated as AB, BC, CD, and DE loops. It has been reported that the A-helix in the AB loop and the E-helix in the DE loop participate in the binding of IFNβ to the IFNAR receptor.

[0122] Two types of IFNβ have been described: interferon-β1 (IFNβ1) and interferon-β3 (IFNβ3) (Schirmer and Neumann, 2019. Cytokines. See: Nijkamp and Parnham's Principles of Immunopharmacology. Springer, Cham.).

[0123] The amino acid sequence of the human IFNβ precursor is listed in GenBank: accession number AAA36040.1, and is reproduced below (with the signal peptide underlined):

[0124] MTNKCLLQIA LLLCFSTTAL SMSYNLLGFL QRSSNFQCQK LLWQLNGRLE YCLKDRMNFDIPEEIKQLQQ FQKEDAALTI YEMLQNIFAI FRQDSSSTGW NETIVENLLA NVYHQINHLK TVLEEKLEKEDFTRGKLMSS LHLKRYYGRI LHYLKAKEYS HCAWTIVRVE ILRNFYFINR LTGYLRN (SEQ ID NO:18)

[0125] In all respects, 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 with a fragment of mature IFNβ1 or a fragment thereof having a truncated amino acid of up to 15 amino acids at its N-terminus and / or C-terminus (e.g., a truncated amino acid of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids at the N-terminus and / or C-terminus of IFNβ1).

[0126] In various embodiments, the IFNβ moiety comprises one or more amino acid substitutions and / or deletions compared to IFNβ1. In some embodiments, the substitution is C17S (refer to mature IFNβ1), and the deletion is one of the C-terminal truncations described in US 2009 / 0025106 A1, such as IFN-Δ1, IFNA2, IFNA3, IFNA4, IFNA5, IFNA6, IFN-Δ7, IFN-Δδ, IFNA9, and IFN-Δ10.

[0127] 6.3.1. General Type I Interferon

[0128] In some respects, the type I IFN moiety is the universal type I IFN (also known as human IFN-α hybrid protein, recombinant human universal type I IFN, or simply "uIFN"), which is a recombinant IFNα moiety constructed from IFNα A and IFNα D. uIFN exhibits biological activity across multiple species.

[0129] The amino acid sequence of universal type I IFN (uIFN) is replicated as follows:

[0130] CDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNVDSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSSLSLSTNLQERLRRKE (SEQ ID NO:137)

[0131] In some embodiments, the IFN 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 with the amino acid sequence of a fragment of uIFN (SEQ ID NO: 137) or a truncated segment of uIFN having up to 15 amino acids at its N-terminus and / or C-terminus (e.g., a truncated segment of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids at its N-terminus and / or C-terminus).

[0132] 6.3.2. Other Type I Interferons

[0133] In some respects, the type I IFN portion differs from the IFNα or IFNβ portions, such as the interferon-ω (IFNω), interferon-ε (IFNε), or interferon-κ (IFNκ) portions.

[0134] 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:

[0135] MALLFPLLAALVMTSYSPVGSLGCD LPQNHGLLSRNTLVLLHQMRRISPFLCLKDRRDFRFPQEMVKGSQLQKAHVMSVLHEMLQQIFSLFHTERSSAAWNMTLLDQLHTGLHQQLQHLETCLLQVVGEGESAGAISSPALTLRRYFQGIRVYLKEKKYSDCAWEVVRMEIMKSLFLSTNMQERLRSKDRDLGSS (SEQ ID NO: 19)

[0136] In all respects, 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 with a fragment of mature IFNω1 or a fragment thereof having a truncated form of up to 15 amino acids at its N-terminus and / or C-terminus (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids at its N-terminus and / or C-terminus).

[0137] Human IFNε is identified by UniProt registry number Q86WN2 and has the amino acid sequence listed below, with the signal sequence underlined:

[0138] MIIKHFFGTVLVLLASTTIFS LDLKLIIFQQRQVNQESLKLLNKLQTLSIQQCLPHRKNFLLPQKSLSPQQYQKGHTLAILHEMLQQIFSLFRANISLDGWEENHTEKFLIQLHQQLEYLEALMGLEAEKLSGTLGSDNLRLQVKMYFRRIHDYLENQDYSTCAWAIVQVEISRCLFFVFSLTEKLSKQGRPLNDMKQELTTEFRSPR (SEQ ID NO:20)

[0139] In all respects, 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 with the mature IFNε or a fragment thereof having a truncated amino acid of up to 15 amino acids at its N-terminus and / or C-terminus (e.g., a truncated amino acid 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 the IFNε).

[0140] Human IFNκ is identified by UniProt registry number Q9P0W0 and has the amino acid sequence listed below, with the signal sequence underlined:

[0141] MSTKPDMIQKCLWLEILMGIFIAGTLSLDCNLLNVHLRRVTWQNLRHLSSMSNSFPVECLRENIAFELPQEFLQYTQPMKRDIKKAFYEMSLQAFNIFSQHTFKYWKERHLKQIQIGLDQQAEYLNQCLEEDKNENEDMKEMKENEMKPSEARVPQLSSLELRRYFHRIDNFLKEKKYSDCAWEIVRVEIRRCLYYFYKFTALFRRK (SEQ ID NO:21)

[0142] In all respects, 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 with respect to the mature IFNκ or a fragment thereof having a truncated form of up to 15 amino acids at its N-terminus and / or C-terminus (e.g., 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 the IFNκ).

[0143] 6.4. IFN Masking Section

[0144] This disclosure provides IFN receptor antagonists in which the IFN moiety is masked by one or more receptor moieties. All human type I interferons bind to a cell surface receptor (IFNα receptor, IFNAR; also known as the "type I interferon receptor"), which is a heterodimer composed of two transmembrane proteins, IFNAR1 and IFNAR2 (see, for example, Novick et al., 1994, Cell 77:391). As described herein, the IFN receptor antagonists of this disclosure, which include an IFN moiety masked by IFNAR1, are able to inhibit IFN signaling in cells expressing type I interferon receptors. Conversely, similar constructs that include an IFN moiety masked by IFNAR2 do not inhibit IFN signaling. Therefore, in certain embodiments, the masking moiety is the IFNAR1 moiety.

[0145] As used herein, “inhibition of IFN signaling” describes a reduction in the signaling, activation, or activity of type I interferon receptors in cells. For example, an IFN receptor antagonist of this disclosure may be considered to inhibit IFN signaling, wherein the molecule reduces interferon signaling by at least 10%, as measured by an activity assay as described in Section 9.1.5.

[0146] An exemplary IFNAR1 portion is disclosed in section 6.4.1.

[0147] 6.4.1. IFNAR1 section

[0148] IFNAR1 is a low-affinity IFN receptor and belongs to the type II helical cytokine receptor family. 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).

[0149] The human IFNAR1 sequence has the UniProt identifier P17181. The human IFNAR1 sequence is replicated as follows:

[0150] MMVVLLGATTLVLVAVAPWVLSAAAGG KNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNFSSLKLNVYEEIKLRIRAEKENTSSWYEVDSFTPFRKAQ IGPPEVHLEAEDKAIVI HISPGTKDSVMWALDGLSFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALLTSWKIGVYSPVHC IKTTVEN ELPPPENIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQafllppvfnirslsdsfhiyigapkqsgntpviqdypliyeiifwentsnaerkiiekktdvt vpnlkpltvycvkarahtmdeklnkssvfsdavcektkpgNTSKIWLIVGICIALFALPFVIYAAKVFLRCINYVFFPSLKPSSSIDEYFSEQPLKNLLLSTSEEQIEKCFIIENISTIATVEETNQTDEDHKKYSSQTSQDSGNYSNEDESESKTSEELQQDFV (SEQ ID NO: 22)

[0151] 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 (italic) 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.

[0152] The IFNAR1 moiety comprises an amino acid sequence having 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) with respect to the IFN-binding moiety of mammalian (e.g., human) IFNAR1. In some embodiments, the IFN-binding moiety comprises an SD2 domain and an SD3 domain. In various embodiments, the IFN binding portion includes (i) 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.

[0153] 6.5. Connector

[0154] In some aspects, this disclosure provides IFN receptor antagonists wherein two or more components of the IFN receptor antagonist are linked to each other via peptide linkers. By way of example and not limitation, linkers can be used to connect a separating portion (e.g., an Fc domain) and an anchoring portion (e.g., a targeting portion), different domains within the anchoring portion (e.g., the VH and VL domains in scFv), a separating domain (e.g., an Fc domain) and an IFN or IFNR1 portion, or an IFN portion and an IFNR1 portion.

[0155] Preferably, all linkers in the IFN receptor are non-cleavable linkers (NCLs).

[0156] The length of the peptide linker can range from 2 amino acids to 60 or more amino acids, and in some respects, the length of the peptide linker ranges from 3 amino acids to 50 amino acids, 4 amino acids to 30 amino acids, 5 amino acids to 25 amino acids, 10 amino acids 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.

[0157] In certain respects, the length of the peptide linker is at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids, and optionally, the length is at most 30 amino acids, at most 40 amino acids, at most 50 amino acids, or at most 60 amino acids.

[0158] In some of the foregoing embodiments, the length of the peptide linker ranges from 5 to 50 amino acids, for example, from 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, or 5 to 20 amino acids. In other foregoing embodiments, the length of the linker ranges from 6 to 50 amino acids, for example, from 6 to 50, 6 to 45, 6 to 40, 6 to 35, 6 to 30, 6 to 25, or 6 to 20 amino acids. In some of the foregoing embodiments, the length of the linker ranges from 7 to 50 amino acids, for example, from 7 to 50, 7 to 45, 7 to 40, 7 to 35, 7 to 30, 7 to 25, or 7 to 20 amino acids.

[0159] Electrically charged (e.g., electrically charged hydrophilic connectors) and / or flexible connectors are particularly preferred.

[0160] Examples of flexible linkers that can be used in the IFN receptor antagonists of this 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 linkers are or contain repeats of glycine and serine, such as G... n S (SEQ ID NO:106) or SG n The monomer or polymer of (SEQ ID NO: 107), where n is an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the non-lytic linker is or contains a monomer or polymer of repeating G4S (SEQ ID NO: 108), for example, (GGGGS). n (SEQ ID NO: 108).

[0161] The polyglycine-independent linker may be suitably used in the IFN receptor antagonists of this disclosure. In some embodiments, the peptide-independent linker comprises two consecutive glycines (2 Gly), three consecutive glycines (3 Gly), four consecutive glycines (4 Gly) (SEQ ID NO:109), five consecutive glycines (5 Gly) (SEQ ID NO:110), six consecutive glycines (6 Gly) (SEQ ID NO:111), seven consecutive glycines (7 Gly) (SEQ ID NO:112), eight consecutive glycines (8 Gly) (SEQ ID NO:113), or nine consecutive glycines (9 Gly) (SEQ ID NO:114).

[0162] Exemplary connector sequences are listed in Table E below.

[0163]

[0164] In some aspects, the IFN receptor antagonists of this disclosure may comprise a polypeptide chain comprising a targeting moiety (or targeting moiety chain), a hinge domain, and an Fc domain oriented from the N-terminus to the C-terminus. Thus, the hinge domain may be said to constitute a type of connector. Exemplary hinge domains are described in Section 6.7.1.3.

[0165] 6.6. Anchoring Part

[0166] The IFN receptor antagonists disclosed herein preferably include one or more anchoring moieties. The incorporation of the anchoring moieties allows the IFN receptor antagonists to be anchored to cells expressing type I interferon receptors, enabling the IFN moieties to bind to the type I interferon receptors and inhibit IFN signaling.

[0167] As a component of an IFN receptor antagonist, the anchoring moiety can be any molecule that binds to a molecule on the surface of a cell expressing a type I interferon receptor. In some respects, the anchoring moiety is a “cell surface protein-binding molecule,” which refers to any molecule capable of binding to a protein present or expressed on the cell surface. Some anchoring moieties considered herein include, for example, ligands that bind to cell surface ligands, receptors or ligand-binding moieties thereof, cell surface protein-binding antibodies or fragments thereof, and lipid-binding antibodies or fragments thereof.

[0168] In some respects, the anchoring portion binds to molecules on the surface of a specific cell or cell type, thereby allowing the IFN portion to both bind to type I interferon receptors and target a specific cell type (e.g., cancer cells, immune cells, etc.). Therefore, in some embodiments, the anchoring portion of this disclosure is a targeting portion.

[0169] Exemplary anchoring portions of this disclosure are further described below.

[0170] 6.6.1. Targeted Component

[0171] In certain embodiments, the anchoring portion of this disclosure is the targeting portion. Any type of target molecule contemplated to be present in an IFN receptor antagonist or capable of driving an IFN receptor antagonist to cells expressing type I interferon receptors may be targeted by the IFN receptor antagonist of this disclosure. In some embodiments, the IFN receptor antagonist is intended to treat cancer, for example, by reducing local autoimmune responses associated with oncolytic virus therapy. Therefore, the targeting molecule may be a tumor-associated antigen-targeting molecule, a checkpoint inhibitor-targeting molecule, or a molecule that targets the cell surface of tumor or viral lymphocytes. In some other embodiments, the IFN receptor antagonist is intended to treat autoimmune inflammatory diseases, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), for example, by reducing local or tissue-specific autoimmune responses. Therefore, the targeting molecule may be an immune cell-targeting molecule, such as a T-cell-targeting molecule, a B-cell-targeting molecule, a dendritic cell-targeting molecule, an antigen-presenting cell-targeting molecule, or a natural killer cell-targeting molecule.

[0172] Target molecules recognized by the targeting portion of the IFN receptor antagonist of this disclosure are typically present, 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, or on the surface of other diseased cells. In various embodiments, the target molecules are tumor-reactive lymphocyte antigens, cell surface molecules of tumor or viral lymphocytes, T-cell antigens (TCAs), checkpoint inhibitors, tumor-associated antigens (TAAs), dendritic cell (DC) or other antigen-presenting cell (APC) antigens, or natural killer (NK) cell antigens. Those skilled in the art will recognize that the foregoing categories of target molecules are not mutually exclusive, and therefore a given target molecule may fall into more than one of the foregoing categories. For example, some molecules may be considered to act as both TCAs and checkpoint inhibitors.

[0173] Exemplary cancer types that can be targeted include acute lymphoblastic leukemia, acute myeloid leukemia, cholangiocarcinoma, B-cell leukemia, B-cell lymphoma, cholangiocarcinoma, bone cancer, brain cancer, breast cancer, triple-negative breast cancer, cervical cancer, Burkitt lymphoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastric cancer, gastrointestinal cancer, glioma, hairy cell leukemia, head and neck cancer, Hodgkin's lymphoma, liver cancer, lung cancer, medullary thyroid carcinoma, melanoma, multiple myeloma, ovarian cancer, non-Hodgkin's 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 virtually any type of cancer.

[0174] Other target molecules are cell surface molecules of tumor or viral lymphocytes, such as T cell costimulatory proteins like CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0175] 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 some embodiments, the target molecule is PDL1.

[0176] Exemplary immune cells that can be targeted include, but are not limited to, T cells (e.g., cytotoxic T cells, regulatory T cells, etc.), B cells, dendritic cells, natural killer (NK) cells, NKT cells, macrophages, and other antigen-presenting cells.

[0177] In some embodiments, the target molecules are 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.

[0178] In a further embodiment, the target molecules are 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.

[0179] A suitable target portion format is described in Section 6.6.1.1. The target portion is preferably an antigen-binding portion, such as an antibody or an antigen-binding portion of an antibody, for example, scFv as described in Section 6.6.1.1.2 or Fab as described in Section 6.6.1.1.1.

[0180] In some embodiments, the targeting portion targets the exemplary target molecules listed in Table F below, while also referring to the exemplary antibody or antibody sequence on which the targeting portion may be based.

[0181]

[0182] In some aspects, the targeting portion competes with the antibodies listed in Table F for binding to the target molecule. In a further aspect, the targeting portion comprises a CDR having the CDR sequence of the antibodies listed in Table F. In some embodiments, the targeting portion comprises all six CDR sequences of the antibodies listed in Table F. In other embodiments, the targeting portion comprises at least the heavy chain CDR sequence (CDR-H1, CDR-H2, CDR-H3 and the light chain CDR sequence of the universal light chain). In a further aspect, the targeting portion comprises a VH that comprises the amino acid sequence of the VH of the antibody shown in Table F. In some embodiments, the targeting portion also comprises a VL that comprises the amino acid sequence of the VL of the antibody listed in Table F. In other embodiments, the targeting portion further comprises a universal light chain VL sequence.

[0183] In some embodiments, the targeting portion targets the PDL1 listed in Table F-1 below, while also referencing exemplary antibodies or antibody sequences on which the targeting portion may be based.

[0184]

[0185] In some aspects, the targeting portion competes with the antibodies listed in Table F-1 for binding to the target molecule. In a further aspect, the targeting portion comprises a CDR having the CDR sequence of the antibody listed in Table F-1. In some embodiments, the targeting portion comprises all six CDR sequences of the antibody listed in Table F. In other embodiments, the targeting portion comprises at least the heavy chain CDR sequence (CDR-H1, CDR-H2, CDR-H3 and the light chain CDR sequence of the universal light chain). In a further aspect, the targeting portion comprises a VH containing the amino acid sequence of the VH of the antibody listed in Table F-1. In some embodiments, the targeting portion further comprises a VL containing the amino acid sequence of the VL of the antibody listed in Table F-1. In other embodiments, the targeting portion further comprises a universal light chain VL sequence.

[0186] In some embodiments, the targeting portion targets PD1 listed in Table F-2 below, while also referring to exemplary antibodies or antibody sequences on which the targeting portion may be based.

[0187]

[0188] In some aspects, the targeting portion competes with the antibodies listed in Table F-2 for binding to the target molecule. In a further aspect, the targeting portion comprises a CDR having the CDR sequence of the antibody listed in Table F-2. In some embodiments, the targeting portion comprises all six CDR sequences of the antibody listed in Table F. In other embodiments, the targeting portion comprises at least the heavy chain CDR sequence (CDR-H1, CDR-H2, CDR-H3 and the light chain CDR sequence of the universal light chain). In a further aspect, the targeting portion comprises a VH that includes the amino acid sequence of the VH of the antibody listed in Table F-2. In some embodiments, the targeting portion further comprises a VL that includes the amino acid sequence of the VL of the antibody listed in Table F-2. In other embodiments, the targeting portion further comprises a universal light chain VL sequence.

[0189] Additional target molecules that can be targeted by IFN receptor antagonists are disclosed in Table I below and, for example, in Hafeez et al., 2020, Molecules 25:4764, doi:10.3390 / molecules25204764 (especially in Table 1). The entire contents of Table 1 by Hafeez et al. are incorporated herein by reference.

[0190] 6.6.1.1. Targeted Part Format

[0191] In some aspects, the targeting portion of the IFN receptor antagonist of this disclosure can be any type of antibody or fragment thereof that retains specific binding to an antigenic determinant. In one embodiment, the targeting portion is an immunoglobulin molecule or fragment thereof, particularly IgG class immunoglobulin molecules, more particularly IgG1 or IgG4 immunoglobulin molecules. 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, microantibodies, biantibodies, triantibodies, and tetraantibodies.

[0192] 6.6.1.1.1.Fab

[0193] Fab domains are traditionally produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. Fab domains can contain constant domains and variable region sequences from any suitable species, and therefore can be mouse, chimeric, human, or humanized.

[0194] The Fab domain typically contains a CH1 domain attached to the VH domain, which pairs with a CL domain attached to the VL domain. In wild-type immunoglobulins, the VH and VL domains pair to form the Fv region, and the CH1 and CL domains pair to further stabilize the binding site. Disulfide bonds between the two constant domains further stabilize the Fab domain.

[0195] For the IFN receptor antagonists of this disclosure, particularly when the light chains of the target moieties are not common or universal light chains, it is advantageous to use a Fab heterodimerization strategy to allow proper association of Fab domains belonging to the same target moieties and minimize anomalous pairing of Fab domains belonging to different target moieties. For example, the Fab heterodimerization strategy shown in Table G below can be used:

[0196] Therefore, in some embodiments, proper association between two peptides of Fab is facilitated by exchanging the VL and VH domains of Fab with each other or by exchanging the CH1 and CL domains with each other, for example, as described in WO 2009 / 080251.

[0197] Proper Fab pairing can also be facilitated by introducing one or more amino acid modifications into the CH1 domain of the Fab, one or more amino acid modifications into the CL domain of the Fab, and / or one or more amino acid modifications into the VH domain, and one or more amino acid modifications into the VL domain. The modified amino acids are typically part of the VH:VL and CH1:CL interfaces, causing Fab components to preferentially pair with each other rather than with components from other Fabs.

[0198] In one embodiment, one or more amino acid modifications are limited to conserved framework residues of 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.

[0199] 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 based on spatial and hydrophobic contacts, electrostatic / charge interactions, or combinations of various interactions. Complementarity between protein surfaces is widely described in the literature as lock-and-key mating, pestle-and-mortise, protrusion-and-cavity, donor-and-acceptor, etc., all of which imply a structural and chemical matching property between two interacting surfaces.

[0200] In one embodiment, one or more of the introduced modifications introduce new hydrogen bonds at the interface of the Fab components. In one embodiment, one or more of the introduced modifications introduce new salt bridges across the interface of the Fab components. Exemplary alternatives are described in WO 2014 / 150973 and WO 2014 / 082179, the contents of which are incorporated herein by reference.

[0201] In some embodiments, the Fab domain includes 192E substitution in the CH1 domain and 114A and 137K substitution in the CL domain, which introduces a salt bridge between the CH1 and CL domains (see, for example, Golay et al., 2016, JImmunol 196:3199-211).

[0202] In some embodiments, the Fab domain includes 143Q and 188V substitutions in the CH1 domain and 113T and 176V substitutions in the CL domain, which are used to exchange the hydrophobic and polar regions of the contact between the CH1 and CL domains (see, for example, Golay et al., 2016, J Immunol 196:3199-211).

[0203] In some embodiments, the Fab domains may include modifications in some or all of the VH, CH1, VL, and CL domains to introduce orthogonal Fab interfaces that promote proper assembly of the Fab domains (Lewis et al., 2014 Nature Biotechnology 32:191-198). In one embodiment, 39K and 62E modifications are introduced in the VH domain, H172A and F174G modifications are introduced in the CH1 domain, 1R, 38D, and (36F) modifications are introduced in the VL domain, and L135Y and S176W modifications are introduced in the CL domain. In another embodiment, 39Y modification is introduced in the VH domain and 38R modification is introduced in the VL domain.

[0204] The Fab domain can also be modified to replace the natural CH1:CL disulfide bonds with engineered disulfide bonds, thereby improving the pairing efficiency of Fab components. For example, engineered disulfide bonds can be introduced by introducing 126C into the CH1 domain and 121C into the CL domain (see, for example, Mazor et al., 2015, MAbs 7:377-89).

[0205] The Fab domain can also be modified by replacing the CH1 and CL domains with alternative domains that promote proper assembly. For example, Wu et al., 2015, MAbs 7:364-76, described replacing the CH1 domain with the constant domain of the T cell receptor and the CL domain with the b domain of the T cell receptor, and replacing these domains with additional charge-charge interaction pairs between the VL and VH domains by introducing 38D modification into the VL domain and 39K modification into the VH domain.

[0206] Instead of or alternatively using a Fab heterodimerization strategy to facilitate proper VH-VL pairing, a VL of a common light chain (also referred to as a universal light chain) can be used for each unique ABD in the IFN receptor antagonists of this disclosure. In various embodiments, employing a common light chain as described herein reduces the amount of inappropriate material in the IFN receptor antagonist compared to employing a pristine homologous VL. In various embodiments, the VL domain of the ABD is identified from monospecific antibodies containing the common light chain. In various embodiments, the VH region of the ABD in the IFN receptor antagonist contains variable gene segments of human heavy chain that are rearranged in vivo in mouse B cells previously engineered to express a limited library of human light chains, or a single human light chain homologous to human heavy chains, and to generate an antibody library containing multiple human VHs homologous to one or two possible human VLs, wherein the antibody library is specific to the antigen of interest, in response to exposure to an antigen of interest. Common light chains are derived from rearranged human Vκ1-39Jκ5 sequences or rearranged human Vκ3-20Jκ1 sequences, and include somatic mutant (e.g., affinity maturation) versions. See, for example, U.S. Patent No. 10,412,940.

[0207] 6.6.1.1.2.scFv

[0208] Single-chain Fv or “scFv” antibody fragments contain the VH and VL domains of the antibody within a single polypeptide chain, enabling them to be expressed as a single polypeptide chain while retaining the specificity of the complete antibody from which they originated. Generally, scFv polypeptides further include a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for target binding. Examples of linkers suitable for connecting the VH and VL chains of scFv are the linkers identified in Section 6.5.

[0209] Unless otherwise stated, as used herein, scFv may have VL and VH variable regions in either order, for example, scFv may contain VL-connector-VH or VH-connector-VL relative to the N-terminus and C-terminus of the polypeptide.

[0210] scFv can contain VH and VL sequences from any suitable species, such as mice, humans, or humanized VH and VL sequences.

[0211] To generate nucleic acids encoding scFv, DNA fragments encoding VH and VL are operatively ligated to another fragment encoding a linker, for example, any linker described in Section 6.5 (typically a repeat of a sequence containing the amino acids glycine and serine, such as the amino acid sequence (Gly4~Ser)3 (SEQ ID NO: 50), such that the VH and VL sequences can be expressed as a continuous single-stranded protein, with the VL and VH regions joined by a flexible linker (see, for example, 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).

[0212] 6.7. Separating Parts

[0213] The IFN receptor antagonists disclosed herein include one or more separatory moieties. As components of the IFN receptor antagonist, the separatory moieties can be any molecule capable of simultaneously binding both the anchoring moieties and the IFN moieties. Therefore, the separatory moieties have specific characteristics (e.g., size, shape, spatial properties, etc.) to provide sufficient separation between the anchoring moieties and the IFN moieties for each moieties to bind to their respective targets. The separatory moieties can be, for example, organic polymers or peptides.

[0214] In addition to being able to simultaneously bind both the anchoring portion and the IFN portion, the separator may also have one or more additional properties. For example, in some embodiments, the separator is a polymerizable portion. "Polymerizable portion" describes any polypeptide or other molecule or portion thereof capable of polymerization (e.g., dimerization). Such polymerization includes, for example, the non-covalent association of two or more polymerizable portions. Various polymerizable portions are well-known in the art and are described herein.

[0215] Exemplary separations of this disclosure are further described below.

[0216] 6.7.1. Fc structural domain and Fc region

[0217] The IFN receptor antagonists disclosed herein typically comprise a pair of Fc domains that associate to form an Fc region. In natural antibodies, the Fc region includes a hinge region at its N-terminus to form a constant domain. Throughout this disclosure, unless otherwise stated, references to Fc domains cover Fc domains having a hinge domain at their N-terminus.

[0218] The Fc domain can be derived from any suitable species that is operatively linked to ABD or a component thereof. In one embodiment, the Fc domain is derived from a human Fc domain. In a preferred embodiment, the targeting portion or a component thereof is fused to an IgG Fc molecule. The targeting portion or a component thereof may be fused to the N-terminus or C-terminus or both of the IgG Fc domain.

[0219] The Fc domain can be derived from any suitable class of antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In one embodiment, the Fc domain is derived from IgG1, IgG2, IgG3, or IgG4. In one embodiment, the Fc domain is derived from IgG1. In one embodiment, the Fc domain is derived from IgG4.

[0220] The two Fc domains within the Fc region may be identical or different from each other. In natural antibodies, the Fc domains are usually identical, but in order to generate multispecific binding molecules, such as the IFN receptor antagonists of this disclosure and the MBM generated by their activation, the Fc domains may advantageously be different to allow heterodimerization, as described in Section 6.7.1.2 below.

[0221] 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.

[0222] In the IFN receptor antagonists disclosed herein, the Fc region and / or the Fc domain within it may contain heavy chain constant domains from one or more different classes (e.g., one, two, or three different classes) of antibodies.

[0223] In one embodiment, the Fc region contains CH2 and CH3 domains derived from IgG1.

[0224] In one embodiment, the Fc region contains CH2 and CH3 domains derived from IgG2.

[0225] In one embodiment, the Fc region includes CH2 and CH3 domains derived from IgG3.

[0226] In one embodiment, the Fc region contains CH2 and CH3 domains derived from IgG4.

[0227] In one embodiment, the Fc region contains a CH4 domain from IgM. The IgM CH4 domain is typically located at the C-terminus of the CH3 domain.

[0228] In one embodiment, the Fc region includes CH2 and CH3 domains derived from IgG and a CH4 domain derived from IgM.

[0229] It should be understood that the heavy chain constant domain of the Fc region used to generate the IFN receptor antagonist of this disclosure may include variants of the aforementioned naturally occurring constant domain. Such variants may contain one or more amino acid variations compared to the wild-type constant domain. In one instance, the Fc region of this disclosure contains at least one constant domain that is sequence-different 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 instance, the variant constant domain is at least 70% identical or similar. In another instance, the variant constant domain is at least 80% identical or similar. In another instance, the variant constant domain is at least 90% identical or similar. In yet another instance, the variant constant domain is at least 95% identical or similar.

[0230] IgM and IgA are naturally occurring covalent polymers of common H2L2 antibody units in the human body. When IgM is incorporated into the J chain, it appears as a pentamer; or when the J chain is absent, it appears as a hexamer. IgA appears as both a monomer and a dimer. The heavy chains of IgM and IgA have 18 amino acids extending to a constant C-terminal domain, called a tail. The tail contains cysteine ​​residues, which form disulfide bonds between the heavy chains of the polymer and are considered to play an important role in polymerization. The tail also contains glycosylation sites. In some embodiments, the IFN receptor antagonists of this disclosure do not contain a tail.

[0231] The Fc domain incorporated into the IFN receptor antagonists of this disclosure may include one or more modifications that alter the functional properties of the protein, such as binding to Fc receptors like FcRn or leukocyte receptors, binding to complement, a modified disulfide bond system architecture, or an altered glycosylation pattern. Exemplary Fc modifications that alter effector function are described in Section 6.7.1.1.

[0232] The Fc domain can also be modified to include, for example, modifications that improve the manufacturability of asymmetric IFN receptor antagonists by allowing heterodimerization, which is the preferential pairing of dissimilar Fc domains relative to identical Fc domains. Heterodimerization allows the generation of IFN receptor antagonists in which different polypeptide components are linked to each other through Fc regions containing Fc domains that are sequence-dissimilar. Examples of heterodimerization strategies are illustrated in Section 6.7.1.2.

[0233] It should be understood that any of the above modifications can be combined in any suitable manner to achieve the desired functional properties and / or combined with other modifications to alter the properties of the IFN receptor antagonist.

[0234] Example Fc structure domain sequences are provided in Table C below.

[0235]

[0236] 6.7.1.1. Fc domain with altered effector function

[0237] In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptor and / or effector function.

[0238] In a particular 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 particular 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 selected from one or more of the group consisting of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and cytokine secretion. In a particular embodiment, the effector function is ADCC.

[0239] In one embodiment, the Fc domain (e.g., the Fc domain of an IFN receptor antagonist hapten) or the Fc region (e.g., one or both Fc domains of an IFN receptor antagonist that can associate to form an Fc region) contains an amino acid substitution at a position selected from the group consisting of E233, L234, L235, N297, P331, and P329 (according to Kabat EU index numbers). In a more specific embodiment, the Fc domain or Fc region contains an amino acid substitution at a position selected from L234, L235, and P329 (according to Kabat EU index numbers). In some embodiments, the Fc domain or Fc region contains amino acid substitutions L234A and L235A (according to Kabat EU index numbers). In one such embodiment, the Fc domain or region is an Igd Fc domain or region, particularly a human Igd Fc domain or region. In one embodiment, the Fc domain or Fc region contains an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (according to the Kabat EU index number). In one embodiment, the Fc domain or Fc region contains an amino acid substitution at position P329 and further amino acid substitutions at positions selected from E233, L234, L235, N297, and P331 (according to the Kabat EU index number). In a more specific embodiment, the further amino acid substitutions are E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a specific embodiment, the Fc domain or Fc region contains amino acid substitutions at positions P329, L234, and L235 (according to the Kabat EU index number). In a more specific embodiment, the Fc domain contains amino acid mutations L234A, L235A, and P329G (“P329G LALA”, “PGLALA”, or “LALAPG”).

[0240] Typically, each of the two Fc domains in the Fc region contains the same one or more amino acid substitutions. Therefore, in a specific embodiment, each Fc domain in the Fc region contains amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbers), meaning that 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) (according to the Kabat EU index number).

[0241] In one embodiment, the Fc domain is the IgG1 Fc domain, particularly the human IgG1 Fc domain. In some embodiments, the IgG1 Fc domain is a variant IgG1 containing D265A, N297A mutations (EU number) to reduce effector function.

[0242] In another embodiment, the Fc domain is an IgG4 Fc domain that reduces binding to the Fc receptor. An exemplary IgG4 Fc domain with reduced binding to the Fc receptor may comprise an amino acid sequence selected from Table H below. In some embodiments, the Fc domain includes only the bolded portion of the sequence shown below:

[0243]

[0244] In a specific embodiment, the reduced effector function IgG4 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.

[0245] For the heterodimer Fc region, combinations of the above-mentioned variant IgG4 Fc sequences can be incorporated, such as an Fc region containing an Fc domain of the amino acid sequence (or its bolded portion) of SEQ ID NO:30 of WO2014 / 121087 and an Fc domain containing an amino acid sequence (or its bolded portion) of SEQ ID NO:37 of WO2014 / 121087, or an Fc region containing an Fc domain of the amino acid sequence (or its bolded portion) of SEQ ID NO:31 of WO2014 / 121087 and an Fc domain containing an amino acid sequence (or its bolded portion) of SEQ ID NO:38 of WO2014 / 121087.

[0246] 6.7.1.2. Fc heterodimer variants

[0247] Some IFN receptor antagonists require dimerization between two Fc domains, which, unlike natural immunoglobulins, are operatively linked to non-identical N-terminal or C-terminal regions. Insufficient heterodimerization of the two Fc domains to form the Fc region can be a barrier to increasing the yield of the desired heterodimer 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 antagonists of this 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.

[0248] In some embodiments, this disclosure provides an IFN receptor antagonist comprising an Fc heterodimer, i.e., an Fc region comprising a heterologous, non-identical Fc domain. Typically, each Fc domain in the Fc heterodimer comprises the CH3 domain of an antibody. The CH3 domain is derived from a constant region of any isotype, class, or subclass of antibody, and preferably from a constant region of IgG (IgG1, IgG2, IgG3, and IgG4) class antibodies, as described in the previous section.

[0249] Heterodimerization of two different heavy chains at the CH3 domain produces the desired IFN receptor antagonist, while homodimerization of the same heavy chain reduces the yield of the desired IFN receptor antagonist. Therefore, in a preferred embodiment, the polypeptide that associates to form the IFN receptor antagonist of this disclosure will contain a CH3 domain modified relative to the unmodified Fc domain to favor heterodimeric association.

[0250] In specific embodiments, the modification that promotes Fc heterodimer formation is a so-called "mortar and pestle" modification, comprising a "mortar" modification in one Fc domain and a "pestle" modification in the other Fc domain. Mortar and pestle techniques are described, for example, in U.S. Patent Nos. 5,731,168; US 7,695,936; Ridgway et al., 1996, Prot Eng 9:617-621; and Carter, 2001, Immunol Meth 248:7-15. Generally, the method involves introducing a protrusion ("pestle") at the interface of a first polypeptide and a corresponding cavity ("mortar") at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity, thereby promoting heterodimer formation and inhibiting homodimer formation. The protrusion is constructed by replacing the small amino acid side chains from the first polypeptide interface with larger side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensating cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide.

[0251] Therefore, in some embodiments, amino acid residues in the CH3 domain of the first subunit of the Fc domain are substituted with amino acid residues having a larger side chain volume, thereby creating a protrusion within the CH3 domain of the first subunit. This protrusion can be positioned in a cavity within the CH3 domain of the second subunit. Conversely, amino acid residues in the CH3 domain of the second subunit of the Fc domain are substituted with amino acid residues having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit. The protrusion within the CH3 domain of the first subunit can be positioned within this cavity. Preferably, the amino acid residues with a larger side chain volume are selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residues with a smaller side chain volume are selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protrusion and cavity can be prepared by altering the nucleic acid encoding the polypeptide, for example, through site-specific mutagenesis or peptide synthesis. An exemplary substitution is Y470T.

[0252] In a specific embodiment of this kind, 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). 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) (according to the Kabat EU index number). 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 glutamate 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) (according to the Kabat EU index number). In a specific embodiment, the first Fc domain contains amino acid substitutions S354C and T366W, and the second Fc domain contains amino acid substitutions Y349C, T366S, L368A, and Y407V (according to Kabat EU index numbers).

[0253] In some implementations, electrostatic redirection (e.g., as described in Gunasekaran et al., 2010, J Biol Chem285(25): 19637-46) can be used to facilitate the bonding of the first and second Fc domains in the Fc region.

[0254] As an alternative or addition to using a modified Fc domain to promote heterodimerization, the Fc domain can be modified to allow for selective purification strategies for Fc heterodimers. In one such embodiment, a polypeptide comprises a modified Fc domain that eliminates its binding to protein A, thereby enabling purification methods that produce heterodimeric proteins. See, for example, U.S. Patent No. 8,586,713. Thus, an IFN receptor antagonist 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 antagonist to protein A compared to a corresponding IFN receptor antagonist lacking this amino acid difference. In one embodiment, the first CH3 domain binds to protein A and the second CH3 domain contains a mutation / modification that reduces or eliminates protein A binding, such as an H95R modification (via IMGT exon numbering; H435R via EU numbering). The second CH3 may further comprise a Y96F modification (via IMGT; Y436F via EU numbering). This type of modification is referred to as the "star" mutation in this paper.

[0255] In some embodiments, Fc may contain one or more mutations (e.g., pestle and mortar mutations) to promote heterodimerization and star mutations to promote purification.

[0256] 6.7.1.3. Hinge Domain

[0257] The IFN receptor antagonists disclosed herein may include an Fc domain comprising a hinge domain at its N-terminus. The hinge region may be natural or modified. 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 naturally occurring or non-naturally occurring hinge sequence that, in the context of a single or monomeric polypeptide chain, is a monomeric hinge domain and, in the context of a dimeric polypeptide (e.g., a homodimeric or heterodimeric IFN receptor antagonist formed by the association of two Fc domains), may comprise two associated hinge sequences located on a separate polypeptide chain. Sometimes, two associated hinge sequences are referred to as "hinge regions." In some embodiments of the IFN receptor antagonist, additional iterations of the hinge region may be incorporated into the polypeptide sequence.

[0258] A natural hinge region is a hinge region typically found between the Fab and Fc domains of a naturally occurring antibody. A modified hinge region is any hinge that differs from the natural hinge region in length and / or composition. Such hinges can include hinge regions derived from other species, such as those of humans, mice, rats, rabbits, sharks, pigs, hamsters, camels, llamas, or goats. Other modified hinge regions may comprise complete hinge regions derived from antibodies of a different class or subclass than the heavy chain Fc domain or Fc region. Alternatively, a modified hinge region may comprise a portion of a natural hinge or repeating unit, where each unit in the repeat originates from the natural hinge region. In another alternative, the natural hinge region can be altered by converting one or more cysteine ​​or other residues to neutral residues, such as serine or alanine, or by converting appropriately placed residues to cysteine ​​residues. In this way, the number of cysteine ​​residues in the hinge region can be increased or decreased. Other modified hinge regions can be fully synthetic and can be designed to have desired properties, such as length, cysteine ​​composition, and flexibility.

[0259] Many modified hinge areas have been described in, for example, U.S. Patent Nos. 5,677,425, WO 99 / 15549, WO2005 / 003170, WO 2005 / 003169, WO 2005 / 003170, WO 98 / 25971 and WO 2005 / 003171, and these contents are incorporated herein by reference.

[0260] In one embodiment, the IFN receptor antagonist of this disclosure comprises an Fc region, wherein one or both Fc domains have a fully hinged domain at their N-terminus.

[0261] In various embodiments, positions 233-236 within the hinge area can be G, G, G and unoccupied; G, G, unoccupied and unoccupied; G, unoccupied, unoccupied and unoccupied; or all unoccupied, wherein the positions are numbered by EU numbering.

[0262] In some embodiments, the IFN receptor antagonists of this disclosure comprise a modified hinge region that reduces the binding affinity to the Fcγ receptor relative to the wild-type hinge region of the same isotype (e.g., human IgG1 or human IgG4).

[0263] In one embodiment, the IFN receptor antagonist of this disclosure comprises an Fc region, wherein each Fc domain has a fully hinged domain at its N-terminus, wherein each Fc domain and hinged domain is derived from IgG4 and each hinged domain contains the modified sequence CPPC (SEQ ID NO: 121). Compared to IgG1 containing the sequence CPPC (SEQ ID NO: 121), the core hinge region of human IgG4 contains the sequence CPSC (SEQ ID NO: 122). The presence of serine residues in the IgG4 sequence increases the flexibility of this region, allowing a portion of the molecule to form disulfide bonds within the same protein chain (intra-chain disulfide bonds) rather than bridging to other heavy chains in the IgG molecule to form inter-chain disulfide bonds (Angel et al., 1993, Mol Immunol 30(1):105-108). Replacing serine residues with proline to obtain the same core sequence as IgG1 allows for complete inter-chain disulfide bond formation in the IgG4 hinge region, thereby reducing heterogeneity in the purified product. This modified isotype is referred to as IgG4P.

[0264] 6.7.1.3.1. Interlocking Hinge Sequence

[0265] The hinge structure domain can be a mating hinge structure domain.

[0266] For example, the chimeric hinge may include an “upper hinge” sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4, which is combined with a “lower hinge” sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4.

[0267] In certain embodiments, the chimeric hinge region comprises the amino acid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 123; previously disclosed as SEQ ID NO: 8 of WO2014 / 121087, which is incorporated herein by reference in its entirety) or ESKYGPPCPPCPAPPVA (SEQ ID NO: 124; previously disclosed as SEQ ID NO: 9 of WO2014 / 121087). Such chimeric hinge sequences may suitably be linked to the IgG4 CH2 region (e.g., by incorporating the IgG4 Fc domain, such as the human or mouse Fc domain, which may be further modified in the CH2 and / or CH3 domains to reduce effector function, for example as described in Section 6.7.1.1).

[0268] 6.7.1.3.2. Hinge sequences with reduced effector functionality

[0269] In further embodiments, the hinge region may be modified to reduce the effector function, for example as described in WO2016161010A2, the entirety of which is incorporated herein by reference. In various embodiments, the modified hinge region positions 233-236 are G, G, G and unoccupied; G, G, unoccupied and unoccupied; G, unoccupied, unoccupied and unoccupied; or all unoccupied, wherein the positions are numbered by EU numbering (as shown in Figure 1 of WO2016161010A2). These segments may be represented as GGG-, GG--, G---, or ----, where "-" indicates an unoccupied position.

[0270] Position 236 is not occupied in canonical human IgG2, but is occupied in other canonical human IgG isoforms. In all four human isoforms, positions 233-235 are occupied by residues other than G (as shown in Figure 1 of WO2016161010A2).

[0271] The hinge modification at 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, 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.

[0272] The exemplary hinge region has residues 226-236, sometimes referred to as the middle (or core) and lower hinge, occupied by modified hinge sequences designated as GGG-(233-236), GG--(233-236), G---(233-236), and without G(233-236). Optionally, the hinge domain amino acid sequence comprises CPCPAPGGG-GPSVF (SEQ ID NO:125; previously disclosed as WO2016161010A2, SEQ ID NO:1), CPCPAPGG--GPSVF (SEQ ID NO:126; previously disclosed as WO2016161010A2, SEQ ID NO:2), CPCPAPG---GPSVF (SEQ ID NO:127; previously disclosed as WO2016161010A2, SEQ ID NO:3), or CPCPAP----GPSVF (SEQ ID NO:128; previously disclosed as WO2016161010A2, SEQ ID NO:4).

[0273] The modified hinge region described above can be incorporated into the heavy chain constant region, which typically includes CH2 and CH3 domains, and may have additional hinge segments (e.g., upper hinges) located on the flanks of the designated region. Such additional constant region segments generally have the same isotype, preferably the human isotype, although they may be hybrids of different isotypes. The isotype of such additional human constant region segments is preferably human IgG4, but may also be human IgG1, IgG2, or IgG3, or hybrids of their domains with different isotypes. Exemplary sequences of human IgG1, IgG2, and IgG4 are shown in Figures 2 through 4 of WO2016161010A2.

[0274] In a particular embodiment, the modified hinge sequence may be linked to the IgG4 CH2 region (e.g., by incorporating an IgG4 Fc domain, such as a human or mouse Fc domain, which may be further modified in the CH2 and / or CH3 domains to reduce effector function, as described in Section 6.7.1.1).

[0275] 6.8. Nucleic Acids and Host Cells

[0276] In another aspect, this disclosure provides nucleic acids encoding the IFN receptor antagonists of this disclosure. In some embodiments, the IFN receptor antagonist is encoded by a single nucleic acid. In other embodiments, the IFN receptor antagonist may be encoded by multiple (e.g., two, three, four or more) nucleic acids.

[0277] A single nucleic acid may encode an IFN receptor antagonist containing a single polypeptide chain, an IFN receptor antagonist containing two or more polypeptide chains, or a portion of an IFN receptor antagonist containing two or more polypeptide chains (e.g., a single nucleic acid may encode two polypeptide chains of an IFN receptor antagonist containing three, four, or more polypeptide chains, or three polypeptide chains of an IFN receptor antagonist containing four or more polypeptide chains). For individual control of expression, open reading frames encoding two or more polypeptide chains may be under the control of individual transcriptional regulatory elements (e.g., promoters and / or enhancers). Open reading frames encoding two or more polypeptides may also be controlled by the same transcriptional regulatory element and separated by internal ribosome entry site (IRES) sequences, thereby allowing translation into individual polypeptides.

[0278] In some embodiments, an IFN receptor antagonist comprising two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding the IFN receptor antagonist may be equal to or less than the number of polypeptide chains in the IFN receptor antagonist (e.g., when more than one polypeptide chain is encoded by a single nucleic acid).

[0279] The nucleic acids in this disclosure can be DNA or RNA (e.g., mRNA).

[0280] In another aspect, this disclosure provides host cells and vectors containing the nucleic acids of this disclosure. The nucleic acids may be present in a single vector or in a single vector within the same host cell or a separate host cell, as described in more detail below.

[0281] 6.8.1. Carrier

[0282] This disclosure provides vectors comprising a nucleotide sequence encoding a nucleotide sequence of an IFN receptor antagonist or a component thereof, such as one or both polypeptide chains of a polypeptide chain of a hapten of an IFN receptor antagonist. Vectors include, but are not limited to, viruses, plasmids, viscera, λ phages, or yeast artificial chromosomes (YACs).

[0283] Various vector systems can be used. For example, one type of vector utilizes DNA elements derived from animal viruses, such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rouse sarcoma virus, MMTV, or MOMLV), or SV40 virus. Another type of vector utilizes RNA elements derived from RNA viruses, such as Semleeki Forest virus, Eastern Equine Encephalitis Virus, and Flavivirosis.

[0284] Additionally, cells that have stably integrated their DNA into their chromosomes can be selected by introducing one or more markers that allow selective transfection of host cells. Markers can provide, for example, tropism against auxotrophic hosts, resistance to biocides (e.g., antibiotics), or resistance to heavy metals (such as copper). The selective marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell via co-transformation. Optimal mRNA synthesis may require additional elements. These elements may include splicing signals, as well as transcription promoters, enhancers, and termination signals.

[0285] Once the expression vector or DNA sequence containing the construct is prepared, it can be transfected or introduced into a suitable host cell. This can be achieved using various techniques, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. The methods and conditions used to culture the resulting transfected cells and to recover the expressed peptide are known to those skilled in the art and can be varied or optimized based on the specific expression vector and mammalian host cells used, according to this specification.

[0286] 6.8.2. Cells

[0287] This disclosure also provides host cells containing nucleic acids from this disclosure.

[0288] In one embodiment, the host cell is genetically engineered to contain one or more nucleic acids as described herein.

[0289] In one embodiment, host cells are genetically engineered using an expression cassette. The phrase "expression cassette" refers to a nucleotide sequence capable of influencing gene expression in a host compatible with such a sequence. Such a cassette may include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or helpful in influencing expression, such as, for example, inducible promoters, may also be used.

[0290] This disclosure also provides host cells containing the vectors described herein.

[0291] The cells can be, but are not limited to, eukaryotic cells, bacterial cells, insect cells, or human cells. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells.

[0292] 6.9. Pharmaceutical Compositions

[0293] The IFN receptor antagonists disclosed herein may be in the form of a composition comprising an IFN receptor antagonist and one or more carriers, excipients, and / or diluents. The composition may be formulated for a specific purpose, such as for veterinary 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 antagonist and, for therapeutic purposes, also on the mode of administration.

[0294] For therapeutic use, the composition may be provided as part of a sterile pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition may be in any suitable form (depending on the method required for its administration to the patient). The pharmaceutical composition may be administered to the patient via a variety of routes, such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, local, or topical. The most appropriate route of administration in any given situation will depend on the specific IFN receptor antagonist, the subject, the nature and severity of the disease, and the subject's physical condition. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

[0295] The pharmaceutical composition can be conveniently available in unit dosage forms containing a predetermined amount of the IFN receptor antagonist of this disclosure per dose. The amount of IFN receptor antagonist contained in a unit dose will depend on the disease being treated and other factors well known in the art. Such unit doses can be in the form of a lyophilized powder containing a predetermined amount of IFN receptor antagonist suitable for a single administration, or in liquid form. The powder unit dosage form can be packaged in a kit with a syringe, a suitable amount of diluent, and / or other components for administration. Liquid unit doses can be conveniently supplied in the form of a syringe pre-filled with a predetermined amount of IFN receptor antagonist suitable for a single administration.

[0296] The pharmaceutical composition can also be supplied in bulk from a certain amount of IFN receptor antagonist suitable for multiple administration.

[0297] Pharmaceutical compositions can be prepared for storage as lyophilized formulations or aqueous solutions by mixing an IFN receptor antagonist of desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (all of which are referred to herein as “carriers”) (i.e., buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, antioxidants, and various other additives) commonly used in the art. See Remington’s Pharmaceutical Sciences, 16th edition (edited by Osol, 1980). Such additives should be non-toxic to the recipient at the dosage and concentration used.

[0298] Buffers help maintain pH values ​​within a range close to physiological conditions. They can be present in a variety of concentrations, but are typically found in concentrations ranging from about 2 mM to about 50 mM. Buffers suitable for use in this disclosure include organic and inorganic acids and their salts, such as citrate buffers (e.g., mixtures of monosodium citrate and disodium citrate, mixtures of citrate and trisodium citrate, mixtures of citrate and monosodium citrate, etc.), succinate buffers (e.g., mixtures of succinate and monosodium succinate, mixtures of succinate and sodium hydroxide, mixtures of succinate and disodium succinate, etc.), tartrate buffers (e.g., mixtures of tartaric acid and sodium tartrate, mixtures of tartaric acid and potassium tartrate, mixtures of tartaric acid and sodium hydroxide, etc.), and fumarate buffers (e.g., mixtures of fumaric acid and monosodium fumarate, etc.). Fumarate-disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, etc., gluconate buffers (e.g., gluconate-sodium gluconate mixtures, gluconate-sodium hydroxide mixtures, gluconate-potassium gluconate mixtures, etc.), oxalate buffers (e.g., oxalate-sodium oxalate mixtures, oxalate-sodium hydroxide mixtures, oxalate-potassium oxalate mixtures, etc.), lactate buffers (e.g., lactate-sodium lactate mixtures, lactate-sodium hydroxide mixtures, lactate-potassium lactate mixtures, etc.), and acetate buffers (e.g., acetate-sodium acetate mixtures, acetate-sodium hydroxide mixtures, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts (such as Tris) can also be used.

[0299] Preservatives may be added to delay microbial growth, and may be added in amounts ranging from about 0.2% to 1% (w / v). Preservatives suitable for use in this disclosure include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyl dimethyl benzyl ammonium chloride, benzalkonium chloride halides (e.g., chlorides, bromides, and iodides), hexamethyl chloride, and alkylparaben esters (such as methylparaben or propylparaben), catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonic agents (sometimes referred to as “stabilizers”) may be added to ensure the isotonicity of the liquid compositions of this disclosure, and isotonic agents include polyols, such as ternary or higher sugar alcohols, such as glycerol, erythritol, arabinitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a large class of excipients whose functional range includes fillers to additives, capable of dissolving therapeutic agents or helping to prevent denaturation or adhesion to container walls. 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 cyclic polyols such as inositol; polyethylene glycol; amino acid polymers; and sulfur-containing reducing agents such as urea. The stabilizer contains glutathione, lipoic acid, sodium thioacetate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight peptides (e.g., peptides with 10 residues or fewer); proteins such as human serum albumin, bovine serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone monosaccharides such as xylose, mannose, fructose, and 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 antagonist.

[0300] Nonionic surfactants or detergents (also known as "wetting agents") can be added to help dissolve glycoproteins and protect them from agitation-induced aggregation. This also allows the formulation to be exposed to shear surface stress without causing protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188, etc.), and Pranic polyols. Nonionic surfactants can be present in the range of about 0.05 mg / mL to about 1.0 mg / mL (e.g., about 0.07 mg / mL to about 0.2 mg / mL).

[0301] Other miscellaneous excipients include fillers (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and cosolvents.

[0302] The IFN receptor antagonists of this disclosure can be formulated into pharmaceutical compositions comprising IFN receptor antagonists, for example, containing one or more pharmaceutically acceptable excipients or carriers. To prepare a pharmaceutical or sterile composition comprising the IFN receptor antagonists of this disclosure, the IFN receptor antagonist preparation can be combined with one or more pharmaceutically acceptable excipients or carriers.

[0303] For example, formulations of IFN receptor antagonists can be prepared by mixing the IFN receptor antagonist with a physiologically acceptable carrier, excipient, or stabilizer, such as a lyophilized powder, slurry, aqueous solution, lotion, or suspension (see, for example, 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. (ed.), 1993, Pharmaceutical Dosage Forms: General Medications, Marcel Dekker, NY; Lieberman et al. (ed.), 1990, Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al. (ed.), 1990, Pharmaceutical Dosage Forms: Disperse Systems, Marcel). Dekker, NY; Weiner and Kotkoskie, 2000, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

[0304] The effective dose for a particular subject can vary depending on a number of factors, such as the disease being treated, the subject’s overall health, the route and dosage of administration, and the severity of side effects (see, for example, 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).

[0305] The compositions of this disclosure can also be administered via one or more routes of administration using one or more of the various methods known in the art. As those skilled in the art will understand, the route of administration and / or mode will vary depending on the desired outcome. Selected routes of administration for the IFN receptor antagonist include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other common routes of administration, such as by injection or infusion. Common administration can represent modes of administration other than enteral and local administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions. Alternatively, the compositions of this disclosure can be administered via non-common routes, such as local, epidermal, or mucosal administration routes, for example, intranasal, oral, vaginal, rectal, sublingual, or local administration. In one embodiment, the IFN receptor antagonist is administered by infusion. In another embodiment, the IFN receptor antagonist of this disclosure is administered subcutaneously.

[0306] 6.9.1. Pharmaceutical compositions for delivering IFN receptor antagonists encoding nucleic acids

[0307] The IFN receptor antagonist disclosed herein can be delivered via nucleic acid encoding the IFN receptor antagonist, for example as a plasmid, DNA, mRNA, or via a viral vector encoding the IFN receptor antagonist under the control of a suitable promoter.

[0308] In one embodiment, the delivery vector is a virus, including a retrovirus, adenovirus, herpes simplex virus, poxvirus, vaccinia virus, lentivirus, or adeno-associated virus. In one embodiment, the delivery vector is adeno-associated virus (AAV), including serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, or engineered or naturally selected variants thereof.

[0309] Exemplary viral vectors include recombinant adenovirus and adeno-associated virus vectors (rAAV). rAAV vectors are based on defective and non-pathogenic parvovirus adeno-associated virus type 2. Most of these vectors are derived from plasmids containing only the inverted terminal repeat sequences of AAVs flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genome of transduced cells are key features of this vector system. AAV serotypes can be used to deliver IL27 transgenes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV8.2, AAV9, and AAV rh10, as well as pseudotyped AAVs such as AAV2 / 8, AAV2 / 5, and AAV2 / 6.

[0310] In one embodiment, the nucleic acid encoding an IFN receptor antagonist (or a component thereof) also contains an adeno-associated virus (AAV) nucleic acid sequence. In one embodiment, the vector is a chimeric adeno-associated virus containing genetic elements from two or more serotypes. For example, an AAV vector having a rep gene from AAV1 and a cap gene from AAV2 (referred to as AAV1 / 2 or AAVRC1 / 2) can be used as a delivery vector to deliver the nucleic acid expressing the IFN receptor antagonist to cells in need or to the patient's cells.In one embodiment, the delivery carrier is AAV1 / 2, AAV1 / 3, AAV1 / 4, AAV1 / 5, AAV1 / 6, AAV1 / 7, AAV1 / 8, AAV1 / 9, AAV1 / 10, AAV1 / 11, AAV2 / 1, AAV2 / 3, AAV2 / 4, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2 / 10, AAV2 / 11, AAV3 / 1, AAV3 / 2, AAV3 / 4, AAV3 / 5, AAV3 / 6, AAV3 / 7, AAV3 / 8, AAV3 / 9, AAV3 / 10, AAV3 / 10, AAV4 / 1, AAV4 / 2, AAV4 / 3, AAV4 / 5, AAV4 / 6, AAV4 / 7, AAV4 / 8, AAV4 / 9, AAV4 / 10. AAV4 / 11, AAV5 / 1, AAV5 / 2,AAV5 / 3, AAV5 / 4, AAV5 / 6, AAV5 / 7, AAV5 / 8, AAV5 / 9, AAV5 / 10, AAV5 / 11, AAV6 / 1,AAV6 / 2, AAV6 / 3, AAV6 / 4, AAV6 / 5, AAV6 / 7, AAV6 / 8, AAV6 / 9, AAV6 / 10, AAV6 / 10,AAV7 / 1, AAV7 / 2, AAV7 / 3, AAV7 / 4, AAV7 / 5, AAV7 / 6, AAV7 / 8, AAV7 / 9, AAV7 / 10,AAV7 / 11, AAV8 / 1, AAV8 / 2, AAV8 / 3, AAV8 / 4, AAV8 / 5, AAV8 / 6, AAV8 / 7, AAV8 / 9, AAV8 / 10, AAV8 / 11, AAV9 / 1, AAV9 / 2, AAV9 / 3, AAV9 / 4, AAV9 / 5, AAV9 / 6, AAV9 / 7, AAV9 / 8, AAV9 / 10, AAV9 / 11, AAV10 / 1, AAV10 / 2, AAV10 / 3, AAV10 / 4, AAV10 / 5, AAV10 / 6, AAV10 / 7, AAV10 / 8, AAV10 / 9, AAV10 / 11, AAV11 / 1, AAV11 / 2, AAV11 / 3, AAV11 / 4, AAV11 / 5, AAV11 / 6, AAV11 / 7, AAV11 / 8, AAV11 / 9, AAV11 / 10, chimeric viral vectors or their derivatives.Gao et al., “Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy,” PNAS 99(18): 11854-11859, September 3, 2002, incorporated herein by reference for AAV vectors and chimeric viral vectors that can be used as delivery vectors, and their construction and uses.

[0311] AAV can be produced on a clinical scale using 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 (by Penaud-Budloo et al., 2018, Mol Ther Methods Clin Dev. 8: 166-180).

[0312] Replication-deficient recombinant adenovirus vectors (Ad) can be produced at high titers and readily infect many different cell types. Most adenovirus vectors are engineered to replace the Ad Ela, Elb, and / or E3 genes with transgenes; subsequently, the replication-deficient vector is propagated in human 293 cells that provide the function of the missing genes in a trans-form. Ad vectors can be transduced in various tissue types in vivo, including non-dividing differentiated cells such as those found in the liver, kidney, and muscle. Conventional Ad vectors have a high carrying capacity.

[0313] Packaging cells are used to form viral particles capable of infecting host cells. These cells include 293 cells, which package adenoviruses, and w2 or PA317 cells, which package retroviruses. Viral vectors used in gene therapy are typically produced by producer cell lines that package nucleic acid vectors into viral particles. The vectors usually contain the minimum viral sequences required for packaging and subsequent integration into the host (if applicable), with other viral sequences replaced by expression cassettes encoding the proteins to be expressed. Missing viral functions are provided trans-by the packaging cell lines. For example, AAV vectors used in gene therapy typically only have inverted terminal repeat (ITR) sequences from the AAV genome, which are essential for packaging and integration into the host genome. Viral DNA is packaged in a cell line containing helper plasmids encoding other AAV genes, namely rep and cap, but lacking the ITR sequences. This 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 plasmids are not packaged in large quantities. Adenovirus contamination can be reduced, for example, by heat treatment, as adenovirus is more sensitive to heat treatment than AAV.

[0314] Nucleic acid molecules (e.g., mRNA) or viruses may be formulated as the sole active pharmaceutical ingredient in a pharmaceutical composition, or may be combined with other active agents for the specific condition being treated. Optionally, the compositions provided herein may contain other pharmaceutical agents, drug agents, carriers, adjuvants, and diluents. For example, wetting agents, emulsifiers, and lubricants (such as sodium dodecyl sulfate and magnesium stearate), as well as colorants, release agents, coating agents, sweeteners, flavorings and aromas, preservatives, antioxidants, chelating agents, and inert gases may also be present in the composition. Other exemplary reagents and excipients that may be included in the composition include, for example: water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; oil-soluble antioxidants such as ascorbate palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0315] 6.10. Indications and Methods of Use

[0316] This disclosure provides methods of use and applications of the IFN receptor antagonists disclosed herein.

[0317] The IFN receptor antagonists disclosed herein can be used to modulate immune responses in a variety of applications.

[0318] In some aspects, this disclosure provides a method for treating cancer or inflammatory or immune (e.g., autoimmune) diseases, the method comprising administering to a subject in need an IFN receptor antagonist or pharmaceutical composition described herein, wherein the IFN receptor antagonist comprises an anchoring portion (e.g., a targeting portion) that binds to a target molecule present on the surface of a target cell expressing a type I interferon receptor and associated with the disease.

[0319] This disclosure further provides a method for locally modulating an immune response in a target tissue, the method comprising administering to a subject an IFN receptor antagonist or pharmaceutical composition as described herein, the IFN receptor agonist or pharmaceutical composition having one or more anchoring portions (e.g., targeting portions) capable of binding to a target molecule expressed in the target tissue.

[0320] In some embodiments, application is not localized to the tissue. For example, when the target tissue is cancerous tissue, application may be systemic or subcutaneous.

[0321] The IFN receptor antagonists disclosed herein can be used, for example, to treat autoimmune inflammatory diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) by reducing local or tissue-specific autoimmune responses.

[0322] The IFN receptor antagonists disclosed herein can be used to treat any proliferative condition (e.g., cancer) that expresses a target molecule (on tumor cells or in the tumor microenvironment, such as the extracellular matrix or tumor lymphocytes). In specific embodiments, the IFN antagonists of this disclosure are administered to subjects who are receiving oncolytic virus therapy, subjects who have previously received oncolytic virus therapy, or subjects who will receive oncolytic virus therapy.

[0323] In some embodiments, cancer includes acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, Burkitt lymphoma, cancer of unknown primary origin, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasm, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, and so on. Tubal cancer, embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, fibrous histiocytoma, Ewing's sarcoma, ocular cancer, germ cell tumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, glioma, head and neck cancer, hairy cell leukemia, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, renal cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cancer, liver cancer, lobularia. Carcinoma, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck carcinoma with occult primary origin, midline carcinoma involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, nasal and paranasal sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oropharyngeal carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, papillomavirus. Paraganglioma, parathyroid carcinoma, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary adenoma, pleural pulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Cezari syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, gastric cancer, T-cell lymphoma, teratoma, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, or Wilms' tumor.

[0324] Table I below shows exemplary indications for the use of IFN receptor antagonists that target specific target molecules.

[0325]

[0326] Additional target molecules and corresponding indications are disclosed, for example, in Hafeez et al., 2020, Molecules 25:4764, doi:10.3390 / molecules25204764, and particularly in Table 1. Table 1 is incorporated herein by reference in its entirety.

[0327] In further embodiments, IFN receptor antagonists can be used to modulate an immune response induced by another agent. Therefore, in some embodiments, the IFN receptor antagonists of this disclosure are administered as adjunctive therapy to an immunogenic agent. In some embodiments, the immunogenic agent is an adjuvanted or unadjuvanted vaccine. Therefore, IFN receptor antagonists can enhance an antigen-specific immune response induced by a vaccine. In various embodiments, the vaccine is a prophylactic or therapeutic cancer vaccine or a prophylactic or therapeutic vaccine against an infectious agent (e.g., a virus, bacteria, or parasite).

[0328] 6.10.1. Combination Therapy

[0329] The IFN receptor antagonists according to this disclosure can be administered in combination with one or more other therapeutic agents in a therapy. For example, the IFN receptor antagonists of this disclosure can be administered co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any pharmaceutical agent administered to treat symptoms or diseases of a subject requiring such treatment. Such additional therapeutic agents may contain any active ingredient suitable for a particular indication being treated, preferably those having complementary activities that do not adversely affect each other.

[0330] Unbound by theory, it is believed that the combined administration of the IFN receptor antagonist of this disclosure with oncolytic virus therapy, by upregulating type I IFN-induced inhibition of IFN signaling in response to oncolytic virus administration, allows for enhanced efficacy and reduced side effects of oncolytic virus therapy. Therefore, in some embodiments, the additional therapeutic agent is an oncolytic virus.

[0331] In some embodiments, the additional therapeutic agent is an immunosuppressant, including but not limited to mycophenolate mofetil (MMF), mycophenolic acid (MPA), cyclosporine A, FK506-like compounds (e.g., FK506, FK506 derivatives, and FK506 analogs), rapamycin compounds (including rapamycin, rapamycin derivatives, and rapamycin analogs), and corticosteroids (e.g., hydrocortisone, hydroxytriamcinolone, α-methyldexamethasone, dexamethasone phosphate, beclomethasone dipropionate, clobetasol valerate, desonide, deoxymethasone acetate, dexamethasone, diclomethasone diacetate, diflubenzuron valerate, fludrosol, fluclofenoxate, fludrosol, and fludrosol). Flumethasone, fluocinolone valerate, fluocinolone acetate, flucodone butyl ester, flucolone, fluprednylidene acetate, flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone, cortisone, todoxacin, fluocinolone, flucodone, difluorocortisone diacetate, flurandrenolone, flucodone diacetate, difluorocortisone diacetate, flurandrenolone acetone compounds, methylphenidate, aminoflavone, acetaminophen, betamethasone and the remainder of esters, chlorprednisolone, chlorprednisolone acetate, chlorcodone, crisinolonone, dichlorpheniramine, difluoroprednisolone ester, fluclofenone Flunisolone, flumethrin, fluperamide, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortisone hydrocortisone, methylprednisolone, peramisone, prednisolone, prednisolone, beclomethasone dipropionate, triamcinolone and mixtures thereof), nonsteroidal anti-inflammatory drugs (e.g., oxacins, such as piroxicam, isoxicam, tenoxicam, sudoxicam; salicylates, such as aspirin, disalicylate, benorilate, triamcinolone acetonide, safapryn, soprin, diflunisal and fendusal; acetic acid derivatives, such as diclofenac, fenchloric acid, indomethacin, sulindac, tometidine, isocolic acid, furofenic acid, thiophene, zidomecin, azithromycin, azithromycin Caprylic acid, fentiic acid, zolpidem, clindamycin, oxipin, biphenylacetic acid, and ketorolac; fenamic acid derivatives, such as mefenamic acid, meclofenamic acid, flufenamic acid, niflunic acid, and tofenamic acid; propionic acid derivatives, such as ibuprofen, naproxen, phenoxprofen, flurbiprofen, ketoprofen, fenofibfen, fenbufen, indoprofen, pirprofen, carprofen, oxaprazin, pranoprofen, miprofen, thioprofen, sulprofen, amiprofen, and tiprofen; pyrazole derivatives, such as phenylbutazone, hydroxyphenylbutazone, feprazolam, azaprofen, and trimethoprim; and anti-inflammatory cytokines or chemokines (e.g., IL-4, IL-6, IL-10, IL-11, and IL-13).

[0332] Other such agents are appropriately available in combinations of amounts effective for the intended purpose. The effective dose of these other agents depends on the amount of IFN receptor antagonist used, the type of patient or treatment, and other factors discussed above. IFN receptor antagonists are generally used at the same doses and routes of administration as described herein, or approximately 1% to 99% of the doses described herein, or at any dose and route of administration as determined empirically / clinically.

[0333] Such combination therapies include combined administration (two or more therapeutic agents contained in the same or separate composition) and single administration, in which case the administration of the IFN receptor antagonist of this disclosure may be performed before, simultaneously with and / or after the administration of additional therapeutic agents and / or adjuvants.

[0334] 7. Sequence

[0335] Some sequences of this disclosure are provided in Table S below.

[0336]

[0337] 8. Numbered Examples

[0338] Although various specific embodiments have been shown and described, it should be understood that various changes can be made without departing from the spirit and scope of this disclosure. This disclosure is illustrated by way of examples with reference to the numbers set forth below.

[0339] 1. A type I interferon (IFN) receptor antagonist, comprising:

[0340] (a) Anchoring portion of cells that express type I interferon receptors;

[0341] (b) Type I interferon α receptor 1 (IFNAR1) portion;

[0342] (c) Type I interferon (IFN) portion;

[0343] (d) Connecting the anchoring portion to the IFNAR1 portion or the partition portion of the IFN portion; and

[0344] (e) Optionally, a connector is used to connect the IFNAR1 portion and the IFN portion.

[0345] 2. The IFN receptor antagonist according to Example 1, wherein the anchoring portion is capable of binding to extracellular matrix (ECM) antigens, tumor reactive lymphocyte antigens, cell surface molecules of tumor or viral lymphocytes, T cell antigens (TCA), checkpoint inhibitors, tumor-associated antigens (TAA), dendritic cell (DC) or other antigen-presenting cell (APC) antigens, or natural killer (NK) cell antigens.

[0346] 3. The IFN receptor antagonist according to Example 1 or 2, wherein the anchoring portion is the anchoring portion of immune cells.

[0347] 4. The IFN receptor antagonist according to Example 2, wherein the anchoring portion is the B cell anchoring portion.

[0348] 5. The IFN receptor antagonist according to Example 1 or 2, wherein the anchoring portion is an anchoring portion for cancer cells.

[0349] 6. The IFN receptor antagonist according to any one of Examples 1 to 5, wherein the anchoring portion is a targeting portion.

[0350] 7. The IFN receptor antagonist according to Example 6, wherein the targeting portion is scFv or Fab.

[0351] 8. The IFN receptor antagonist according to Example 6 or 7, wherein the targeting portion is capable of binding to the target molecule identified in Section 6.6.1.

[0352] 9. An IFN receptor antagonist according to any one of Examples 6 to 8, wherein the targeting portion (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.

[0353] 10. The IFN receptor antagonist according to any one of Examples 6 to 9, wherein the targeting portion is capable of binding to a checkpoint inhibitor.

[0354] 11. The IFN receptor antagonist according to Example 10, 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.

[0355] 12. The IFN receptor antagonist according to Example 11, wherein the checkpoint inhibitor is PDL1.

[0356] 13. The IFN receptor antagonist according to Example 12, wherein the targeting portion is an antigen-binding fragment of an anti-PDL1 antibody.

[0357] 14. The IFN receptor antagonist according to any one of Examples 1 to 5, wherein the anchoring portion is a cell surface protein binding molecule.

[0358] 15. The IFN receptor antagonist according to Example 14, wherein the anchoring portion is PD1 or its PDL1 binding portion.

[0359] 16. The IFN receptor antagonist according to any one of Examples 1 to 15, wherein the septum is an Fc domain.

[0360] 17. The IFN receptor antagonist according to Example 16, wherein the Fc domain comprises a hinge domain.

[0361] 18. The IFN receptor antagonist according to any one of Examples 1 to 17, wherein the linker is a non-cleavable linker (NCL).

[0362] 19. The IFN receptor antagonist according to Example 18, wherein the NCL is the NCL described in Table E.

[0363] 20. The IFN receptor antagonist according to any one of Examples 1 to 19, further comprising an additional connector for connecting the septum portion to the IFNAR1 portion.

[0364] 21. The IFN receptor antagonist according to any one of Examples 1 to 19, further comprising an additional connector for connecting the septum portion to the IFN portion.

[0365] 22. The IFN receptor antagonist according to Example 20 or 21, wherein the additional linker is NCL.

[0366] 23. The IFN receptor antagonist according to Example 22, wherein the additional linker is NCL as described in Table E.

[0367] 24. The IFN receptor antagonist according to any one of Examples 1 to 23, further comprising an additional anchoring portion.

[0368] 25. The IFN receptor antagonist according to Example 24, wherein the additional anchoring portion is an additional targeting portion.

[0369] 26. The IFN receptor antagonist according to Example 25, wherein the additional targeting portion is scFv or Fab.

[0370] 27. The IFN receptor antagonist according to Example 26, wherein the additional targeting portion specifically binds to the same target as the anchoring portion.

[0371] 28. The IFN receptor antagonist according to any one of Examples 25 to 27, wherein the additional targeting portion is capable of binding to any target molecule identified in Section 6.6.1.

[0372] 29. An IFN receptor antagonist according to any one of Examples 25 to 28, wherein the additional targeting portion (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.

[0373] 30. The IFN receptor antagonist according to any one of Examples 25 to 29, wherein the additional targeting portion is capable of binding to a checkpoint inhibitor.

[0374] 31. The IFN receptor antagonist according to Example 30, 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.

[0375] 32. The IFN receptor antagonist according to Example 31, wherein the checkpoint inhibitor is PDL1.

[0376] 33. The IFN receptor antagonist according to Example 32, wherein the additional targeting portion is an antigen-binding fragment of an anti-PD1 antibody.

[0377] 34. The IFN receptor antagonist according to Example 24, wherein the anchoring portion is a cell surface protein binding molecule.

[0378] 35. The IFN receptor antagonist according to Example 34, wherein the anchoring portion is PD1 or its PDL1 binding portion.

[0379] 36. The IFN receptor antagonist according to any one of Examples 24 to 35, further comprising an additional septum operatively connected to the additional anchoring portion.

[0380] 37. The IFN receptor antagonist according to Example 36, wherein the additional septum is an additional Fc domain.

[0381] 38. The IFN receptor antagonist according to Example 37, wherein the additional Fc domain comprises a hinge domain.

[0382] 39. The IFN receptor antagonist according to any one of Examples 1 to 38, wherein the IFN portion is masked by the IFNAR1 portion.

[0383] 40. The IFN receptor antagonist according to any one of Examples 1 to 39, wherein the IFN receptor antagonist comprises a first polypeptide chain and a second polypeptide chain.

[0384] 41. The IFN receptor antagonist according to Example 40, wherein the first polypeptide chain comprises:

[0385] (a) the anchoring portion or its components;

[0386] (b) the dividing portion; and

[0387] (c) The IFNAR1 portion.

[0388] 42. The IFN receptor antagonist according to Example 41, wherein the second polypeptide chain comprises:

[0389] (a) additional anchoring portions or components thereof; and

[0390] (b) Additional separators.

[0391] 43. The IFN receptor antagonist according to Example 41 or 42, wherein the first polypeptide chain further comprises the IFN moiety.

[0392] 44. The IFN receptor antagonist according to Example 43, wherein the IFN portion is located at the C-terminus of the IFNAR1 portion.

[0393] 45. The IFN receptor antagonist according to Example 43, wherein the IFN portion is located at the N-terminus of the IFNAR1 portion.

[0394] 46. ​​The IFN receptor antagonist according to any one of Examples 41 to 45, wherein the IFNAR1 portion is located at the C-terminus of the septum.

[0395] 47. The IFN receptor antagonist according to any one of Examples 40 to 43, wherein the first polypeptide chain comprises, in an N-terminal to C-terminal orientation:

[0396] (a) the anchoring portion or its components;

[0397] (b) the dividing portion;

[0398] (c) the IFNAR1 portion; and

[0399] (d) The IFN portion.

[0400] 48. The IFN receptor antagonist according to any one of Examples 40 to 43, wherein the first polypeptide chain comprises, in an N-terminal to C-terminal orientation:

[0401] (a) the anchoring portion or its components;

[0402] (b) the dividing portion;

[0403] (c) the IFN portion; and

[0404] (d) The IFNAR1 portion.

[0405] 49. The IFN receptor antagonist according to Example 42, wherein the second polypeptide chain further comprises the IFN moiety.

[0406] 50. The IFN receptor antagonist according to Example 49, wherein the second polypeptide chain comprises, in an N-terminal to C-terminal orientation:

[0407] (a) Additional anchoring portions or components thereof;

[0408] (b) Additional dividing sections; and

[0409] (c) The IFN portion.

[0410] 51. The IFN receptor antagonist according to any one of Examples 40 to 48, wherein the second polypeptide chain further comprises an additional IFNAR1 moiety and an additional IFN moiety.

[0411] 52. An IFN receptor antagonist according to any one of Examples 1 to 51, wherein the IFN portion comprises an amino acid sequence having at least about 90% sequence identity with: (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ, or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

[0412] 53. The IFN receptor antagonist according to Example 52, wherein the IFN portion comprises an amino acid sequence having at least about 90% sequence identity with: (a) full-length mature human IFNα2b, or (b) mature human IFNα2b having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

[0413] 54. An IFN receptor antagonist according to any one of Examples 1 to 51, wherein the IFN portion comprises an amino acid sequence having at least about 95% sequence identity with: (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ, or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

[0414] 55. The IFN receptor antagonist according to Example 54, wherein the IFN portion comprises an amino acid sequence having at least about 95% sequence identity with: (a) full-length mature human IFNα2b, or (b) mature human IFNα2b having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

[0415] 56. An IFN receptor antagonist according to any one of Examples 1 to 51, wherein the IFN moiety comprises an amino acid sequence having at least about 98% sequence identity with: (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ, or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

[0416] 57. The IFN receptor antagonist according to Example 56, wherein the IFN portion comprises an amino acid sequence having at least about 98% sequence identity with: (a) full-length mature human IFNα2b, or (b) mature human IFNα2b having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

[0417] 58. An IFN receptor antagonist according to any one of Examples 1 to 51, wherein the IFN moiety is (a) a full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ, or (b) a mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

[0418] 59. The IFN receptor antagonist according to Example 58, wherein the IFN portion is: (a) a full-length mature human IFNα2b, or (b) a mature human IFNα2b having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

[0419] 60. An IFN receptor antagonist according to any one of Examples 1 to 59, wherein the IFNAR1 portion comprises or consists of an amino acid sequence having at least 90% sequence identity with: (i) the SD2 and SD3 domains of human IFNAR1, (ii) the SD1, SD2, and SD3 domains of human IFNAR1, or (iii) the SD1, SD2, SD3, and SD4 domains of human IFNAR1.

[0420] 61. The IFN receptor antagonist according to any one of Examples 1 to 59, wherein the IFNAR1 portion comprises or consists of an amino acid sequence having at least 95% sequence identity with: (i) the SD2 and SD3 domains of human IFNAR1, (ii) the SD1, SD2, and SD3 domains of human IFNAR1, or (iii) the SD1, SD2, SD3, and SD4 domains of human IFNAR1.

[0421] 62. The IFN receptor antagonist according to any one of Examples 1 to 59, wherein the IFNAR1 portion comprises or consists of an amino acid sequence having at least 98% sequence identity with: (i) the SD2 and SD3 domains of human IFNAR1, (ii) the SD1, SD2, and SD3 domains of human IFNAR1, or (iii) the SD1, SD2, SD3, and SD4 domains of human IFNAR1.

[0422] 63. The IFN receptor antagonist according to any one of Examples 1 to 59, wherein the IFNAR1 portion is: (i) the SD2 and SD3 domains of human IFNAR1, (ii) the SD1, SD2, and SD3 domains of human IFNAR1, or (iii) the SD1, SD2, SD3, and SD4 domains of human IFNAR1.

[0423] 64. The IFN receptor antagonist according to Example 51, wherein the additional IFN portion comprises an amino acid sequence having at least about 90% sequence identity with: (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ, or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ, having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

[0424] 65. The IFN receptor antagonist according to Example 64, wherein the additional IFN portion comprises an amino acid sequence having at least about 90% sequence identity with: (a) full-length mature human IFNα2b, or (b) mature human IFNα2b having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

[0425] 66. The IFN receptor antagonist according to Example 51, wherein the additional IFN portion comprises an amino acid sequence having at least about 95% sequence identity with: (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ, or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ, having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

[0426] 67. The IFN receptor antagonist according to Example 66, wherein the additional IFN portion comprises an amino acid sequence having at least about 95% sequence identity with: (a) full-length mature human IFNα2b, or (b) mature human IFNα2b having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

[0427] 68. The IFN receptor antagonist according to Example 51, wherein the additional IFN portion comprises an amino acid sequence having at least about 98% sequence identity with: (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ, or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ, having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

[0428] 69. The IFN receptor antagonist according to Example 68, wherein the additional IFN portion comprises an amino acid sequence having at least about 98% sequence identity with: (a) full-length mature human IFNα2b, or (b) mature human IFNα2b having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

[0429] 70. The IFN receptor antagonist according to Example 51, wherein the additional IFN moiety is (a) a full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ, or (b) a mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

[0430] 71. The IFN receptor antagonist according to Example 70, wherein the additional IFN portion is: (a) a full-length mature human IFNα2b, or (b) a mature human IFNα2b having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

[0431] 72. The IFN receptor antagonist according to Example 51, wherein the additional IFNAR1 portion comprises or consists of an amino acid sequence having at least 90% sequence identity with: (i) the SD2 and SD3 domains of human IFNAR1, (ii) the SD1, SD2, and SD3 domains of human IFNAR1, or (iii) the SD1, SD2, SD3, and SD4 domains of human IFNAR1.

[0432] 73. The IFN receptor antagonist according to Example 51, wherein the additional IFNAR1 portion comprises or consists of an amino acid sequence having at least 95% sequence identity with: (i) the SD2 and SD3 domains of human IFNAR1, (ii) the SD1, SD2, and SD3 domains of human IFNAR1, or (iii) the SD1, SD2, SD3, and SD4 domains of human IFNAR1.

[0433] 74. The IFN receptor antagonist according to Example 51, wherein the additional IFNAR1 portion comprises or consists of an amino acid sequence having at least 98% sequence identity with: (i) the SD2 and SD3 domains of human IFNAR1, (ii) the SD1, SD2, and SD3 domains of human IFNAR1, or (iii) the SD1, SD2, SD3, and SD4 domains of human IFNAR1.

[0434] 75. The IFN receptor antagonist according to Example 51, wherein the additional IFNAR1 portion is: (i) the SD2 and SD3 domains of human IFNAR1, (ii) the SD1, SD2, and SD3 domains of human IFNAR1, or (iii) the SD1, SD2, SD3, and SD4 domains of human IFNAR1.

[0435] 76. The IFN receptor antagonist according to any one of Examples 1 to 75, wherein the IFN receptor antagonist does not contain the IFNAR2 portion.

[0436] 77. The IFN receptor antagonist according to Example 76, wherein the IFNAR2 portion comprises or consists of an amino acid sequence having at least 90% sequence identity with: (i) the D1 domain of human IFNAR2, or (ii) the D1 and D2 domains of human IFNAR2.

[0437] 78. The IFN receptor antagonist according to Example 76, wherein the IFNAR2 portion comprises or consists of an amino acid sequence having at least 95% sequence identity with: (i) the D1 domain of human IFNAR2, or (ii) the D1 and D2 domains of human IFNAR2.

[0438] 79. The IFN receptor antagonist according to Example 76, wherein the IFNAR2 portion comprises or consists of an amino acid sequence having at least 98% sequence identity with: (i) the D1 domain of human IFNAR2, or (ii) the D1 and D2 domains of human IFNAR2.

[0439] 80. The IFN receptor antagonist according to Example 76, wherein the IFNAR2 portion is (i) the D1 domain of human IFNAR2 or (ii) the D1 and D2 domains of human IFNAR2.

[0440] 81. An IFN receptor antagonist according to any one of Examples 1 to 80, comprising any one-and-a-half antibody as depicted in Table 2.

[0441] 82. An IFN receptor antagonist, optionally an IFN receptor antagonist according to any one of Examples 1 to 80, comprising having Figure 1B The polypeptide chains of the two half-antibodies shown are in the configuration described.

[0442] 83. An IFN receptor antagonist, optionally an IFN receptor antagonist according to any one of Examples 1 to 80, comprising having Figure 1C The polypeptide chains of the two half-antibodies shown are in the configuration described.

[0443] 84. An IFN receptor antagonist, optionally an IFN receptor antagonist according to any one of Examples 1 to 80, comprising having Figure 1D The polypeptide chains of the two half-antibodies shown are in the configuration described.

[0444] 85. An IFN receptor antagonist, optionally an IFN receptor antagonist according to any one of Examples 1 to 80, comprising having Figure 1E The polypeptide chains of the two half-antibodies shown are in the configuration described.

[0445] 86. An IFN receptor antagonist, optionally an IFN receptor antagonist according to any one of Examples 1 to 80, comprising having Figure 1F The polypeptide chains of the two half-antibodies shown are in the configuration described.

[0446] 87. A type I interferon (IFN) receptor antagonist, optionally an IFN receptor antagonist according to any one of Examples 1 to 86, comprising:

[0447] (a) A first polypeptide, which comprises, in the orientation from the N-terminus to the C-terminus:

[0448] (i) First target portion;

[0449] (ii) The first Fc structural domain;

[0450] (iii) First connector;

[0451] (iv) IFNAR1 section;

[0452] (v) the second joint; and

[0453] (vi) The IFN part; and

[0454] (b) A second polypeptide, which comprises, in the orientation from the N-terminus to the C-terminus:

[0455] (i) the second target portion; and

[0456] (ii) A second Fc structural domain, which is associated with the first Fc structural domain.

[0457] 88. A type I interferon (IFN) receptor antagonist, optionally an IFN receptor antagonist according to any one of Examples 1 to 86, comprising:

[0458] (a) A first polypeptide, which comprises, in the orientation from the N-terminus to the C-terminus:

[0459] (i) First target portion;

[0460] (ii) The first Fc structural domain;

[0461] (iii) First connector;

[0462] (iv) IFN section;

[0463] (v) the second joint; and

[0464] (vi) IFNAR1 section; and

[0465] (b) A second polypeptide, which comprises, in the orientation from the N-terminus to the C-terminus:

[0466] (i) the second target portion; and

[0467] (ii) A second Fc structural domain, which is associated with the first Fc structural domain.

[0468] 89. A type I interferon (IFN) receptor antagonist, optionally an IFN receptor antagonist according to any one of Examples 1 to 86, comprising:

[0469] (a) A first polypeptide, which comprises, in the orientation from the N-terminus to the C-terminus:

[0470] (i) First target portion;

[0471] (ii) The first Fc structural domain;

[0472] (iii) First connector;

[0473] (iv) First IFNAR1 section;

[0474] (v) the second joint; and

[0475] (vi) The first IFN part; and

[0476] (b) A second polypeptide, which comprises, in the orientation from the N-terminus to the C-terminus:

[0477] (i) Second target portion;

[0478] (ii) A second Fc structural domain, which is associated with the first Fc structural domain;

[0479] (iii) Third connector;

[0480] (iv) Part 2 IFNAR1;

[0481] (v) Fourth connector; and

[0482] (vi) Second IFN part.

[0483] 90. A type I interferon (IFN) receptor antagonist, optionally an IFN receptor antagonist according to any one of Examples 1 to 86, comprising:

[0484] (a) A first polypeptide, which comprises, in the orientation from the N-terminus to the C-terminus:

[0485] (i) First target portion;

[0486] (ii) The first Fc structural domain;

[0487] (iii) First connector;

[0488] (iv) First IFN section;

[0489] (v) the second joint; and

[0490] (vi) The first IFNAR1 part; and

[0491] (b) A second polypeptide, which comprises, in the orientation from the N-terminus to the C-terminus:

[0492] (i) Second target portion;

[0493] (ii) A second Fc structural domain, which is associated with the first Fc structural domain;

[0494] (iii) Third connector;

[0495] (iv) Second IFN section;

[0496] (v) Fourth connector; and

[0497] (vi) Second IFNAR1 part.

[0498] 91. One or more nucleic acids encoding an IFN receptor antagonist according to any one of Examples 1 to 90.

[0499] 92. A host cell engineered to express the IFN receptor antagonist according to any one of Examples 1 to 90 or the nucleic acid according to Example 70.

[0500] 93. A method for producing an IFN receptor antagonist according to any one of Examples 1 to 90, the method comprising culturing a host cell according to Example 92 and recovering the IFN receptor antagonist expressed by the host cell.

[0501] 94. A pharmaceutical composition comprising an IFN receptor antagonist according to any one of Examples 1 to 90 and an excipient.

[0502] 95. The pharmaceutical composition according to Example 94 further comprises an oncolytic virus.

[0503] 96. A method for inhibiting IFN signaling in cells expressing (a) an IFN receptor and (b) a molecule specifically binding to an anchoring moiety, the method comprising contacting the cells with an IFN receptor antagonist according to any one of Examples 1 to 90 or a pharmaceutical composition according to Example 94.

[0504] 97. The method according to Example 96, wherein the cell is an immune cell.

[0505] 98. The method according to Example 96 or 97, wherein the cell is a B cell.

[0506] 99. The method according to Example 96, wherein the cell is a cancer cell.

[0507] 100. The method according to any one of Examples 96 to 99, wherein the method is an in vitro method.

[0508] 101. The method according to any one of Examples 96 to 99, wherein the method comprises administering the IFN receptor antagonist to a subject in need of it.

[0509] 102. The method according to Example 101, wherein the subject is a patient with cancer.

[0510] 103. The method according to Example 102, further comprising: administering an oncolytic virus to the subject.

[0511] 104. The method according to Example 103, wherein the oncolytic virus is oncolytic vesicular stomatitis virus (VSV).

[0512] 105. The method according to Example 101, wherein the subject is a patient suffering from an immune disorder or condition.

[0513] 106. The method according to Example 105, wherein the immune disorder or condition is systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSc), or Sjögren's syndrome (SS).

[0514] 107. A method for reducing inflammation, the method comprising administering to a subject in need an IFN receptor antagonist according to any one of Examples 1 to 90 or a pharmaceutical composition according to Example 94.

[0515] 108. A method of treating a subject with cancer, the method comprising administering to the subject in need an IFN receptor antagonist according to any one of Examples 1 to 90 or a pharmaceutical composition according to Example 94.

[0516] 109. The method according to Example 108, further comprising: administering an oncolytic virus to the subject.

[0517] 110. The method according to Example 109, wherein the oncolytic virus is oncolytic vesicular stomatitis virus (VSV).

[0518] 111. A method of treating a subject suffering from an immune disorder or condition, the method comprising administering to the subject an IFN receptor antagonist according to any one of Examples 1 to 90 or a pharmaceutical composition according to Example 94.

[0519] 112. The method according to Example 111, wherein the immune disorder or condition is systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSc), or Sjögren's syndrome (SS).

[0520] 9. Example

[0521] 9.1. Materials and Methods

[0522] 9.1.1. Design of IFN receptor antagonist construction

[0523] The IFN receptor antagonist construct shown in Figure 1 was designed as a dimer (e.g., homodimer or heterodimer) comprising two polypeptide chains. Table E-1 below provides the core polypeptide sequences used in the studies described herein. The core polypeptide sequences in Table E-1 can be used to generate IFN receptor antagonist and control constructs, thereby allowing the addition of a targeting moiety, such as Fab or scFv, to the N-terminus of each polypeptide chain.

[0524]

[0525] 9.1.2. Production of IFN receptor antagonist constructs

[0526] Constructs encoding IFN receptor antagonists were generated in standard mammalian protein expression DNA vectors (pcDNA3.4 or similar vectors). These vectors are suitable for high-yield protein production and contain standard elements such as promoter sequences, polyA sequences, regulatory elements, and resistance genes. Codon optimization was performed on the sequences where applicable. A 29-amino acid signal sequence from the mouse inactivated tyrosine protein kinase transmembrane receptor ROR1 (mROR1) was added to the N-terminus of the constructs as a signal for secretion. All IFN receptor antagonists were expressed as preproteins containing the signal sequence, which was cleaved intracellularly to produce the mature protein. Constructs were expressed in Expi293F™ cells via transient transfection (Thermo Fisher Scientific). Proteins in the Expi293F supernatant were purified using the ProteinMaker system (Protein BioSolutions, Gaithersburg, MD) and either a HiTrap™ Protein G HP or a MabSelect SuRe pcc column (Cytiva). After single-step elution, 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 substances relative to the substance of interest.

[0527] 9.1.3. Engineering of KG-1a reporter cells

[0528] The promyelocytic macrophage cell line KG-1a was transduced using an interferon-stimulated response element (ISRE)-driven luciferase reporter gene construct, and the promyelocytic macrophage cell line was maintained in Iscove-modified Dulbecco medium supplemented with 2 mM L-glutamine / penicillin / streptomycin + 20% FBS + 1 μg / mL puromycin. A single-cell clone KG-1a / ISRE-Luc, highly responsive to IFNα2b, was isolated. PDL1 in this clone was knocked out using CRISPR-Cas9 technology, and the resulting cell line KG-1a / ISRE-Luc / PDL1 KO was verified by flow cytometry. KG-1a / ISRE-Luc was engineered to overexpress PDL1 (amino acid M1-T290 of accession number #NP_054862.1), and the resulting cell line KG-1a / ISRE-Luc / hPDL1 was verified by flow cytometry.

[0529] 9.1.4. Staining and Flow Cytometry

[0530] KG-1a / ISRE-Luc cells were used at 2.5 x 10⁻⁶ cells per well. 5Cells were plated at high density and incubated on ice for 30 minutes with serially diluted IFN receptor antagonists. Cells were washed twice and counterstained on ice for 30 minutes with goat anti-human IgG F(ab')2 AF647. After staining, cells were washed and fixed, and data were acquired on iQue Plus (Sartorius).

[0531] 9.1.5. Luciferase assay device for IFN signal transduction assessment

[0532] Cell suspensions and dilutions of IFN receptor antagonists or control constructs were prepared using RPMI 1640 medium supplemented with 2 mM L-glutamine / penicillin / streptomycin + 10% FBS as the assay medium.

[0533] On the day of the assay, KG-1a / ISRE-Luc reporter cells were centrifuged and measured at 5 x 10⁻⁶. 5 The concentration was resuspended in assay medium at a density of / mL. IFNα2b, IFN receptor antagonist, and control constructs were diluted 1:5 according to the 11-point dilution range, with point 12 containing no recombinant protein. 2.5 x 10 4 Reporter cells were added to 96-well white flat-bottomed plates and incubated with serially diluted IFNα2b, IFN receptor antagonists, and control constructs in the presence or absence of 200 pM IFNα2b, 45 pM IFNα2b, or 90 pM IFNβ. The plates were incubated at 37°C and 5% CO2 for 5 hours, followed by cell lysis with 100 μL of ONE-Glo™ (Promega) reagent and detection of luciferase activity. Emitted light was captured in relative light units (RLU) on an Envision (PerkinElmer) multi-label plate reader. All serial dilutions were tested in duplicate.

[0534] 9.1.6. Assessing cell viability by luminescence assay

[0535] Cell suspensions and dilutions of recombinant IFNα2b, IFNβ, or control constructs were prepared using Iskoff modified Durbeco medium supplemented with 2 mM L-glutamine / penicillin / streptomycin + 20% fetal bovine serum (FBS) as the assay medium.

[0536] On the day of the assay, KG-1a / ISRE-Luc reporter cells were centrifuged at 1 x 10⁻⁶. 5 The solution was resuspended in the assay medium at a density of 5 x 10⁻⁶ mL. 3Reporter cells / wells were seeded in 96-well white flat-bottomed plates and incubated with titrated recombinant IFNα2b, IFNβ, or control constructs in combination with constant amounts of IFNα2b (900 pM) or IFNβ (30 pM). All constructs were serially diluted (1:5) within a 9-point titration range (50 nM to 5.12 fM), with no recombinant protein or constant IFNα2b or IFNβ at point 10. The plates were incubated at 37°C and 5% CO2 for 5 days. On day 5, 100 mL of RealTime-Glo™ (Promega) reagent was added to each well, and the plates were incubated at 37°C and 5% CO2 for 1 h, followed by NanoLuc assay. ® Luciferase activity. Emission was measured in relative optical units (RLU) on a multi-label plate reader, Envision (PerkinElmer). GraphPadPrism was used. TM The software determines the EC of the test construct on a 10-point dose-response curve using a four-parameter logistic equation. 50 Value. The percentage inhibition of cytotoxic response for each test construct was calculated using the following formula: % inhibition = ((maximum luminescence (construct) - minimum luminescence (construct)) / (maximum IFN luminescence - minimum IFN luminescence)) x 100. All series dilutions were tested in duplicate.

[0537] 9.2. Example 1: In vitro binding affinity of IFN receptor antagonists

[0538] As described in Sections 9.1.1 and 9.1.2, targeting monovalent and bivalent IFN receptor antagonists and control constructs were designed and manufactured. The binding affinity of the IFN receptor antagonists and control constructs was evaluated as described in Section 9.1.4, thereby using data obtained from live cells as a measure of binding affinity. The strongest binding affinity was observed with the cis-masked bivalent Fc-R1-IFNα2b and monovalent Fc-R1-IFNα2b constructs. Figure 2A ).

[0539] The activity of IFN receptor antagonists and control constructs was evaluated in KG-1a / ISRE-Luc reporter cells as described in Section 9.1.4. Both monovalent and divalent Fc-IFNa2b showed reduced potency compared to IFNa2b. Cis-masked divalent Fc-R1-IFNα2b (bivalent Fc-R1-IFNα2b) and monovalent Fc-R1-IFNα2b (Fc-R1-IFNα2b), as well as the less trans-masked monovalent Fc-IFNα2b x R1 (Fc-IFNα2b x R1) construct, showed reduced potency compared to Fc-IFNα2b.

[0540] In summary, these results indicate that cis-masking constructs containing the IFNAR1 masking portion occupy IFNAR2 receptors on the cell membrane and prevent free IFN from binding to these receptors, thereby weakening downstream intracellular cascades.

[0541] 9.3. Example 2: In vitro signal transduction activity of IFN receptor antagonists

[0542] The monovalent and bivalent IFN receptor antagonists and control constructs were designed and manufactured as described in Sections 9.1.1 and 9.1.2. The signal transduction activity of the IFN receptor antagonists and control constructs was evaluated using KG-1a / ISRE-Luc reporter cells as described in Sections 9.1.3 and 9.1.5.

[0543] In the first evaluation, the activity of PDL1-targeting monovalent IFN receptor antagonists and isotype control constructs was assessed in PDL1-overexpressing (OE) KG-1a / ISRE-Luc reporter cells in the absence of IFNα2b in buffer. Figures 3A-3C Compared to IFNα2b, the untargeted Iso-IFNα2b construct exhibits lower potency. Figure 3A Compared to untargeted Ab-IFNα2b, masking untargeted constructs have lower efficiency, regardless of their masking components, with monovalent cis-masking (monovalent Iso-R1-IFNα2b) and bivalent masking (bivalent Iso-R1-IFNα2b) having the lowest efficiency. Figure 3B and Figure 3C The construct aPDL1-IFNα2b exhibited the strongest activity, exceeding the activity shown by the IFNα2b control. Figure 3A The study highlighted the effectiveness of PDL1 targeting. Another target-dependent activation of IFNAR was observed in the IFNAR1-masked construct using inverse masking, where the activity of monovalent aPDL1-R1 x IFNα2b was more potent than that of Iso-R1 x IFNα2b. No increase in target-dependent activity was observed in the constructs using cis-masked monovalent and divalent R1 masks.

[0544] Next, the activity of PDL1-targeting IFN receptor antagonists and isotype control constructs was evaluated in PDL1 KO cells in the presence of 200 pM IFNα2b. Figures 3D-3F Due to the presence of 200 pM IFNα2b, all PDL1-targeting monovalent and bivalent IFN receptor antagonists and isotype control constructs were associated with relatively constant reporter activity levels, and none of the constructs showed any concentration-dependent activity changes, indicating that IFN receptor antagonists do not have target-independent antagonistic activity.

[0545] In the next evaluation, the activity of PDL1 targeting IFN receptor antagonists and isotype control constructs was assessed in PDL1-overexpressing (OE) cells in the presence of 200 pM IFNα2b. FIGS. 3G-3I Comparable concentration-response curves were generated using IFNα2b titration. FIG. 3D and FIG. 3F When PDL1-targeting monovalent IFN receptor antagonists and isotype control constructs were evaluated using PDL1 OE cells, the PDL1-targeting construct aPDL1-R1-IFNα2b with an IFNAR1 masking motif showed concentration-dependent reduced activity, indicating target-dependent IFNAR blockade. FIG. 3H Furthermore, the structure of aPDL1-R1-IFNα2b appears to be associated with its strongly reduced activity. The masking and IFN moieties of aPDL1-R1-IFNα2b are located on the same polypeptide chain and linked by a linker, as shown below. FIG. 1B As shown. A similar but trans-masked monovalent construct (PDL1-IFNα2b x R1), where the masking portion and the IFN portion are located on different polypeptide chains, as shown. FIG. 1D (As shown) is associated with incomplete IFNAR blockade.

[0546] The divalent construct aPDL1-R1-IFNα2b was associated with a similar decrease in activity as the monovalent cis-masked construct. FIG. 3I Both monovalent aPDL1-R1-IFNα2b and divalent aPDL1-R1-IFNα2b achieved complete blocking of IFNAR activity, while the trans-masked aPDL1-IFNα2b x PDL1-R1 achieved partial blocking of IFNAR activity. FIG. 3H and FIG. 3I ).

[0547] Next, in PDL1 KO ( FIGS. 4A-4C and FIGS. 4G-4I ) or OE cells ( FIGS. 4D-4F and FIGS. 4J-4L In the presence of 45 pM IFNα2b ( FIGS. 4A-4F ) or 95 pM IFNβ ( FIGS. 4G-4L In the case of ), the activity of PDL1-targeting IFN receptor antagonists and isotype control constructs was evaluated compared with exemplary anti-IFNAR1 and anti-IFNAR2 antibodies. Titration with masked monovalent or bivalent PDL1-R1-IFN resulted in targeting IFNα2b ( FIGS. 4A-4F ) and IFNβ ( FIGS. 4G-4LThe dose-dependent PDL1-dependent inhibition response curves of the anti-IFNAR1 and anti-IFNAR2 antibodies were observed. In contrast, the anti-IFNAR1 and anti-IFNAR2 antibodies exhibited lower potency blocking of IFNα2b or IFNβ and were not PDL1-dependent. In summary, these data demonstrate that the monovalent and bivalent masking formats of Ab-R1-IFNa2b can potently target and block IFN receptors.

[0548] 9.4. Example 3: In vitro cell protective activity of an IFN receptor-targeting antagonist

[0549] The monovalent and bivalent IFN receptor antagonists and control constructs were designed and manufactured as described in Sections 9.1.1 and 9.1.2. The cytoprotective activity of the IFN receptor antagonists and control constructs was evaluated using KG-1a / ISRE-Luc reporter cells as described in Sections 9.1.3 and 9.1.6.

[0550] In the first set of evaluations, the cytoprotective activity of the IFN receptor antagonist and control constructs was assessed in PDL1 overexpression (OE) and PDL1 KOKG-1a / ISRE-Luc reporter cells co-treated with 900 pM IFNα2b for five days. This IFNα2b treatment resulted in either a cytotoxic response (detected as a flat luminescence curve due to lack of cell division) or cytotoxicity (detected as a reduced luminescence signal due to decreased cell number). Neither the PDL1-targeting unmasked construct nor the allotype control unmasked construct blocked the cytotoxic response to IFNα2b treatment. FIG. 5A and FIG. 6A (and Table E-2). Conversely, PDL1-targeting monovalent and bivalent masking IFN receptor antagonists were associated with a concentration-dependent increase in luminescence in PDL1 OE KG-1a / ISRE-Luc reporter cells, while isotype controls showed relatively constant luminescence levels ( FIGS. 5B-5C The percentage of inhibition of IFNα2b-induced cell inhibition / cytotoxicity was correlated with the IFNα2b treatment-induced cytotoxic response (Table E-2). In PDL1 KO KG-1a / ISRE-Luc reporter cells, neither monovalent nor bivalent IFN receptor antagonists were associated with luminescence (IFNα2b). FIGS. 6B-6C An increase in either the percentage of inhibition or the percentage of inhibition (Table E-2) is associated with this, indicating that the cytoprotective activity of IFN receptor antagonists is target-dependent. Anti-IFNAR1 and anti-IFNAR2 antibodies produce relatively modest cytoprotective activity, which is not PDL1-dependent. FIG. 5D and FIG. 6D (and Table E-2).

[0551] In the second assessment, IFNβ was used to trigger a cell-inhibiting / cytotoxic response, and the cytoprotective activity of the IFN receptor antagonists and control constructs was evaluated in PDL1 OE and PDL1 KO KG-1a / ISRE-Luc reporter cells co-treated with 30 pM IFNβ for 5 days. As observed in the first assessment, PDL1-targeting monovalent and bivalent masked IFN receptor antagonists (but not the control constructs) showed a concentration-dependent increase in luminescence in PDL1 OE KG-1a / ISRE-Luc reporter cells. FIGS. 7A-7C and FIGS. 8A-8C The percentage of inhibition of IFNα2b-induced cell inhibition / cytotoxicity was correlated with the results (Table E-2). Again, anti-IFNAR1 and anti-IFNAR2 antibodies produced a relatively modest increase in luminescence independent of PDL1. FIG. 7D and FIG. 8D (and Table E-2). In summary, these data demonstrate that monovalent and bivalent masked IFN receptor antagonists can achieve potent target-dependent cytoprotective activity.

[0552] 9.5. Example 4: Blocking IFN-induced cell inhibition activity with IFN receptor-targeting antagonists

[0553] Monovalent and bivalent IFNAR1 or IRNAR2 masking constructs were designed and manufactured as described in Sections 9.1.1 and 9.1.2. Cell viability with and without IFN receptor antagonists was evaluated in PDL1 KO and PDL1 overexpression (OE) KG-1a / ISRE-Luc reporter cells in the presence of IFNa2b or IFNβ, as described in Section 9.1.6.

[0554] In PDL1-KO cells, IFNα2b exhibits cell-inhibiting effects in a dose-dependent manner. FIG. 9A In the presence of IFNα2b, all isotypes or PDL1-targeting monovalent IFN receptor antagonists and control constructs showed relatively constant luminescence levels ( FIG. 9B ). Targeting and isotype-specific bivalent IFN receptor antagonists were associated with weak blockade of cytoseptic activity in the absence of PD-L1 expression. FIG. 9C ).

[0555] Next, cell viability of PD-L1 OE KG-1a / ISRE-Luc cells was assessed in the presence of 900 pM IFNα2b, an IFN receptor antagonist, and a control construct. All control molecules were associated with relatively constant luminescence levels. FIG. 9D However, PD-L1-targeting monovalent and bivalent IFN receptor antagonists are associated with concentration-dependent IFNα2b-blocking cytoseptic effects. FIG. 9E and FIG. 9F Both the monovalent and divalent cis-masking molecule (aPDL1-R1-IFNα2b) significantly blocked the inhibitory activity of IFNα2b in cells. FIG. 9E and FIG. 9F The trans-masking molecule (aPDL1-IFNα2b x R1) also achieved significant blocking, although at a lower level compared to the monovalent aPDL1-R1-IFNα2b of the cis-masking molecule. FIG. 9E ).

[0556] In the next evaluation, cell viability of PDL1 KO KG-1a / ISRE-Luc cells was assessed in the presence of IFNβ. Similarly, IFNβ itself also exhibits cell-inhibiting effects in a dose-dependent manner. FIG. 9G In the presence of IFNβ, all targeting monovalent IFN receptor antagonists and control constructs showed relatively constant luminescence levels. FIG. 9H ). Targeting and isotype-specific bivalent IFN receptor antagonists are associated with weak blockade of cell inhibition. FIG. 9I ).

[0557] Next, cell viability of PDL1 OE KG-1a / ISRE-Luc cells was assessed in the presence of 30 pM IFNβ. All control molecules correlated with relatively constant luminescence levels. FIGS. 9J-9L However, PD-L1-targeting monovalent and bivalent IFN receptor antagonists are associated with concentration-dependent blockade of IFNβ activity. Both the monovalent and bivalent cis-masking molecules (aPDL1-R1-IFNα2b) significantly block IFNβ cellular inhibitory activity. FIG. 9K and FIG. 9L The trans-masking molecule (aPDL1-IFNα2b x R1) also achieved significant blocking, although at a lower level compared to the cis-masking monovalent aPDL1-R1-IFNα2b. FIG. 9K ).

[0558] 9.6. Example 5: Effect of IFN receptor antagonists on IP10 release

[0559] Monovalent and bivalent IFNAR1 or IFNAR2 masking constructs were designed and manufactured as described in Sections 9.1.1 and 9.1.2. The effectiveness of the IFNAR1 and IFNAR2 masking constructs was evaluated in PDL1-expressing mononuclear cell-derived DCs (MoDcCs). MoDcCs were incubated with individual constructs for three days, after which the supernatant was collected and IP10 was measured using an AlphaLISA.

[0560] PDL1 expression in MoDC was confirmed. FIG. 10B The IFNAR1 masking construct exhibited target-dependent blocking activity against IFNα2b-mediated IP10 release in MoDC. FIG. 10A Conversely, the IFNAR2 masking construct does not block IP10 release ( FIG. 10A Furthermore, compared to the IP10 release blocking efficacy demonstrated by the bivalent IFNAR1-masked PDL1-targeting construct, the monovalent IFNAR1-masked PDL1-targeting construct was associated with higher IP10 release blocking efficacy. FIG. 10A ).

[0561] 9.7. Example 6: In vitro signal transduction activity of IFN receptor antagonists

[0562] Allotype or PDL1-targeting IFN receptor antagonists and control constructs were designed and manufactured as described in Sections 9.1.1 and 9.1.2. The signaling activity of the allotype or PDL1-targeting IFN receptor antagonists and control constructs was assessed using PDL1-KO and PDL1-overexpressing (OE) KG-1a / ISRE-Luc reporter cells as described in Sections 9.1.3 and 9.1.5.

[0563] In the first evaluation, the activity of allotype or PDL1-targeting bivalent IFN receptor antagonists and control constructs was assessed in PDL1 KO KG-1a / ISRE-Luc reporter cells. In the absence of hIFNα2b or hIFNβ, all allotype and PDL1-targeting bivalent IFN receptor antagonists showed relatively low levels of ISRE-Luc activity (…). FIG. 11A In the presence of 20 pMhIFNα2b, all bivalent IFN receptor antagonists were associated with low levels of antagonism. FIG. 11B In the presence of 10 pM hIFNβ, a target-independent antagonistic effect was observed in the IFNβ-containing construct. FIG. 11C ).

[0564] Next, the activities of allotype or PDL1-targeting bivalent IFN receptor antagonists and control constructs were evaluated in PDL1 OE KG-1a / ISRE-Luc reporter cells. Again, in the absence of hIFNα2b or hIFNβ, all allotype and PDL1-targeting bivalent IFN receptor antagonists showed relatively low levels of ISRE-Luc activity. FIG. 11D In the presence of 20 pMhIFNα2b, bivalent PDL1-targeting IFN receptor antagonists containing both IFNα2b and IFNβ were associated with significant antagonistic effects. FIG. 11E In the presence of 10 pM hIFNβ, bivalent PDL1-targeting IFN receptor antagonists containing both IFNα2b and IFNβ were associated with significant antagonistic effects. FIG. 11F ).

[0565] In all experiments, the aPDL1-R1 construct lacking any IFN molecules did not exhibit any antagonistic effect. FIGS. 11A-11F ).

[0566] 9.1. Example 7: In vitro signal transduction activity of PDL1 and EGFR-targeting IFN receptor antagonists containing uIFN.

[0567] As described in Sections 9.1.1 and 9.1.2, isotype, PDL1, or EGFR-targeting IFN receptor antagonists and control constructs comprising a universal type I interferon (uIFN) moiety are designed and manufactured. As described in Sections 9.1.3 and 9.1.5, the signal transduction activity of the isotype or PDL1-targeting IFN receptor antagonists and control constructs is evaluated using PDL1-KO and PDL1-OE KG-1a / ISRE-Luc reporter cells or EGFR OE and EGFR-free cells.

[0568] In the absence of hIFNα2b or hIFNβ, the isotype and PDL1-targeting IFN antagonist constructs exhibited similar ISRE-Luc activity in PDL1KO cells. FIG. 12A The isotype control construct showed similar activity in PDL1 OE cells, while the PDL1-targeting IFN antagonist showed no activity in these cells. FIG. 12B In the presence of 20 pMhIFNα2b, both constructs exhibited stable levels of ISRE-Luc activity in PDL1 KO cells. FIG. 12C PDL1-targeting IFN antagonists inhibit this activity in PDL1 OE cells. FIG. 12DHowever, no inhibition was observed in the isotype control. Similarly, in the presence of 10 pM hIFNβ, both constructs showed stable levels of ISRE-Luc activity in PDL1 KO cells. FIG. 12E However, in PDL1 OE cells, PDL1-targeting IFN antagonists exhibit strong antagonistic effects.

[0569] Next, the EGFR-targeting construct and the allotype construct were evaluated. In the absence of hIFNα2b or hIFNβ, the allotype construct and the EGFR-targeting IFN antagonist construct showed similar ISRE-Luc activity in EGFR-free cells. FIG. 13A The isotype control construct showed similar activity in PDL1 OE cells, while the PDL1-targeting IFN antagonist showed reduced activity in these cells. FIG. 13B In the presence of 20 pM hIFNα2b, both constructs showed stable levels of ISRE-Luc activity in EGFR-free cells. FIG. 13C In EGFR OE cells, EGFR-targeting IFN antagonists exhibit significant antagonistic effects. FIG. 13D However, the isotype control did not. Similarly, in the presence of 10 pMhIFNβ, both constructs showed stable levels of ISRE-Luc activity in "EGFR-free" cells. FIG. 13E FIG. 13E Furthermore, EGFR-targeting IFN antagonists showed significant antagonistic effects in EGFR OE cells.

[0570] 10. Citation of References

[0571] All publications, patents, patent applications and other documents cited in this application are incorporated herein by reference in their entirety for all purposes, as if each individual publication, patent, patent application or other document were individually cited and incorporated herein by reference for all purposes. In the event of any inconsistency between the teachings of one or more of the references incorporated herein and the contents of this disclosure, the teachings of this specification shall prevail.

Claims

1. A type I interferon (IFN) receptor antagonist, comprising: (a) Anchoring portion of cells that express type I interferon receptors; (b) Type I interferon α receptor 1 (IFNAR1) portion; (c) Type I interferon (IFN) portion; (d) Connect the anchoring portion to the IFNAR1 portion or the partition portion of the IFN portion; as well as (e) Optionally, a connector is used to connect the IFNAR1 portion and the IFN portion.

2. The IFN receptor antagonist of claim 1, wherein the anchoring portion is capable of binding to extracellular matrix (ECM) antigens, tumor reactive lymphocyte antigens, cell surface molecules of tumor or viral lymphocytes, T cell antigens (TCA), checkpoint inhibitors, tumor-associated antigens (TAA), dendritic cell (DC) or other antigen-presenting cell (APC) antigens, or natural killer (NK) cell antigens.

3. The IFN receptor antagonist of claim 1, wherein the anchoring portion is a targeting portion.

4. The IFN receptor antagonist of claim 3, wherein the targeting portion is scFv or Fab.

5. The IFN receptor antagonist of claim 3, wherein the targeting portion is capable of binding to a checkpoint inhibitor, optionally 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.

6. The IFN receptor antagonist of claim 5, wherein the checkpoint inhibitor is PDL1.

7. The IFN receptor antagonist of claim 6, wherein the targeting portion is an antigen-binding fragment of an anti-PDL1 antibody.

8. The IFN receptor antagonist according to any one of claims 1 to 7, wherein the septum is an Fc domain.

9. The IFN receptor antagonist according to any one of claims 1 to 8, wherein the linker is a non-cleavable linker (NCL).

10. The IFN receptor antagonist of claim 9, wherein the NCL is the NCL described in Table E.

11. The IFN receptor antagonist according to any one of claims 1 to 10, further comprising an additional connector connecting the septum portion to the IFNAR1 portion.

12. The IFN receptor antagonist according to any one of claims 1 to 10, further comprising an additional connector connecting the septum portion to the IFN portion.

13. The IFN receptor antagonist according to any one of claims 1 to 12, further comprising an additional anchoring portion.

14. The IFN receptor antagonist of claim 13, wherein the additional anchoring portion is an additional targeting portion.

15. The IFN receptor antagonist of claim 14, wherein the additional targeting moiety is scFv or Fab.

16. The IFN receptor antagonist of claim 15, wherein the additional targeting portion specifically binds to the same target as the anchoring portion.

17. The IFN receptor antagonist according to any one of claims 14 to 16, wherein the additional targeting portion is capable of binding to a checkpoint inhibitor, said checkpoint inhibitor optionally being CTLA-4, PD1, PDL1, PDL2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, or CHK2.

18. The IFN receptor antagonist of claim 17, wherein the checkpoint inhibitor is PDL1.

19. The IFN receptor antagonist of claim 18, wherein the additional targeting portion is an antigen-binding fragment of an anti-PD1 antibody.

20. The IFN receptor antagonist according to any one of claims 13 to 19, further comprising an additional septum operatively connected to the additional anchoring portion.

21. The IFN receptor antagonist according to any one of claims 1 to 20, wherein the IFN portion is masked by the IFNAR1 portion.

22. The IFN receptor antagonist according to any one of claims 1 to 21, wherein the IFN receptor antagonist comprises a first polypeptide chain and a second polypeptide chain.

23. The IFN receptor antagonist of claim 22, wherein the first polypeptide chain comprises: (a) the anchoring portion or its components; (b) the dividing portion; and (c) The IFNAR1 portion.

24. The IFN receptor antagonist of claim 23, wherein the second polypeptide chain comprises: (a) additional anchoring portions or components thereof; and (b) Additional separators.

25. The IFN receptor antagonist of claim 23 or 24, wherein the first polypeptide chain further comprises the IFN moiety.

26. The IFN receptor antagonist of claim 24, wherein the second polypeptide chain further comprises the IFN moiety.

27. The IFN receptor antagonist of claim 26, wherein the second polypeptide chain comprises, in an N-terminal to C-terminal orientation: (a) Additional anchoring portions or components thereof; (b) Additional dividing sections; and (c) The IFN portion.

28. The IFN receptor antagonist according to any one of claims 22 to 27, wherein the second polypeptide chain further comprises an additional IFNAR1 moiety and an additional IFN moiety.

29. The IFN receptor antagonist according to any one of claims 1 to 28, wherein the IFN portion comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or 100% sequence identity with: (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ, or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

30. The IFN receptor antagonist of claim 29, wherein the IFN portion comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or 100% sequence identity with: (a) full-length mature human IFNα2b, or (b) mature human IFNα2b having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

31. The IFN receptor antagonist of claim 30, wherein the IFN moiety is: (a) a full-length mature human IFNα2b, or (b) a mature human IFNα2b having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

32. The IFN receptor antagonist according to any one of claims 1 to 31, wherein the IFNAR1 moiety comprises or is composed of an amino acid sequence having at least 90%, at least 95%, at least 98%, or 100% sequence identity with, or consisting of, the following: (i) the SD2 and SD3 domains of human IFNAR1, (ii) the SD1, SD2 and SD3 domains of human IFNAR1, or (iii) the SD1, SD2, SD3 and SD4 domains of human IFNAR1.

33. The IFN receptor antagonist of claim 28, wherein the additional IFN portion comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or 100% sequence identity with: (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ, or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ, having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

34. The IFN receptor antagonist of claim 33, wherein the additional IFN portion comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or 100% sequence identity with: (a) full-length mature human IFNα2b, or (b) mature human IFNα2b having a truncation of up to 15 amino acids at its N-terminus and / or its C-terminus.

35. The IFN receptor antagonist of claim 28, wherein the additional IFNAR1 moiety comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity with, or is composed of, the following amino acid sequences: (i) the SD2 and SD3 domains of human IFNAR1, (ii) the SD1, SD2 and SD3 domains of human IFNAR1, or (iii) the SD1, SD2, SD3 and SD4 domains of human IFNAR1.

36. The IFN receptor antagonist of claim 28, wherein the additional IFNAR1 portion is: (i) the SD2 and SD3 domains of human IFNAR1, (ii) the SD1, SD2, and SD3 domains of human IFNAR1, or (iii) the SD1, SD2, SD3, and SD4 domains of human IFNAR1.

37. The IFN receptor antagonist according to any one of claims 1 to 36, wherein the IFN receptor antagonist does not contain the IFNAR2 portion.

38. The IFN receptor antagonist of claim 37, wherein the IFNAR2 moiety comprises or is composed of an amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity with, or consisting of, the following: (i) the D1 domain of human IFNAR2, or (ii) the D1 and D2 domains of human IFNAR2.

39. An IFN receptor antagonist, optionally an IFN receptor antagonist according to any one of claims 1 to 38, comprising a polypeptide chain having the following configurations: (a) the configuration of the two halves shown in FIG. 1B, (b) the configuration of the two halves shown in FIG. 1C, (c) the configuration of the two halves shown in FIG. 1D, (d) the configuration of the two halves shown in FIG. 1E, and (e) the configuration of the two halves shown in FIG. 1F.

40. One or more nucleic acids encoding an IFN receptor antagonist according to any one of claims 1 to 39.

41. A host cell engineered to express an IFN receptor antagonist according to any one of claims 1 to 39 or a nucleic acid according to claim 40.

42. A method for producing an IFN receptor antagonist according to any one of claims 1 to 39, the method comprising culturing a host cell according to claim 41 and recovering the IFN receptor antagonist expressed by the host cell.

43. A pharmaceutical composition comprising an IFN receptor antagonist according to any one of claims 1 to 39 and an excipient, optionally further comprising an oncolytic virus.

44. A method for inhibiting IFN signaling in cells expressing (a) an IFN receptor and (b) a molecule specifically binding to an anchoring moiety, the method comprising contacting the cells with an IFN receptor antagonist according to any one of claims 1 to 39 or a pharmaceutical composition according to claim 43.

45. The method of claim 44, wherein the cell is: (a) an immune cell, optionally a B cell, or (b) a cancer cell.

46. ​​The method according to claim 44 or 45, wherein the method is an in vitro method.

47. A method of treating a subject suffering from cancer, the method comprising administering to the subject in need an IFN receptor antagonist according to any one of claims 1 to 39 or a pharmaceutical composition according to claim 43.

48. The method of claim 47, further comprising: The oncolytic virus was administered to the subject.

49. A method of treating a subject suffering from an immune disorder or condition, the method comprising administering to the subject an IFN receptor antagonist according to any one of claims 1 to 39 or a pharmaceutical composition according to claim 43, optionally wherein the immune disorder or condition is systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSc), or Sjögren's syndrome (SS).

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