Interferon receptor antagonists and their use

Abstract

This disclosure provides interferon (IFN) receptor antagonists. The IFN receptor antagonists disclosed herein include an anchoring moiety, an IFN masking moiety, an IFN moiety, and a separator moiety, for example, a targeting moiety that recognizes an antigen associated with cells expressing type 1 interferon receptors and anchors the IFN receptor antagonist to such cells. This disclosure further provides methods of using IFN receptor antagonists in IFN signaling inhibition, including pharmaceutical compositions comprising IFN receptor antagonists and methods of treatment. Nucleic acids encoding IFN receptor antagonists, recombinant cells expressing IFN receptor antagonists, and methods for producing IFN receptor antagonists are also disclosed.

Description

[Technical Field]

[0001] 1. Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 501,840, filed on 12 May 2023, and U.S. Provisional Application No. 63 / 597,502, filed on 9 November 2023, the contents of each of those applications being incorporated herein by reference in their entirety.

[0002] 2. Sequence Listing This application includes a sequence listing submitted electronically in XML format, which is incorporated herein by reference in its entirety. The sequence listing, created on May 6, 2024, is named RGN-035WO_SL.xml and has a size of 146,865 bytes. [Background technology]

[0003] 3.Background Type I interferons (IFNs) are a family of cytokines that function as immunomodulators in innate and adaptive immune responses. Most cell types constitutively produce low levels of type I IFNs; however, infection and other triggers can stimulate the production of these molecules, which then bind to the IFNAR1 / IFNAR2 receptor complex and signal through the IFNAR1 / IFNAR2 receptor complex.

[0004] Several pieces of evidence strongly suggest elevated type I IFN and increased IFN signaling in the pathogenesis of various autoimmune inflammatory diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSc), and Sjögren's syndrome (SS). Inhibitors of IFN signaling are currently being tested in several clinical trials (Chasset et al., 2021, Front.Pharmacol.12:633821.doi:10.3389 / fphar.2021.633821).

[0005] Furthermore, type I IFNs are also upmodulated in response to oncolytic virus (OV) therapy. OVs are biotherapeutic agents effective against solid tumors. Unfortunately, upmodulation of type I IFNs enhances autoimmune side effects in patients, increases resistance of tumor cells to oncolytic viruses, and reduces the therapeutic effectiveness of this approach (El-Sayes et al., 2022, Oncolytics 25:16-30). Preclinical studies have shown that the combination of OV therapy with IFN signaling inhibitors is associated with improved 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):eaao1613).

[0006] Nevertheless, the harm associated with treatments that indiscriminately inhibit type I IFN signaling may outweigh the benefits, given that the IFNAR1 / IFNAR2 receptor complex is expressed throughout the body, and systemic suppression of type I IFN signaling can weaken the body's immune response, such as its ability to fight infection.

[0007] Therefore, there is a need in the art for novel therapies that specifically target and modulate inappropriate or prolonged type I IFN signaling in specific cells. [Overview of the Initiative]

[0008] 4. Overview This disclosure relates to novel molecular constructs referred to herein as “IFN receptor antagonists,” which comprise an IFN moiety masked by an IFNAR1 moiety and are capable of robust IFN signaling inhibition. Surprisingly, although they contain an IFN molecule, the IFN receptor antagonists described herein do not activate IFN signaling in cells, but function solely as IFN signaling blockers, competing for binding with exogenous IFN and preventing or significantly reducing IFN signaling in cells via the type I interferon receptor.

[0009] IFN receptor antagonists generally include, in addition to the IFN and IFNAR1 moieties, an anchor moiety (e.g., a targeting moiety), a separator moiety (e.g., an Fc domain), and optionally, a linker connecting the IFNAR1 moiety to the IFN moiety. IFN receptor antagonists may further include one or more linkers connecting one or more components (e.g., a separator moiety connecting the IFNAR1 or IFN moiety). The anchor moiety can bind to a target molecule present on the surface of a cell expressing the type I interferon receptor. For example, the anchor moiety may be a targeting moiety containing an antigen-binding domain capable of binding to such a target molecule. The separator moiety allows for the simultaneous binding of the anchor moiety to the target molecule and the simultaneous binding of the IFN moiety to the type I interferon receptor on the cell. For example, the separator moiety may be an Fc domain.

[0010] Exemplary IFN moieties that can be used in the IFN receptor antagonists of this disclosure are described in Section 6.3.

[0011] Exemplary masking portions that can be used in the IFN receptor antagonists of this disclosure are described in Section 6.4.

[0012] Linkers that can be used with the IFN receptor antagonists of this disclosure are described in Section 6.5.

[0013] The anchor portions that can be used in the IFN receptor antagonists of this disclosure are described in Section 6.6. The targeting portions are described in Section 6.6.1, while the targeting portion format is disclosed in Section 6.6.1.1.

[0014] The separator portion that can be incorporated into the IFN receptor antagonist of this disclosure is described in Section 6.7. The Fc domain is described in Section 6.7.1.

[0015] Exemplary IFN receptor antagonists of this disclosure are described in Section 6.2 and in numbered embodiments 1 to 90.

[0016] This disclosure further provides nucleic acids encoding the IFN receptor antagonists of this disclosure. The nucleic acids encoding the IFN receptor antagonists may be a single nucleic acid (e.g., a vector encoding all 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). This disclosure further provides host cells and cell lines engineered to express the nucleic acids and IFN receptor antagonists of this disclosure. This disclosure further provides methods for producing the IFN receptor antagonists of this disclosure. Exemplary nucleic acids, host cells, and cell lines, as well as methods for producing the IFN receptor antagonists, are described in Section 6.8 and Numbered Embodiments 91-93.

[0017] 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 numbered embodiments 94 and 95.

[0018] For example, methods of using the IFN receptor antagonists and pharmaceutical compositions of this disclosure to treat cancer or autoimmune conditions are further provided herein. Exemplary methods are described in Section 6.10 and numbered embodiments 96-112. [Brief explanation of the drawing]

[0019] 5. Brief description of the drawing [Figure 1A] Figures 1A to 1F are schematic diagrams representing targeted IFN receptor antagonists. Figure 1A shows the components used to generate a masked IFN receptor antagonist (TM = targeting moiety, IFN = interferon, IFNAR1 = interferon alpha / beta receptor 1). In use in Figures 1A to 1F, "IFN" generally refers to any IFN moiety (e.g., IFNα2b), and "IFNAR1" generally refers to any IFNAR1 moiety. The targeting moiety in Figures 1A to 1F is shown as Fab, but other targeting moieties (e.g., scFv) can be used instead of Fab. [Figure 1B] Figures 1A to 1F are schematic diagrams representing targeted IFN receptor antagonists. Figures 1B to 1F show the structures of targeted IFN receptor antagonist constructs. In use in Figures 1A to 1F, "IFN" generally refers to any IFN moiety (e.g., IFNα2b), and "IFNAR1" generally refers to any IFNAR1 moiety. The targeted moiety in Figures 1A to 1F is shown as Fab, but other targeted moieties (e.g., scFv) can be used instead of Fab. [Figure 1C] Figures 1A to 1F are schematic diagrams representing targeted IFN receptor antagonists. Figures 1B to 1F show the structures of targeted IFN receptor antagonist constructs. In use in Figures 1A to 1F, "IFN" generally refers to any IFN moiety (e.g., IFNα2b), and "IFNAR1" generally refers to any IFNAR1 moiety. The targeted moiety in Figures 1A to 1F is shown as Fab, but other targeted moieties (e.g., scFv) can be used instead of Fab. [Figure 1D]Figures 1A - 1F are schematic diagrams representing targeted IFN receptor antagonists. Figures 1B - 1F show the structures of the targeted IFN receptor antagonist constructs. When used in Figures 1A - 1F, "IFN" generally refers to any IFN moiety (e.g., IFNα2b), and "IFNAR1" generally refers to any IFNAR1 moiety. The targeted moieties in Figures 1A - 1F are shown as Fab, but other targeted moieties (e.g., scFv) can be used in place of Fab. [Figure 1E] Figures 1A - 1F are schematic diagrams representing targeted IFN receptor antagonists. Figures 1B - 1F show the structures of the targeted IFN receptor antagonist constructs. When used in Figures 1A - 1F, "IFN" generally refers to any IFN moiety (e.g., IFNα2b), and "IFNAR1" generally refers to any IFNAR1 moiety. The targeted moieties in Figures 1A - 1F are shown as Fab, but other targeted moieties (e.g., scFv) can be used in place of Fab. [Figure 1F] Figures 1A - 1F are schematic diagrams representing targeted IFN receptor antagonists. Figures 1B - 1F show the structures of the targeted IFN receptor antagonist constructs. When used in Figures 1A - 1F, "IFN" generally refers to any IFN moiety (e.g., IFNα2b), and "IFNAR1" generally refers to any IFNAR1 moiety. The targeted moieties in Figures 1A - 1F are shown as Fab, but other targeted moieties (e.g., scFv) can be used in place of Fab. [Figure 2A] Shows the staining and bioassay activity profiles of an untargeted construct containing an IFNAR1 (R1) masking moiety. It is a graph showing the staining profile of a bivalent masked construct. [Figure 2B] Shows the staining and bioassay activity profiles of an untargeted construct containing an IFNAR1 (R1) masking moiety. It is a graph showing the in vitro activity of a bivalent masked construct. [Figure 3A]This shows the in vitro activity of exemplary targeted IFN receptor antagonists in the presence or absence of a certain amount of IFNα2b. PDL1-targeted constructs and isotype control constructs (Figures 3A, 3D, and 3G) were evaluated in PDL1-overexpressing (OE) KG1a / ISRE-Luc cells (Figures 3A-3C and 3G-3I) and titrated in the absence of 200 pM IFNα2b (Figures 3A-3C). [Figure 3B] The in vitro activity of exemplary targeted IFN receptor antagonists in the presence or absence of a certain amount of IFNα2b is shown. PDL1-targeted and masked monovalent constructs (Figures 3B, 3E, and 3H) were evaluated in PDL1-overexpressing (OE) KG1a / ISRE-Luc cells (Figures 3A–3C and 3G–3I) and titrated in the absence of 200 pM IFNα2b (Figures 3A–3C). [Figure 3C] The in vitro activities of exemplary targeted IFN receptor antagonists in the presence or absence of a certain amount of IFNα2b are shown. The in vitro activities of PDL1-targeted constructs and masked bivalent (Figure 3C, Figure 3F, and Figure 3I) constructs were evaluated in PDL1-overexpressing (OE) KG1a / ISRE-Luc cells (Figures 3A-3C and 3G-3I) and titrated in the absence of 200 pM IFNα2b (Figures 3A-3C). [Figure 3D] The in vitro activity of exemplary targeted IFN receptor antagonists in the presence or absence of a certain amount of IFNα2b is shown. PDL1-targeted constructs and isotype control constructs (Figures 3A, 3D, and 3G) were evaluated in PDL1 knockout (KO) KG1a / ISRE-Luc cells (Figures 3D-3F) and titrated in the presence of 200 pM IFNα2b (Figures 3D-3I). [Figure 3E]The in vitro activity of exemplary targeted IFN receptor antagonists in the presence or absence of a certain amount of IFNα2b is shown. PDL1-targeted and masked monovalent constructs (Figures 3B, 3E, and 3H) were evaluated in PDL1 knockout (KO) KG1a / ISRE-Luc cells (Figures 3D-3F) and titrated in the presence of 200 pM IFNα2b (Figures 3D-3I). [Figure 3F] The in vitro activities of exemplary targeted IFN receptor antagonists in the presence or absence of a certain amount of IFNα2b are shown. The in vitro activities of PDL1-targeted constructs and masked bivalent (Figures 3C, 3F, and 3I) constructs were evaluated in PDL1 knockout (KO) KG1a / ISRE-Luc cells (Figures 3D-3F) and titrated in the presence of 200 pM IFNα2b (Figures 3D-3I). [Figure 3G] The in vitro activity of exemplary targeted IFN receptor antagonists in the presence or absence of a certain amount of IFNα2b is shown. PDL1-targeted constructs and isotype control constructs (Figures 3A, 3D, and 3G) were evaluated in PDL1-overexpressing (OE) KG1a / ISRE-Luc cells (Figures 3A-3C and 3G-3I) and titrated in the presence of 200 pM IFNα2b (Figures 3D-3I). [Figure 3H] The in vitro activity of exemplary targeted IFN receptor antagonists in the presence or absence of a certain amount of IFNα2b is shown. PDL1-targeted and masked monovalent constructs (Figures 3B, 3E, and 3H) were evaluated in PDL1-overexpressing (OE) KG1a / ISRE-Luc cells (Figures 3A-3C and 3G-3I) and titrated in the presence of 200 pM IFNα2b (Figures 3D-3I). [Figure 3I]The in vitro activities of exemplary targeted IFN receptor antagonists in the presence or absence of a certain amount of IFNα2b are shown. The in vitro activities of PDL1-targeted constructs and masked bivalent (Figures 3C, 3F, and 3I) constructs were evaluated in PDL1-overexpressing (OE) KG1a / ISRE-Luc cells (Figures 3A-3C and 3G-3I) and titrated in the presence of 200 pM IFNα2b (Figures 3D-3I). [Figure 4A] The in vitro activities of exemplary targeted IFN receptor antagonists and anti-IFNAR1 or anti-IFNAR2 antibodies in the presence of a certain amount of IFNα2b or IFNβ are shown. The in vitro activities of PDL1-targeted constructs and isotype control constructs, masked monovalent constructs, masked bivalent constructs, and anti-IFNAR1 or anti-IFNAR2 antibodies were evaluated in PDL1 KO (Figures 4A-4C and 4G-4I) KG1a / ISRE-Luc cells and titrated in the presence of 45 pM IFNα2b (Figures 4A-4F). [Figure 4B] The in vitro activities of exemplary targeted IFN receptor antagonists and anti-IFNAR1 or anti-IFNAR2 antibodies in the presence of a certain amount of IFNα2b or IFNβ are shown. The in vitro activities of PDL1-targeted constructs and isotype control constructs, masked monovalent constructs, masked bivalent constructs, and anti-IFNAR1 or anti-IFNAR2 antibodies were evaluated in PDL1 KO (Figures 4A-4C and 4G-4I) KG1a / ISRE-Luc cells and titrated in the presence of 45 pM IFNα2b (Figures 4A-4F). [Figure 4C]The in vitro activities of exemplary targeted IFN receptor antagonists and anti-IFNAR1 or anti-IFNAR2 antibodies in the presence of a certain amount of IFNα2b or IFNβ are shown. The in vitro activities of PDL1-targeted constructs and isotype control constructs, masked monovalent constructs, masked bivalent constructs, and anti-IFNAR1 or anti-IFNAR2 antibodies were evaluated in PDL1 KO (Figures 4A-4C and 4G-4I) KG1a / ISRE-Luc cells and titrated in the presence of 45 pM IFNα2b (Figures 4A-4F). [Figure 4D] The in vitro activities of exemplary targeted IFN receptor antagonists and anti-IFNAR1 or anti-IFNAR2 antibodies in the presence of a certain amount of IFNα2b or IFNβ are shown. The in vitro activities of PDL1-targeted constructs and isotype control constructs, masked monovalent constructs, masked bivalent constructs, and anti-IFNAR1 or anti-IFNAR2 antibodies were evaluated in PDL1 OE (Figures 4D-4F and 4J-4L) KG1a / ISRE-Luc cells and titrated in the presence of 45 pM IFNα2b (Figures 4A-4F). [Figure 4E] The in vitro activities of exemplary targeted IFN receptor antagonists and anti-IFNAR1 or anti-IFNAR2 antibodies in the presence of a certain amount of IFNα2b or IFNβ are shown. The in vitro activities of PDL1-targeted constructs and isotype control constructs, masked monovalent constructs, masked bivalent constructs, and anti-IFNAR1 or anti-IFNAR2 antibodies were evaluated in PDL1 OE (Figures 4D-4F and 4J-4L) KG1a / ISRE-Luc cells and titrated in the presence of 45 pM IFNα2b (Figures 4A-4F). [Figure 4F]The in vitro activities of exemplary targeted IFN receptor antagonists and anti-IFNAR1 or anti-IFNAR2 antibodies in the presence of a certain amount of IFNα2b or IFNβ are shown. The in vitro activities of PDL1-targeted constructs and isotype control constructs, masked monovalent constructs, masked bivalent constructs, and anti-IFNAR1 or anti-IFNAR2 antibodies were evaluated in PDL1 OE (Figures 4D-4F and 4J-4L) KG1a / ISRE-Luc cells and titrated in the presence of 45 pM IFNα2b (Figures 4A-4F). [Figure 4G] The in vitro activities of exemplary targeted IFN receptor antagonists and anti-IFNAR1 or anti-IFNAR2 antibodies in the presence of a certain amount of IFNα2b or IFNβ are shown. The in vitro activities of PDL1-targeted constructs and isotype control constructs, masked monovalent constructs, masked bivalent constructs, and anti-IFNAR1 or anti-IFNAR2 antibodies were evaluated in PDL1 KO (Figures 4A-4C and 4G-4I) KG1a / ISRE-Luc cells and titrated in the presence of 95 pM IFNβ (Figures 4G-4L). [Figure 4H] The in vitro activities of exemplary targeted IFN receptor antagonists and anti-IFNAR1 or anti-IFNAR2 antibodies in the presence of a certain amount of IFNα2b or IFNβ are shown. The in vitro activities of PDL1-targeted constructs and isotype control constructs, masked monovalent constructs, masked bivalent constructs, and anti-IFNAR1 or anti-IFNAR2 antibodies were evaluated in PDL1 KO (Figures 4A-4C and 4G-4I) KG1a / ISRE-Luc cells and titrated in the presence of 95 pM IFNβ (Figures 4G-4L). [Figure 4I]The in vitro activities of exemplary targeted IFN receptor antagonists and anti-IFNAR1 or anti-IFNAR2 antibodies in the presence of a certain amount of IFNα2b or IFNβ are shown. The in vitro activities of PDL1-targeted constructs and isotype control constructs, masked monovalent constructs, masked bivalent constructs, and anti-IFNAR1 or anti-IFNAR2 antibodies were evaluated in PDL1 KO (Figures 4A-4C and 4G-4I) KG1a / ISRE-Luc cells and titrated in the presence of 95 pM IFNβ (Figures 4G-4L). [Figure 4J] The in vitro activities of exemplary targeted IFN receptor antagonists and anti-IFNAR1 or anti-IFNAR2 antibodies in the presence of a certain amount of IFNα2b or IFNβ are shown. The in vitro activities of PDL1-targeted constructs and isotype control constructs, masked monovalent constructs, masked bivalent constructs, and anti-IFNAR1 or anti-IFNAR2 antibodies were evaluated in PDL1 OE (Figures 4D-4F and 4J-4L) KG1a / ISRE-Luc cells and titrated in the presence of 95 pM IFNβ (Figures 4G-4L). [Figure 4K] The in vitro activities of exemplary targeted IFN receptor antagonists and anti-IFNAR1 or anti-IFNAR2 antibodies in the presence of a certain amount of IFNα2b or IFNβ are shown. The in vitro activities of PDL1-targeted constructs and isotype control constructs, masked monovalent constructs, masked bivalent constructs, and anti-IFNAR1 or anti-IFNAR2 antibodies were evaluated in PDL1 OE (Figures 4D-4F and 4J-4L) KG1a / ISRE-Luc cells and titrated in the presence of 95 pM IFNβ (Figures 4G-4L). [Figure 4L]The in vitro activities of exemplary targeted IFN receptor antagonists and anti-IFNAR1 or anti-IFNAR2 antibodies in the presence of a certain amount of IFNα2b or IFNβ are shown. The in vitro activities of PDL1-targeted constructs and isotype control constructs, masked monovalent constructs, masked bivalent constructs, and anti-IFNAR1 or anti-IFNAR2 antibodies were evaluated in PDL1 OE (Figures 4D-4F and 4J-4L) KG1a / ISRE-Luc cells and titrated in the presence of 95 pM IFNβ (Figures 4G-4L). [Figure 5A] This shows exemplary in vitro cytoprotective activity of the unmasked control (Figure 5A) construct in PDL1 OE KG1a / ISRE-Luc cells. [Figure 5B] This shows exemplary in vitro cytoprotective activity of targeted constructs and isotype monovalent (Figure 5B) constructs in PDL1 OE KG1a / ISRE-Luc cells. [Figure 5C] This shows exemplary in vitro cytoprotective activity of targeted constructs and isotype-divalent (Figure 5C) constructs in PDL1 OE KG1a / ISRE-Luc cells. [Figure 5D] This shows exemplary in vitro cytoprotective activity of anti-IFNAR1 and anti-IFNAR2 antibodies in PDL1 OE KG1a / ISRE-Luc cells (Figure 5D). [Figure 6A] This shows exemplary in vitro cytoprotective activity of the unmasked control (Figure 6A) construct in PDL1 KO KG1a / ISRE-Luc cells. [Figure 6B] This demonstrates the exemplary in vitro cytoprotective activity of targeted and isotype-monovalent (Figure 6B) constructs in PDL1 KO KG1a / ISRE-Luc cells. [Figure 6C] This demonstrates the exemplary in vitro cytoprotective activity of targeted and isotype-divalent (Figure 6C) constructs in PDL1 KO KG1a / ISRE-Luc cells. [Figure 6D]This shows exemplary in vitro cytoprotective activity of anti-IFNAR1 and anti-IFNAR2 antibodies in PDL1 KO KG1a / ISRE-Luc cells (Figure 6D). [Figure 7A] This shows exemplary in vitro cytoprotective activity of the unmasked control construct (Figure 7A) in PDL1 OE KG1a / ISRE-Luc cells. [Figure 7B] This shows exemplary in vitro cytoprotective activity of targeted and isotype-monovalent (Figure 7B) constructs in PDL1 OE KG1a / ISRE-Luc cells. [Figure 7C] This shows exemplary in vitro cytoprotective activity of targeted and isotype-divalent (Figure 7C) constructs in PDL1 OE KG1a / ISRE-Luc cells. [Figure 7D] This shows exemplary in vitro cytoprotective activity of anti-IFNAR1 and anti-IFNAR2 antibodies in PDL1 OE KG1a / ISRE-Luc cells (Figure 7D). [Figure 8A] This shows exemplary in vitro cytoprotective activity of the unmasked control construct (Figure 8A) in PDL1 KO KG1a / ISRE-Luc cells. [Figure 8B] This shows exemplary in vitro cytoprotective activity of targeted and isotype-monovalent (Figure 8B) constructs in PDL1 KO KG1a / ISRE-Luc cells. [Figure 8C] This demonstrates the exemplary in vitro cytoprotective activity of targeted and isotype-divalent (Figure 8C) constructs in PDL1 KO KG1a / ISRE-Luc cells. [Figure 8D] This shows exemplary in vitro cytoprotective activity of anti-IFNAR1 and anti-IFNAR2 antibodies in PDL1 KO KG1a / ISRE-Luc cells (Figure 8D). [Figure 9A]The in vitro activity of exemplary targeted IFN receptor antagonists is shown in the presence or absence of a certain amount of IFNα2b (Figures 9A–9F). PDL1-targeted and isotype-controlled (Figures 9A, 9D, 9G, and 9J) were evaluated in PDL1 knockout (KO) KG1a / ISRE-Luc cells (Figures 9A–9C and 9G–9I). [Figure 9B] The in vitro activity of exemplary targeted IFN receptor antagonists is shown in the presence or absence of a certain amount of IFNα2b (Figures 9A–9F). PDL1-targeted and masked monovalents (Figures 9B, 9E, 9H, and 9K) were evaluated in PDL1 knockout (KO) KG1a / ISRE-Luc cells (Figures 9A–9C and 9G–9I). [Figure 9C] The in vitro activities of exemplary targeted IFN receptor antagonists are shown in the presence or absence of a certain amount of IFNα2b (Figures 9A–9F). The in vitro activities of PDL1-targeted and masked bivalent (Figures 9C, 9F, 9I, and 9L) constructs were evaluated in PDL1 knockout (KO) KG1a / ISRE-Luc cells (Figures 9A–9C and 9G–9I). [Figure 9D] The in vitro activity of exemplary targeted IFN receptor antagonists is shown in the presence or absence of a certain amount of IFNα2b (Figures 9A–9F). PDL1-targeted and isotype-controlled (Figures 9A, 9D, 9G, and 9J) were evaluated in PDL1-overexpressing (OE) KG1a / ISRE-Luc cells (Figures 9D–9F and 9J–9L). [Figure 9E] The in vitro activity of exemplary targeted IFN receptor antagonists is shown in the presence or absence of a certain amount of IFNα2b (Figures 9A–9F). PDL1-targeted and masked monovalents (Figures 9B, 9E, 9H, and 9K) were evaluated in PDL1-overexpressing (OE) KG1a / ISRE-Luc cells (Figures 9D–9F and 9J–9L). [Figure 9F]The in vitro activity of exemplary targeted IFN receptor antagonists in the presence or absence of a certain amount of IFNα2b (Figures 9A–9F) is shown. The in vitro activity of PDL1-targeted and masked bivalent (Figures 9C, 9F, 9I, and 9L) constructs was evaluated in PDL1-overexpressing (OE) KG1a / ISRE-Luc cells (Figures 9D–9F and 9J–9L). [Figure 9G] The in vitro activity of exemplary targeted IFN receptor antagonists is shown in the presence or absence of a certain amount of IFNβ (Figures 9G–9L). PDL1-targeted and isotype-controlled (Figures 9A, 9D, 9G, and 9J) were evaluated in PDL1 knockout (KO) KG1a / ISRE-Luc cells (Figures 9A–9C and 9G–9I). [Figure 9H] The in vitro activity of exemplary targeted IFN receptor antagonists is shown in the presence or absence of a certain amount of IFNβ (Figures 9G–9L). PDL1-targeted and masked monovalents (Figures 9B, 9E, 9H, and 9K) were evaluated in PDL1 knockout (KO) KG1a / ISRE-Luc cells (Figures 9A–9C and 9G–9I). [Figure 9I] The in vitro activity of exemplary targeted IFN receptor antagonists in the presence or absence of a certain amount of IFNβ (Figures 9G–9L) is shown. The in vitro activity of PDL1-targeted and masked bivalent (Figures 9C, 9F, 9I, and 9L) constructs was evaluated in PDL1 knockout (KO) KG1a / ISRE-Luc cells (Figures 9A–9C and 9G–9I). [Figure 9J] The in vitro activity of exemplary targeted IFN receptor antagonists is shown in the presence or absence of a certain amount of IFNβ (Figures 9G–9L). PDL1-targeted and isotype-controlled (Figures 9A, 9D, 9G, and 9J) were evaluated in PDL1-overexpressing (OE) KG1a / ISRE-Luc cells (Figures 9D–9F and 9J–9L). [Figure 9K]The in vitro activity of exemplary targeted IFN receptor antagonists is shown in the presence or absence of a certain amount of IFNβ (Figures 9G–9L). PDL1-targeted and masked monovalents (Figures 9B, 9E, 9H, and 9K) were evaluated in PDL1-overexpressing (OE) KG1a / ISRE-Luc cells (Figures 9D–9F and 9J–9L). [Figure 9L] The in vitro activity of exemplary targeted IFN receptor antagonists in the presence or absence of a certain amount of IFNβ (Figures 9G–9L) is shown. The in vitro activity of PDL1-targeted and masked bivalent (Figures 9C, 9F, 9I, and 9L) constructs was evaluated in PDL1-overexpressing (OE) KG1a / ISRE-Luc cells (Figures 9D–9F and 9J–9L). [Figure 10A] The effects of exemplary targeted and isotyped IFN receptor antagonist constructs on IFNα2b-mediated IP10 release by monocyte-derived dendritic cells (MoDCs) (Figure 10A) and PDL1 expression in MoDCs are shown (Figure 10B). [Figure 10B] The effects of exemplary targeted and isotyped IFN receptor antagonist constructs on IFNα2b-mediated IP10 release by monocyte-derived dendritic cells (MoDCs) (Figure 10A) and PDL1 expression in MoDCs are shown (Figure 10B). [Figure 11A] The in vitro activity of exemplary IFN receptor antagonist constructs in the presence or absence of certain amounts of hIFNα2b or hIFNβ is shown. The in vitro activity of PDL1-targeted masked bivalent constructs and control constructs was evaluated in PDL1 knockout (KO) KG1a / ISRE-Luc cells (Figures 11A-11C). [Figure 11B] The in vitro activity of exemplary IFN receptor antagonist constructs in the presence or absence of certain amounts of hIFNα2b or hIFNβ is shown. The in vitro activity of PDL1-targeted masked bivalent constructs and control constructs was evaluated in PDL1 knockout (KO) KG1a / ISRE-Luc cells (Figures 11A-11C). [Figure 11C] The in vitro activity of exemplary IFN receptor antagonist constructs in the presence or absence of certain amounts of hIFNα2b or hIFNβ is shown. The in vitro activity of PDL1-targeted masked bivalent constructs and control constructs was evaluated in PDL1-overexpressing (OE) KG1a / ISRE-Luc cells (Figures 11D-11F). [Figure 11D] The in vitro activity of exemplary IFN receptor antagonist constructs in the presence or absence of certain amounts of hIFNα2b or hIFNβ is shown. The in vitro activity of PDL1-targeted masked bivalent constructs and control constructs was evaluated in PDL1-overexpressing (OE) KG1a / ISRE-Luc cells (Figures 11D-11F). [Figure 11E] The in vitro activity of exemplary IFN receptor antagonist constructs in the presence or absence of certain amounts of hIFNα2b or hIFNβ is shown. The in vitro activity of PDL1-targeted masked bivalent constructs and control constructs was evaluated in PDL1-overexpressing (OE) KG1a / ISRE-Luc cells (Figures 11D-11F). [Figure 11F] The in vitro activity of exemplary IFN receptor antagonist constructs in the presence or absence of certain amounts of hIFNα2b or hIFNβ is shown. The in vitro activity of PDL1-targeted masked bivalent constructs and control constructs was evaluated in PDL1-overexpressing (OE) KG1a / ISRE-Luc cells (Figures 11D-11F). [Figure 12A] This shows the in vitro activity of exemplary IFN receptor antagonist constructs containing a universal type I interferon (uIFN) moiety in the presence or absence of a certain amount of hIFNα2b or hIFNβ. The in vitro activity of isotype or PDL1-targeted masked bivalent constructs was evaluated in PDL1 knockout (KO) KG1a / ISRE-Luc cells (Figure 12A, Figure 12C, and Figure 12E). [Figure 12B]The in vitro activity of exemplary IFN receptor antagonist constructs containing a universal type I interferon (uIFN) moiety is shown in the presence or absence of a certain amount of hIFNα2b or hIFNβ. The in vitro activity of isotype or PDL1-targeted masked bivalent constructs was evaluated in PDL1-overexpressing (OE) KG1a / ISRE-Luc cells (Figure 12B, Figure 12D, and Figure 12F). [Figure 12C] This shows the in vitro activity of exemplary IFN receptor antagonist constructs containing a universal type I interferon (uIFN) moiety in the presence or absence of a certain amount of hIFNα2b or hIFNβ. The in vitro activity of isotype or PDL1-targeted masked bivalent constructs was evaluated in PDL1 knockout (KO) KG1a / ISRE-Luc cells (Figure 12A, Figure 12C, and Figure 12E). [Figure 12D] The in vitro activity of exemplary IFN receptor antagonist constructs containing a universal type I interferon (uIFN) moiety is shown in the presence or absence of a certain amount of hIFNα2b or hIFNβ. The in vitro activity of isotype or PDL1-targeted masked bivalent constructs was evaluated in PDL1-overexpressing (OE) KG1a / ISRE-Luc cells (Figure 12B, Figure 12D, and Figure 12F). [Figure 12E] This shows the in vitro activity of exemplary IFN receptor antagonist constructs containing a universal type I interferon (uIFN) moiety in the presence or absence of a certain amount of hIFNα2b or hIFNβ. The in vitro activity of isotype or PDL1-targeted masked bivalent constructs was evaluated in PDL1 knockout (KO) KG1a / ISRE-Luc cells (Figure 12A, Figure 12C, and Figure 12E). [Figure 12F]The in vitro activity of exemplary IFN receptor antagonist constructs containing a universal type I interferon (uIFN) moiety is shown in the presence or absence of a certain amount of hIFNα2b or hIFNβ. The in vitro activity of isotype or PDL1-targeted masked bivalent constructs was evaluated in PDL1-overexpressing (OE) KG1a / ISRE-Luc cells (Figure 12B, Figure 12D, and Figure 12F). [Figure 13A] This shows the in vitro activity of exemplary IFN receptor antagonist constructs containing a universal type I interferon (uIFN) moiety in the presence or absence of a certain amount of hIFNα2b or hIFNβ. The in vitro activity of isotypes or EGFR-targeted masked bivalent constructs was evaluated in EGFR-deficient KG1a / ISRE-Luc cells ("EGFR-free"; Figures 13A, 13C, and 13E). [Figure 13B] The in vitro activity of exemplary IFN receptor antagonist constructs containing a universal type I interferon (uIFN) moiety is shown in the presence or absence of a certain amount of hIFNα2b or hIFNβ. The in vitro activity of isotypes or EGFR-targeted masked bivalent constructs was evaluated in EGFR-overexpressing (OE) KG1a / ISRE-Luc cells (Figures 13B, 13D, and 13F). [Figure 13C] This shows the in vitro activity of exemplary IFN receptor antagonist constructs containing a universal type I interferon (uIFN) moiety in the presence or absence of a certain amount of hIFNα2b or hIFNβ. The in vitro activity of isotypes or EGFR-targeted masked bivalent constructs was evaluated in EGFR-deficient KG1a / ISRE-Luc cells ("EGFR-free"; Figures 13A, 13C, and 13E). [Figure 13D]The in vitro activity of exemplary IFN receptor antagonist constructs containing a universal type I interferon (uIFN) moiety is shown in the presence or absence of a certain amount of hIFNα2b or hIFNβ. The in vitro activity of isotypes or EGFR-targeted masked bivalent constructs was evaluated in EGFR-overexpressing (OE) KG1a / ISRE-Luc cells (Figures 13B, 13D, and 13F). [Figure 13E] This shows the in vitro activity of exemplary IFN receptor antagonist constructs containing a universal type I interferon (uIFN) moiety in the presence or absence of a certain amount of hIFNα2b or hIFNβ. The in vitro activity of isotypes or EGFR-targeted masked bivalent constructs was evaluated in EGFR-deficient KG1a / ISRE-Luc cells ("EGFR-free"; Figures 13A, 13C, and 13E). [Figure 13F] The in vitro activity of exemplary IFN receptor antagonist constructs containing a universal type I interferon (uIFN) moiety is shown in the presence or absence of a certain amount of hIFNα2b or hIFNβ. The in vitro activity of isotypes or EGFR-targeted masked bivalent constructs was evaluated in EGFR-overexpressing (OE) KG1a / ISRE-Luc cells (Figures 13B, 13D, and 13F). [Modes for carrying out the invention]

[0020] 6. Detailed explanation 6.1.Definition As used herein, the following terms are intended to have the following meanings:

[0021] ABD chain, targeted subchain: Targeted subchains and the antigen-binding sites (ABDs) within them may exist as a single polypeptide chain (e.g., in the case of scFv or scFab) or may be formed through the association of two or more polypeptide chains (e.g., in the case of Fab or Fv). As used herein, the terms “ABD chain” and “targeted subchain” refer to all or part of an ABD or targeted subchain present on a single polypeptide chain. The use of the terms “ABD chain” or “targeted subchain” is for convenience and descriptive purposes only and does not imply any particular composition or method of production. Furthermore, references to ABDs or targeted subchains when describing IFN receptor agonists include ABD chains or targeted subchains unless otherwise indicated by the context. Therefore, when describing an IFN receptor antagonist in which the Fc domain is operably linked to the targeting portion, the Fc domain may be covalently linked, directly or indirectly (e.g., via a linker), via peptide bonds to, for example, (1) the first ABD or targeting subchain of Fab or Fv (with other components of Fab or Fv on the second associated ABD or targeting subchain), or (2) the ABD or targeting subchain containing scFv or scFab.

[0022] Approximately, roughly: Terms such as "approximately" and "roughly" are used throughout the specification before numerical values ​​to indicate that the numerical values ​​are not necessarily exact (for example, to account for fractions, measurement accuracy and / or accuracy variations, timing, etc.). It should be understood that a disclosure of "approximately X" or "roughly X," where X is a numerical value, is also a disclosure of "X." Therefore, for example, a disclosure of an embodiment in which one sequence has "approximately X% sequence identity" with another sequence is also a disclosure of an embodiment in which that sequence has "X% sequence identity" with the other sequence.

[0023] Anchor portion: As used herein, the term “anchor portion” refers to any molecule or portion thereof that can bind to a cell. Examples of anchor portions as used herein include, but are not limited to, cell surface protein-binding molecules (e.g., ligands and other protein-binding partners, such as those described in Section 6.6) and targeting portions (e.g., antibody and antigen-binding fragments, such as those described in Section 6.6.1). As used herein, an anchor portion “for” a particular cell means that the anchor portion can bind to a particular cell. Binding does not need to be selective or specific.

[0024] And and or: Unless otherwise indicated, the conjunction "or" is intended to be used in its correct sense as a Boolean logical operator, encompassing both the selection of a feature in a choice (choice A is mutually exclusive from choice B, A or B) and the selection of a combined feature (both A and B are selected, A or B). In some parts of the text, the terms "and / or" are used for the same purpose and should not be interpreted as meaning that "or" is used to refer to mutually exclusive choices.

[0025] Antagonistic: As used herein with respect to IFN receptor antagonists, the terms “antagonistic” or “antagonist” refer to the ability to reduce the 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% in the presence of IFNα2b, as measured by an activity assay such as those described in Section 9.1.4.

[0026] Antibody: As used herein, the term “antibody” refers to a polypeptide (or set of polypeptides) of the immunoglobulin family that can bind noncovalently, reversibly, and specifically to an antigen. For example, a naturally occurring “antibody” of the IgG type is a tetramer containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL 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 region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term “antibody” includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, bispecific or multispecific antibodies, and anti-idiotype (anti-id) antibodies. Antibodies can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Both the light and heavy chains are divided into regions of structural and functional homology. The terms "stationary" and "variable" are used functionally. In this regard, it will be understood that both the variable domain (VL) of the light chain portion and the variable domain (VH) of the heavy chain portion determine antigen recognition and specificity.Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, placental translocation, Fc receptor binding, and complement binding. By convention, the numbering of constant region domains increases as they move further away from the antigen-binding domain or amino terminus of the antibody. The N-terminus is the variable region, the C-terminus is the constant region, and the CH3 and CL domains represent the carboxyl termins of the heavy and light chains of native antibodies, respectively. For convenience, and unless otherwise indicated by context, reference to an antibody also refers to antibody fragments, as well as engineered antibodies that contain antigen-binding domains with antigen-binding domains and / or non-native configurations that do not exist naturally.

[0027] Antigen-binding domain: As used herein, the term “antigen-binding domain” or “ABD” refers to a portion of an antibody or antibody fragment (e.g., a targeting portion) that has the ability to bind to an antigen noncovalently, reversibly, and specifically. Examples of antibody fragments that may contain an ABD include, but are not limited to, single-strand Fv(scFv), Fab fragments, monovalent fragments consisting of a VL domain, VH domain, CL domain, and CH1 domain, F(ab)2 fragments, bivalent fragments containing two Fab fragments linked by disulfide crosslinks at a hinge region, Fd fragments consisting of a VH domain and a CH1 domain, Fv fragments consisting of a single arm of the antibody consisting of a VL domain and a VH domain, dAb fragments consisting of a VH domain (Ward et al., 1989, Nature 341:544-546), and isolated complementarity-determining regions (CDRs). Therefore, the term “antibody fragment” encompasses both proteolytic fragments of antibodies (e.g., Fab fragments and F(ab)2 fragments) and engineered proteins containing one or more parts of an antibody (e.g., scFv). Antibody fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology 23:1126–1136).

[0028] Associated: In the context of IFN receptor antagonists, the term “associated” refers to a functional relationship between two or more polypeptide chains. Specifically, “associated” means that two or more polypeptides associate with each other, for example, non-covalently via molecular interactions or covalently via one or more disulfide crosslinks or chemical crosslinks, in order to produce a functional IFN receptor antagonist. Examples of associations that may be present in the IFN receptor antagonists of this disclosure include, but are not limited to, associations between Fc domains forming an Fc region (homodimers or heterodimers as described in Section 6.7), associations between VH and VL regions in Fab or Fv, and associations between CH1 and CL in Fab.

[0029] Cancer: The term "cancer" refers to a disease characterized by the uncontrolled (and often rapid) growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers described herein include, but are not limited to, any of the above types of TAA-positive cancers, including, for example, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, adrenal cancer, autonomic ganglion cancer, biliary tract cancer, bone cancer, endometrial cancer, eye cancer, fallopian duct 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 gastrointestinal cancer, urinary tract cancer, vaginal cancer, vulvar cancer, lymphoma, leukemia, lung cancer, etc.

[0030] Complementarity-determining regions: The term “complementarity-determining regions” or “CDR” as used herein refers to sequences of amino acids within the antibody variable region that confer antigen specificity and binding affinity. For example, generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, and CDR-H3), and each light chain variable region has three CDRs (CDR-L1, CDR-L2, and CDR-L3). The precise amino acid sequence boundaries of a given CDR are as follows: Kabat et al., 1991, “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., 1997, JMB 273:927-948 (“Chothia” numbering scheme), and ImMunoGenTics (IMGT) numbering (Lefranc, 1999, The Immunologist 7:132-136, Lefranc et al. It can be determined using one of several well-known schemes, including the one described by al., 2003, Dev. Comp. Immunol. 27:55-77 ("IMGT" numbering scheme). For example, for the classical format, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3), and the light chain variable domain The CDR amino acid residues in (VL) are numbered 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3). Under Chothia, the CDR amino acids in (VH) are numbered 26-32 (CDR-H1), 52-56 (CDR-H2), and 95-102 (CDR-H3), and the amino acid residues in (VL) are numbered 26-32 (CDR-L1), 50-52 (CDR-L2), and 91-96 (CDR-L3).By combining the CDR definitions of both Kabat and Chothia, the 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-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3) in human VL. Under IMGT, the CDR amino acid residues in VH are numbered approximately 26-35 (CDR-H1), 51-57 (CDR-H2), and 93-102 (CDR-H3), and the CDR amino acid residues in VL are numbered approximately 27-32 (CDR-L1), 50-52 (CDR-L2), and 89-97 (CDR-L3) (numbering by "Kabat"). Under IMGT, the CDR region of the antibody can be determined using the IMGT / DomainGap Align program.

[0031] Constant domain: The term "constant domain" refers to the CH1, CH2, CH3, or CL domain of an immunoglobulin.

[0032] The term “CH1 domain” refers to the heavy chain constant region that links the variable domain to a hinge within the heavy chain constant domain. In some embodiments, the term “CH1 domain” refers to the region of an immunoglobulin molecule extending from amino acids 118 to 215 (EU numbering). The term “CH1 domain” encompasses wild-type CH1 domains and their variants (e.g., CH1 domains that do not exist naturally or modified CH1 domains). For example, the term “CH1 domain” includes wild-type IgG1, IgG2, IgG3, and IgG4 CH1 domains, as well as their variants, having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5, and / or up to 5, 4, 3, 2, or 1 mutation, e.g., substitution, deletion, and / or addition. Exemplary CH1 domains include CH1 domains with mutations that modify the biological activity of an antibody, such as ADCC, CDC, or half-life.

[0033] The term “CH2 domain” refers to the heavy chain constant region that ligates a hinge to the CH3 domain within the heavy chain constant domain. In some embodiments, the term “CH2 domain” refers to the region of an immunoglobulin molecule extending from amino acids 238 to 340 (EU numbered). The term “CH2 domain” encompasses wild-type CH2 domains and their variants (e.g., CH2 domains that do not exist naturally or modified CH2 domains). For example, the term “CH2 domain” includes wild-type IgG1, IgG2, IgG3, and IgG4 CH2 domains, as well as their variants, having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5, and / or up to 5, 4, 3, 2, or 1 mutation, e.g., substitution, deletion, and / or addition. Exemplary CH2 domains include CH2 domains with mutations that modify the biological activity of an antibody, such as ADCC, CDC, purification, dimerization, and half-life.

[0034] The term “CH3 domain” refers to the heavy chain constant region at the C-terminus of the CH2 domain within the heavy chain constant domain. In some embodiments, the term “CH3 domain” refers to the region of an immunoglobulin molecule extending from amino acids 341 to 447 (EU numbering). The term “CH3 domain” encompasses wild-type CH3 domains and their variants (e.g., CH3 domains that do not exist naturally or modified CH3 domains). For example, the term “CH3 domain” includes wild-type IgG1, IgG2, IgG3, and IgG4 CH3 domains, as well as their variants, having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5, and / or up to 5, 4, 3, 2, or 1 mutation, e.g., substitution, deletion, and / or addition. Exemplary CH3 domains include CH3 domains with mutations that modify the biological activity of an antibody, such as ADCC, CDC, purification, dimerization, and half-life.

[0035] The term "CL domain" refers to the constant region of an immunoglobulin light chain. The term "CL domain" encompasses wild-type CL domains (e.g., kappa or lambda light chain constant regions) and their variants (e.g., naturally occurring or modified CL domains). For example, the term "CL domain" includes wild-type kappa and lambda constant domains with 1, 2, 3, 4, 5, 1-3, 1-5, 3-5, and / or up to 5, 4, 3, 2, or 1 mutation, e.g., substitution, deletion, and / or addition, as well as their variants.

[0036] Effector Function: The term "effector function" typically refers to the activity of an antibody molecule mediated by binding via an antibody domain other than the antigen-binding domain, which is mediated by the binding of an effector molecule. Effector function includes, for example, complement-mediated effector function, which is mediated by the binding of the complement C1 component to the antibody. Complement activation is important for opsonization and lysis of cellular pathogens. Complement activation also stimulates inflammatory responses and may be involved in autoimmune hypersensitivity. Effector function also includes Fc receptor (FcR)-mediated effector function, which can be triggered by the binding of the constant domain of an antibody to an Fc receptor (FcR). Binding of antibodies to Fc receptors on the cell surface triggers a number of important and diverse biological responses, including engulfment and destruction of antibody-coated particles, removal of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and regulation of immunoglobulin production. The effector function of an antibody can be altered by changing the antibody's affinity for effector molecules, such as Fc receptors or complement components, for example, by enhancing or reducing it. Binding affinity is generally altered by modifying the effector molecule binding site, in which case it is appropriate to position the site of interest and modify at least a portion of it in a suitable manner. Furthermore, changes to the binding site on the antibody for effector molecules do not necessarily need to significantly alter the overall binding affinity, but it is conceivable that they may alter the shape of interactions that invalidate the effector mechanism, such as in unproductive binding. It is also conceivable that effector function can be altered by modifying sites that are not directly involved in effector molecule binding but are involved in the performance of effector function in other ways.

[0037] Epitope: An epitope, or antigenic determinant, is a portion of an antigen recognized by an antibody or other antigen-binding moiety described herein. Epitopes can be linear or three-dimensional.

[0038] Fab: The term "Fab" refers to a pair of polypeptide chains, the first polypeptide chain comprising a variable heavy (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 comprising a variable light (VL) domain of the antibody N-terminus operably linked (typically at the N-terminus) to a second constant domain (referred to herein as C2) that can pair with the first constant domain. In natural antibodies, VH is located at the N-terminus of the first constant domain (CH1) of the heavy chain, and VL is located at the N-terminus of the constant domain (CL) of the light chain. The Fabs of this disclosure may be positioned according to their natural orientation or may include domain substitutions or swaps to facilitate correct VH and VL pairing. For example, in a heterodimer molecule, the CH1 and CL domain pairs in the Fab may be replaced with CH3 domain pairs to facilitate modified correct Fab-chain pairing. It is also possible to reverse the CH1 and CL configurations, with CH1 attached to VL and CL attached to VH, which is a generally known configuration as Crossmab. The term "Fab" encompasses single-stranded Fab.

[0039] Fc domains and Fc regions: 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, an Fc domain includes a CH2 domain, followed by a CH3 domain, with or without a hinge region at the N-terminus of the CH2 domain. The term “Fc region” refers to 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 one another. In natural antibodies, Fc domains are typically identical, but one or both Fc domains may be modified to enable heterodimerization, for example, via knob-in-hole interactions.

[0040] Fv: The term "Fv" refers to the smallest antibody fragment that can be derived from immunoglobulin containing a complete target recognition and binding site. This region consists of a dimer (VH-VL dimer) of one heavy-chain variable domain and one light-chain variable domain in a close, non-covalent association. In this configuration, three CDRs of each variable domain interact to define the target binding site on the surface of the VH-VL dimer. Often, six CDRs confer target-binding specificity to the antibody. However, in some examples, even a single variable domain (or half of an Fv containing only three target-specific CDRs) can have the ability to recognize and bind to a target. References to VH-VL dimers herein are not intended to convey any particular configuration. When present on a single polypeptide chain (e.g., scFv), VH and VL are the N-terminus or C-terminus.

[0041] Semi-antibodies: The term "semi-antibodies" refers to molecules that contain at least one Fc domain and can associate with another molecule containing an Fc domain, for example, via disulfide crosslinking or molecular interactions. Semi-antibodies can consist of one polypeptide chain or two or more polypeptide chains (e.g., two polypeptide chains of Fab). An example of a semi-antibody is a molecule containing the heavy and light chains of an antibody (e.g., an IgG antibody). Another example of a semi-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, where the VL and VH domains form an ABD. Yet another example of a semi-antibody is a polypeptide containing scFv, CH2, and CH3 domains.

[0042] The IFN receptor antagonists of this disclosure typically comprise two half-antibodies, each comprising one or two receptor moieties, e.g., an IFN moiety masked by an IFN masking moiety. The one or two masking moieties may be present in the same half-antibody as the IFN moiety, or in the other half-antibody separate from the IFN moiety, as illustrated in Figures 1B–1G and illustrated in the embodiments described in Table 2. Exemplary locations of linkers (e.g., non-cleavable linkers) in the half-antibodies are shown in Table 1. One or both half-antibodies in the IFN receptor antagonist may further comprise a targeting moiety, such as scFv or Fab. Exemplary IFN receptor antagonists comprising targeting moieties are illustrated in Figures 1B–1G and described in Table 2.

[0043] The term "half-antibody" is for descriptive purposes only and does not imply any specific composition or method of production. The designations of half-antibodies as "first half-antibody," "second half-antibody," "left half-antibody," "right half-antibody," etc., are merely for convenience and descriptive purposes.

[0044] Host cells or recombinant host cells: The terms “host cells” or “recombinant host cells” refer to, for example, cells that have been genetically modified through the introduction of heterologous nucleic acids. It should be understood that such terms are intended to refer not only to specific target cells but also to the offspring of such cells. Such offspring may not be identical to the parent cells in practice, as certain modifications may occur in later generations due to mutation or environmental influences, but they are still included within the scope of the term “host cells” as used herein. Host cells may harbor heterologous nucleic acids, for example, transiently on extrachromosomal heterologous expression vectors, or stably, for example, by incorporating heterologous nucleic acids into the host cell genome. For the purpose of expressing the IFN receptor antagonists of this disclosure, the host cells are preferably mammalian-derived or mammalian-like cell lines such as monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293), baby hamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2 / 0, HeLa, Madin-Darby bovine kidney (MDBK), myeloma and lymphoma cells, or derivatives and / or engineered variants thereof. Examples of engineered variants include derivatives that grow at a higher density than the original cell line, as well as / or glycan-profile-modified derivatives and / or site-specific integration site derivatives.

[0045] Interferon: As used herein, the term “interferon” refers to full-length interferon or modified interferon, e.g., 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% activity in the in vitro luciferase reporter assay described in Section 9.1.5). In some embodiments, the modified interferon is attenuated by any two of the aforementioned values, e.g., 0.05%–50%, 0.1%–20%, 0.1%–10%, 0.05%–5%, 1%–20%, etc. In other embodiments, modified interferons substantially retain the biological activity of the corresponding wild-type interferon (e.g., retaining at least 50% of the activity in the in vitro luciferase reporter assay 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 encompasses synthetic or engineered proteins that have the biological activity of wild-type interferon (e.g., retaining at least 50% of the activity in the in vitro luciferase reporter assay described in Section 9.1.5), such as the universal type I interferon described in Section 6.3.1.

[0046] Linker: As used herein, the term "linker" refers to a connecting peptide between two parts. For example, a linker can connect an IFN moiety and an IFN masking moiety.

[0047] Non-cleavable linkers: As used herein, non-cleavable linkers refer to peptides whose amino acid sequence lacks the substrate sequence of a protease. Exemplary non-cleavable linkers are described in Section 6.5.

[0048] Oncolytic viruses: The term “oncolytic viruses” refers to viruses that replicate in tumor cells. These include viruses that naturally and preferentially replicate and accumulate in tumor cells, such as poxviruses, as well as viruses that have been engineered to do so. Some oncolytic viruses can kill tumor cells after infection. For example, oncolytic viruses can cause tumor cell death by lysing tumor cells or by inducing tumor cell death. Exemplary oncolytic viruses include, but are not limited to, poxviruses, herpesviruses, adenoviruses, adeno-associated viruses (AAVs), lentiviruses, retroviruses, rhabdoviruses, papillomaviruses, papillomaviruses, varicella-stomatitis viruses (VSVs), measles viruses, Newcastle disease viruses, picornaviruses, Sindbisviruses, parvoviruses, reoviruses, and coxsackieviruses. In certain embodiments, the oncolytic virus of this disclosure is a VSV. Oncolytic viruses and their use in treating cancer are further described, for example, in Chiocca and Rabkin Cancer Immunol Res (2014) 2(4):295-300.

[0049] Operablely linked: The term “operable 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, “operable linked” means that two or more amino acid segments are linked to produce a functional polypeptide. For example, in the context of the IFN receptor antagonist of this disclosure, separate components (e.g., the Fc domain and the IFN portion) can be operable linked directly or via a peptide linker sequence. In the context of nucleic acids encoding fusion proteins, such as the half-antibody of the IFN receptor antagonist of this disclosure, “operable linked” means that two nucleic acids are linked such that the amino acid sequence encoded by those two nucleic acids remains in frame. In the context of transcriptional regulation, the term refers to the functional relationship of a transcriptional regulatory sequence to a transcriptional sequence. For example, a promoter or enhancer sequence is operable linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in a suitable host cell or other expression system.

[0050] Polypeptides, peptides, and proteins: The terms “polypeptides,” “peptides,” and “proteins” are used interchangeably herein to refer to polymers of amino acid residues.

[0051] Recognize: As used herein, the term “recognize” refers to an antibody or antibody fragment (e.g., a targeting moiety) that finds its epitope and interacts with (e.g., binds to) that epitope.

[0052] Single-stranded Fab or scFab: As used herein, the terms “single-stranded Fab” or “scFab” refer 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 an N-terminal to C-terminal orientation in one of the following orders: (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 32 to 50 amino acids. The single-stranded Fab fragment is typically stabilized via a native disulfide bond between the CL domain and the CH1 domain. In addition, these single-chain Fab molecules can be further stabilized by the formation of interchain disulfide bonds via the insertion of cysteine ​​residues (e.g., at position 44 of the VH domain and position 100 of the VL domain according to Kabat numbering).

[0053] Single-stranded Fv or scFv: As used herein, the terms “single-stranded Fv” or “scFv” refer to an antibody ABD containing the VH and VL domains, which are present in a single polypeptide chain. Preferably, the Fv polypeptide further includes a polypeptide linker between the VH and VL domains, which allows the scFv to form a desired structure for antigen binding. For an overview of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. (1994), Springer-Verlag, New York, pp. 269–315. Typically, the VH and VL and VL are arranged in an N-terminal to C-terminal order of VH-VL or VL-VH, separated by a linker, e.g., the linkers listed in Table E.

[0054] Separator parenchyma: As used herein, the term “separator parenchyma” refers to an amino acid sequence that provides sufficient spatial separation of the anchor parenchyma and the IFN parenchyma as components of an IFN receptor antagonist, including the anchor parenchyma and the IFN parenchyma, thereby enabling their simultaneous binding to the same cell, for example, the cells described in Section 9.1.3. In some embodiments, the separator parenchyma is a polypeptide with a length of at least about 100 amino acids. In certain embodiments, the separator parenchyma of this disclosure comprises an Fc domain or a fragment thereof.

[0055] Specific (or selective) binding: The term “specifically (or selectively) binding” to an antigen or epitope refers to a binding reaction that determines the presence of a congener antigen or epitope in a heterogeneous population of proteins and other molecules. The binding reaction may, but does not need to be, mediated by an antibody or antibody fragment. The term “specifically binding” does not exclude interspecies reactivity. For example, an antigen-binding domain that “specifically binds” 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. Thus, such interspecies reactivity itself does not change the classification of antigen-binding domains as “specific” binders. In certain embodiments, an antigen-binding domain of this disclosure that specifically binds to a human antigen has interspecies reactivity with one or more non-human mammalian species, e.g., primate species (including, but not limited to, one or more of Macaca fascicularis, Macaca mulatta, and Macaca nemestrina), or rodent species, e.g., Mus musculus.

[0056] Subject: The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. In preferred embodiments, the subject is humans.

[0057] Target Molecules: As used herein, the term “target molecule” refers to any biomolecule expressed on the cell surface or in the extracellular matrix (e.g., proteins, carbohydrates, lipids, or combinations thereof) that can be specifically bound by the targeting moiety in the IFN receptor antagonists of this disclosure.

[0058] Targeting moiety: As used herein, the term “targeting moiety” refers to any molecule or its binding portion (e.g., immunoglobulin or antigen-binding fragment) that can bind to a cell surface molecule on a cell to which the IFN receptor antagonist of this disclosure is localized, for example, on a cell expressing type I interferon receptor (e.g., on lymphocytes involved in an autoimmune state). Targeting moieties may also have functional activity in addition to localizing the IFN receptor antagonist to a specific site. For example, a targeting moiety that binds to a checkpoint inhibitor such as PDL1 may also exhibit antitumor activity, for example, by inhibiting PD1 / PDL1 signaling.

[0059] T-cell antigens, TCAs: The term “T-cell antigen” or “TCA” refers to molecules (typically proteins, carbohydrates, lipids, or some combination thereof) expressed on the surface of T lymphocytes that are useful for preferential targeting of pharmacological agents to specific sites. In some embodiments, the site is cancer tissue, and / or the T-cell antigen is a tumor-reactive lymphocyte antigen, a cell surface molecule of tumor or viral lymphocytes, or a checkpoint inhibitor expressed on T lymphocytes.

[0060] Tumor: The term “tumor” is used herein interchangeably with the term “cancer,” and both terms, for example, encompass solid and liquid tumors, such as diffuse or circulating tumors. As used herein, the terms “cancer” or “tumor” include precancerous and malignant cancers and tumors.

[0061] Tumor-associated antigens, TAAs: The term “tumor-associated antigen” or “TAA” refers to molecules (typically proteins, carbohydrates, lipids, or some combination thereof) that are expressed on the surface of cancer cells, either as a whole or as fragments (e.g., MHC / peptides), and are useful for preferential targeting of pharmacological agents against cancer cells. In some embodiments, TAAs are markers expressed by both normal and cancer cells, e.g., lineage markers. In some embodiments, TAAs are cell surface molecules that are overexpressed in cancer cells compared to normal cells, e.g., 1x overexpression, 2x overexpression, 3x overexpression, or more compared to normal cells. In some embodiments, TAAs are cell surface molecules that are improperly synthesized in cancer cells, e.g., molecules containing deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, TAAs are exclusively expressed on the cell surface of cancer cells, either as a whole or as fragments (e.g., MHC / peptides), and are not synthesized or expressed on the surface of normal cells. Therefore, the term "TAA" encompasses antigens specific to cancer cells, which may be known in the field as tumor-specific antigens (TSAs).

[0062] To treat, to treat, to treat: As used herein, the terms “to treat,” “to treat,” and “to treat” mean a reduction or improvement in the progression, severity, and / or duration of a disorder, e.g., an inflammatory disorder, an autoimmune disorder, or a proliferative disorder, or improvement of one or more symptoms (preferably one or more identifiable symptoms) of a disorder resulting from the administration of one or more IFN receptor antagonists of the Disclosure. In some embodiments, the disorder is an inflammatory disorder, an autoimmune disorder, or a proliferative disorder, and the terms “to treat,” “to treat,” and “to treat” mean improvement in at least one measurable physical parameter of the inflammatory disorder, autoimmune disorder, or proliferative disorder, which is not necessarily identifiable by the patient. In other embodiments, the terms “to treat,” “to treat,” and “to treat” mean, for example, inhibition of the progression of a disorder, either physical or physiological, by stabilization of an identifiable symptom, e.g., a physical parameter. In other embodiments, the terms “to treat,” “to treat,” and “to treat” mean a reduction or stabilization of tumor size or cancer cell number in a proliferative disorder.

[0063] 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 the heavy chain variable region (VL). In the context of a targeted moiety, the term “universal light chain” or “ULC” refers to a light chain polypeptide that can pair with the heavy chain region of the targeted moiety and can also pair with other heavy chain regions. A ULC may also include a constant domain, such as the CL domain of an antibody. A universal light chain is also known as a “common light chain.”

[0064] VH: The term "VH" refers to the variable region of the immunoglobulin heavy chain of an antibody, which includes the Fv, scFv, dsFv, or Fab heavy chains.

[0065] VL: The term "VL" refers to the variable region of an immunoglobulin light chain, including the Fv, scFv, dsFv, or Fab light chains.

[0066] 6.2. IFN receptor antagonists This disclosure relates to an IFN receptor antagonist comprising an anchor portion, a separator portion, a type I interferon (IFN) portion, and a type I interferon alpha receptor 1 (IFNAR1) portion.

[0067] The IFN receptor antagonists of this disclosure generally comprise one or more anchoring moieties that bind to cells expressing type I interferon receptors. Depending on the desired indication or use, the anchoring moieties may be designed to specifically bind to a particular cell type expressing type I interferon receptors. Accordingly, in some embodiments, the IFN receptor antagonists of this disclosure comprise one or more targeting moieties, such as an antigen-binding domain of an antibody, that target the IFN receptor antagonist to cells expressing type I interferon receptors, such as cancer cells or lymphocytes involved in autoimmune disorders.

[0068] The IFN receptor antagonists of this disclosure generally include one or more separator moieties that separate the anchor moiety from the IFN moiety and the IFNAR1 moiety. The separator moieties may be any molecule or polypeptide that allows the simultaneous binding of the anchor moiety and the IFN moiety to the same cell. In some cases, the separator moiety is a multimerizing moiety that allows the multimerization (e.g., dimerization) of two or more individual components. Thus, in embodiments in which the IFN receptor antagonist is dimerized, the IFN receptor antagonist generally consists of two half-antibodies containing a pair of multimerizing domains, such as Fc domains that associate to form an Fc region (typically including a hinge sequence). In the IFN receptor antagonists of this disclosure, the two half-antibodies together contain at least one IFN moiety, but may contain two or more IFN moieties. Each IFN moiety in the IFN receptor antagonist may be masked by an interferon alpha receptor 1 (IFNAR1) moiety.

[0069] Examples of IFN receptor antagonists are illustrated in Figures 1B to 1G.

[0070] Table 1 below lists exemplary half-antibodies that can be incorporated into the IFN receptor antagonists of this disclosure. As is evident from Table 1, each half-antibody may contain one or more polypeptide chains. For convenience in describing the half-antibody combinations in the IFN receptor antagonists of this disclosure, each half-antibody listed in Table 1 is often referred to herein as an “exemplary monomer.” [Table 1-1] [Table 1-2]

[0071] 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 the hinge domains described in Section 6.7.1.3.

[0072] 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 separator portion is an Fc domain (e.g., as described in Section 6.7.1). While not intended to be theoretically bound, the inventors believe that in this configuration, the anchoring portion anchors the IFN receptor antagonist to the cell, thereby enabling the binding of the IFNAR1-masked IFN portion to the type I interferon receptor on the cell and the inhibition of IFN signaling.

[0073] Table 2 below shows additional exemplary monomer pairings that may be available with the IFN receptor antagonists of this disclosure. The IFN receptor antagonists identified in Table 2 include two targeting moieties. [Table 2]

[0074] The sequences and lengths of the hinge and linker sequences can be modified, and the sequence of the IFN moiety (either a full-length IFN sequence or an N-terminal and / or C-terminal truncated IFN sequence, including amino acid substitutions) can also be modified. Exemplary IFN moieties are described in Section 6.3 and include the IFNα-based and IFNβ-based moieties described in Sections 6.3.1 and 6.3.2 below, as well as other type I IFN-based moieties described in Section 6.3.1. Exemplary IFNAR1 moieties 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 moieties are disclosed in Section 6.6. Exemplary Fc domains, including Fc domains suitable for heterodimerization when two half-antibodies of an IFN receptor antagonist are not identical, are described in Section 6.7.

[0075] 6.3. IFN part IFNs have two main classes: 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.

[0076] The IFN moiety of this disclosure 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, which consists of IFNAR1 and IFNAR2, and is ubiquitously expressed in all nucleated cells. Ligand binding is initiated by the high-affinity receptor subunit IFNAR2 (Piehler et al., 2012, Immunological Reviews, doi.org / 10.1111 / imr.12001). Thus, type I IFNs can act on substantially all cells of the body. Sixteen type I interferon subtypes have been identified, which differ in their inherent variability in affinity for IFNAR2 and activity.

[0077] In some embodiments, the type I IFN portion is the interferon-α (IFNα) portion. In other embodiments, the type I IFN portion is the interferon-β (IFNβ) portion.

[0078] In other embodiments, the type I IFN portion is an interferon-ω (IFNω), interferon-ε (IFNε), or interferon-κ (IFNκ) portion.

[0079] The type I IFN moiety may contain one or more mutations, such as substitutions, deletions, or insertions, that differ from the wild-type IFN sequence. Substitutions that reduce receptor binding and thereby attenuate IFN activity may be preferred. N-terminal or C-terminal deletions (or cleavages), such as amino acids having cleavages 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 a mature type I IFN, may also be used.

[0080] Further details of an exemplary Type I IFN portion are provided below.

[0081] 6.3.1. Interferon-α portion The IFNα gene is a member of the alpha interferon gene cluster on chromosome 9. The encoded cytokine is a member of the type I interferon family, produced in response to viral infection as a key part of the innate immune response with potent antiviral, antiproliferative, and immunomodulatory properties. IFNα refers to a family of proteins with at least 15 known subtypes of human IFNα. The major subtypes identified are IFNα1, IFNα2, IFNα8, IFNα10, IFNα14, and IFNα21.

[0082] The IFNα1 gene has two allele 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. [ka]

[0083] The human IFNα1b gene differs from the IFNα1a allele variant by a single base change within the coding region, resulting in a single change in the amino acid sequence (Val114 instead of Ala114 in the mature protein, which corresponds to Val137 instead of Ala137 in the full-length polypeptide).

[0084] The IFNα2 allele has three allele variants: IFNα2a, IFNα2b, and IFNα2c. The IFNα2b allele is dominant, while the IFNα2a allele is less dominant, and IFNα2c is only a minor allele variant. The amino acid sequence of human IFNα2 is assigned UniProtKB accession number P01563. The sequence of the IFNα2b allele is reproduced below, with the signal peptide underlined. [ka]

[0085] IFNα2b has arginine (R) at position 23 of the mature protein, and IFNα2a has lysine (K). Therefore, in some embodiments, the IFNα2 moiety has arginine at the position corresponding to position 23 of the mature protein. In other embodiments, the IFNα2 moiety has lysine at the position corresponding to position 23 of the mature protein.

[0086] In various embodiments, the IFNα moiety includes 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 mature IFNα1a, IFNα1b, and / or IFNα2b, IFNα2a, or IFNα2c, or fragments thereof, having up to 15 amino acid cleavages 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 acid cleavages from the N-terminus and / or C-terminus of mature IFNα1a, IFNα1b, and / or IFNα2b, IFNα2a, or IFNα2c, or fragments thereof.

[0087] In some embodiments, the IFNα moiety has one or more amino acid substitutions, e.g., substitutions that alter the IFNAR bond and / or agonism. Exemplary substitutions are found in WO2013 / 107791, U.S. Patent No. 8,258,263, WO2007 / 000769A2, WO2008 / 124086, WO2010 / 030671, WO2018 / 144999A1, and WO2015 / 007520, WO2013 / 059885, WO2020156467A1, WO2021 / 126929A1. In some embodiments, the IFNα moiety includes: a) L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K or R33A or R33Q, H34A, D35A, Q40A, H57Y, E58N, Q61S, F64A, N65A, T69A, L80A, D82E, Y85A, T86I, Y89A, D1 One or more substitutions selected from 14R or D114A, L117A, R120A or R120E or R120K, K121E, R125A, K133A, K134A, R144A, A145G or A145M, M148A, R149A, R149K, S152A, L153A, N156A, and / or b) One or more substitutions at amino acids 57-89 and 159-165 as described in WO2007000769A2, and / or c) One or more amino acid substitutions at 9, 17, 47, 65, 66, 117, 123, 128, 147, and 157 for alanine, glycine, or threonine as described in WO2021126929A1.

[0088] The amino acid positions of the aforementioned substitutions are given by referring to mature IFNα2b.

[0089] In further embodiments, the IFNα moiety includes one or more amino acid substitutions listed in Table 3. Table 3 lists IFNα substitutions identified by reference to amino acid positions in the sequence of IFNα2. [Table 3-1] [Table 3-2] [Table 3-3]

[0090] In some embodiments, the IFNα portion includes an amino acid sequence containing the amino acid substitution R33A or R33K, Q90A, E96A, R120A, R120E, A145M, R149A or R149K, S152A, or any combination of two or more of the above, for example, Q90A+R120A or A145M+R149K.

[0091] Examples of IFNα moiety sequences that can be used in the IFN receptor antagonists of this disclosure are listed in Table 4 below. [Table 4-1] [Table 4-2] [Table 4-3]

[0092] 6.3.2. Interferon-β portion Interferon-beta (IFNβ) is a cytokine spontaneously produced by the immune system in response to biological and chemical stimuli. IFNβ is a glycosylated, secreted monomer with a molecular weight of approximately 22 kDa, produced in large quantities by fibroblasts, and is therefore also known as fibroblast interferon. IFNβ binds to the IFNAR receptor, which is composed of IFNAR1 and IFNAR2 dimers, and induces signaling via the JAK / STAT pathway and other pathways. IFNβ can also function by binding only to IFNAR1 and signaling independently of the Jak-STAT pathway (Ivashkiv and Donlin, 2014, Nat Rev Immunol. 14(1):36-49).

[0093] 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. The A helix of the AB loop and the E helix of the DE loop have been reported to be involved in the binding of IFNβ to the IFNAR receptor.

[0094] Two types of IFNβ are described: interferon-β1 (IFNβ1) and interferon-β3 (IFNβ3) (Schirmer and Neumann, 2019. Cytokines. In: Nijkamp and Parnham's Principles of Immunopharmacology. Springer, Cham.).

[0095] The amino acid sequence of the human IFNβ precursor is listed under GenBank accession number AAA36040.1 and is reproduced below (with the signal peptide underlined). [ka]

[0096] In various embodiments, the IFNβ moiety comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with the amino acid sequence of a mature IFNβ1 or fragment having up to 15 amino acid cleavages 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 acid cleavages from the N-terminus and / or C-terminus of IFNβ1).

[0097] In various embodiments, the IFNβ moiety includes one or more amino acid substitutions and / or deletions compared to IFNβ1. In some embodiments, the substitution is C17S (referencing mature IFNβ1), and the deletion is one of the C-terminal cleavages described in US2009 / 0025106A1 as IFN-ΔI, IFNA2, IFNA3, IFNA4, IFNA5, IFNA6, IFN-Δ7, IFN-Δδ, IFNA9, and IFN-ΔIO.

[0098] 6.3.1. Universal Type I Interferon In certain embodiments, the type I IFN moiety is a universal type I IFN (also referred to as human IFN-alpha hybrid protein, recombinant human universal type I IFN, or simply "uIFN"), which is a recombinant IFNα moiety constructed from IFNαA and IFNαD. uIFN exhibits bioactivity across multiple species.

[0099] The amino acid sequence of universal type I IFN (uIFN) is reproduced below: CDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNVDSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE (Sequence ID 137)

[0100] In some embodiments, the IFN portion includes 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 uIFN (SEQ ID NO: 137) or a fragment thereof, having up to 15 amino acid cleavages 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 acid cleavages from the N-terminus and / or C-terminus of uIFN).

[0101] 6.3.2. Other Type I Interferons In certain embodiments, the type I IFN moiety is other than the IFNα or IFNβ moiety, for example, the interferon-ω (IFNω), interferon-ε (IFNε), or interferon-κ (IFNκ) moiety.

[0102] Human IFNω is identified by UniProt accession number P05000, and the IFNω1 allele has the amino acid sequence described below, with the signal sequence underlined. [ka]

[0103] In various embodiments, the IFNω moiety includes 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 mature IFNω1 or fragment having up to 15 amino acid cleavages 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 acid cleavages from the N-terminus and / or C-terminus of IFNω1).

[0104] Human IFNε has been identified with UniProt accession number Q86WN2 and has the amino acid sequence described below, with the signal sequence underlined. [ka]

[0105] In various embodiments, the IFNε moiety includes 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 mature IFNε or fragment having up to 15 amino acid cleavages 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 acid cleavages from the N-terminus and / or C-terminus of IFNε).

[0106] Human IFNκ has been identified with UniProt accession number Q9P0W0 and has the amino acid sequence described below, with the signal sequence underlined. [ka]

[0107] In various embodiments, the IFNκ moiety includes 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 mature IFNκ or fragment having up to 15 amino acid cleavages 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 acid cleavages from the N-terminus and / or C-terminus of IFNκ).

[0108] 6.4. IFN Masking Area This disclosure provides IFN receptor antagonists having an IFN moiety masked by one or more receptor moieties. All human type I interferons bind to a cell surface receptor (IFN alpha receptor, IFNAR; also known as the "type I interferon receptor") which is a heterodimer consisting of two transmembrane proteins, IFNAR1 and IFNAR2 (see, e.g., Novick et al., 1994, Cell 77:391). As described herein, IFN receptor antagonists of this disclosure comprising an IFN moiety masked by IFNAR1 can inhibit IFN signaling in cells expressing the type I interferon receptor. In contrast, similar constructs comprising an IFN moiety masked by IFNAR2 cannot inhibit IFN signaling. Therefore, in certain embodiments, the masking moiety is the IFNAR1 moiety.

[0109] As used herein, “inhibiting IFN signaling” describes the reduction of signaling, activation, or activity of type I interferon receptors in cells. For example, the IFN receptor antagonists of this disclosure may be said to inhibit IFN signaling such that the molecule reduces interferon signaling by at least 10%, as measured by the activity assay described in Section 9.1.5.

[0110] An exemplary portion of IFNAR1 is disclosed in Section 6.4.1.

[0111] 6.4.1. IFNAR1 part IFNAR1 is a low-affinity IFN receptor and belongs to the type II helical cytokine receptor class. 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).

[0112] The human IFNAR1 sequence has the UniProt identifier P17181. The human IFNAR1 sequence is reproduced below. [ka]

[0113] The signal sequence (single underline) corresponds to amino acids 1-27 of the full-length human IFNAR1 protein reproduced above, the SD1 domain (bold) corresponds to amino acids 28-127, the SD2 domain (double underline) corresponds to amino acids 128-227, the SD3 domain (italic) corresponds to amino acids 231-329, the SD4 domain (lowercase) corresponds to amino acids 330-432, and the extracellular domain corresponds to amino acids 28-436.

[0114] The IFNAR1 portion is an amino acid sequence having at least 70% sequence identity with the IFN-binding portion of mammalian (e.g., human) IFNAR1, for example, 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. In some embodiments, the IFN-binding portion includes an SD2 domain and an SD3 domain. In various embodiments, the IFN-binding moiety includes (i) only the SD2 and SD3 domains, (ii) the SD1, SD2, and SD3 domains, (iii) the SD2, SD3, and SD4 domains, (iv) the SD1, SD2, SD3, and SD4 domains, or (v) all extracellular domains of IFNAR1.

[0115] 6.5. Linker In certain embodiments, the disclosure provides IFN receptor antagonists in which two or more components of an IFN receptor antagonist are linked to one another by a peptide linker. For example, but not limited to, a linker may be used to link a separator portion (e.g., an Fc domain) and an anchor portion (e.g., a targeting portion), different domains within the anchor portion (e.g., the VH domain and VL domain in scFv), a separator portion (e.g., an Fc domain) and an IFN portion or an IFNR1 portion, or an IFN portion and an IFNR1 portion.

[0116] Preferably, all linkers in the IFN receptor are non-cleavable linkers (NCLs).

[0117] The peptide linker can be in the range of 2 amino acids to 60 amino acids or more, and in certain embodiments, the peptide linker may be in the range of 3 to 50 amino acids, 4 to 30 amino acids, 5 to 25 amino acids, 10 to 25 amino acids, 10 to 60 amino acids, 12 to 20 amino acids, 20 to 50 amino acids, or 25 to 35 amino acids in length.

[0118] In certain embodiments, the peptide linker is at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids long, and optionally up to 30 amino acids, at least 40 amino acids, at least 50 amino acids, or at least 60 amino acids long.

[0119] In some of the embodiments described above, the peptide linker is in the range of 5 to 50 amino acid lengths, for example, 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, or 5 to 20 amino acid lengths. In other embodiments described above, the linker is in the range of 6 to 50 amino acid lengths, for example, 6 to 50, 6 to 45, 6 to 40, 6 to 35, 6 to 30, 6 to 25, or 6 to 20 amino acid lengths. In yet another embodiment described above, the linker is in the range of 7 to 50 amino acid lengths, for example, 7 to 50, 7 to 45, 7 to 40, 7 to 35, 7 to 30, 7 to 25, or 7 to 20 amino acid lengths.

[0120] Charged (e.g., charged hydrophilic linkers) and / or flexible linkers are particularly preferred.

[0121] Examples of flexible linkers that can be used with the IFN receptor antagonists of this disclosure include those disclosed in Chen et al., 2013, Adv Drug Deliv Rev. 65(10):1357-1369 and Klein et al., 2014, Protein Engineering, Design & Selection 27(10):325-330. Particularly useful flexible linkers are glycine and serine repeats, e.g., G n S (Sequence ID 106) or SG n The monomer or polymer of (SEQ ID NO: 107), or containing thereof, where n is an integer from 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the non-cuttable linker is G4S (SEQ ID NO: 108), e.g., (GGGGS) n It is a monomer or polymer of the repeat of (SEQ ID NO: 108), or contains such a repeat.

[0122] Polyglycine non-cleavable linkers can be suitably used in the IFN receptor antagonists of this disclosure. In some embodiments, the peptide non-cleavable linkers include two consecutive glycine molecules (2Gly), three consecutive glycine molecules (3Gly), four consecutive glycine molecules (4Gly) (SEQ ID NO: 109), five consecutive glycine molecules (5Gly) (SEQ ID NO: 110), six consecutive glycine molecules (6Gly) (SEQ ID NO: 111), seven consecutive glycine molecules (7Gly) (SEQ ID NO: 112), eight consecutive glycine molecules (8Gly) (SEQ ID NO: 113), or nine consecutive glycine molecules (9Gly) (SEQ ID NO: 114).

[0123] An example linker sequence is shown in Table E below. [Table 5-1] [Table 5-2]

[0124] In certain embodiments, the IFN receptor antagonists of this disclosure may comprise a polypeptide chain oriented from N-terminus to C-terminus, comprising a targeting moiety (or targeting sub-chain), a hinge domain, and an Fc domain. Thus, the hinge domain can be said to constitute a certain type of linker. Exemplary hinge domains are described in Section 6.7.1.3.

[0125] 6.6. Anchor section The IFN receptor antagonists of this disclosure preferably include one or more anchoring moieties. Incorporation of anchoring moieties enables anchoring of the IFN receptor antagonist to cells expressing type I interferon receptors, allowing the binding of the IFN moiety to type I interferon receptors and inhibition of IFN signaling.

[0126] As a component of an IFN receptor antagonist, the anchor moiety can be any molecule that binds to a molecule on the surface of a cell expressing a type I interferon receptor. In certain embodiments, the anchor moiety is a “cell surface protein-binding molecule,” which refers to any molecule that is present on or can bind to a protein expressed on the surface of a cell. Certain anchor moieties contemplated herein include, for example, ligands, receptors, or their ligand-binding moieties that bind to cell surface ligands, cell surface protein-binding antibodies or fragments thereof, and lipid-binding antibodies or fragments thereof.

[0127] In certain embodiments, the anchor portion binds to molecules on the surface of specific cells or cell types, enabling the binding of the IFN portion to type I interferon receptors and targeting the IFN portion to specific cell types (e.g., cancer cells, immune cells, etc.). Thus, in some embodiments, the anchor portion of this disclosure is the targeting portion.

[0128] Exemplary anchor portions of this disclosure are further described below.

[0129] 6.6.1. Targeting part In certain embodiments, the anchor portion of the Disclosure is the targeting portion. Any type of target molecule present in cells expressing type I interferon receptors, or capable of driving an IFN receptor antagonist to express type I interferon receptors, is expected to be targeted by the IFN receptor antagonists of the 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. Thus, the targeting molecule may be a tumor-associated antigen targeting molecule, a checkpoint inhibitor targeting molecule, or a molecule that targets cell surface molecules 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 can 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.

[0130] The target molecules recognized by the targeting moieties of the IFN receptor antagonists of this disclosure are generally found, for example, on the surface of activated T cells, tumor cells, dendritic cells or other antigen-presenting cells, natural killer (NK) cells, virus-infected cells, or other diseased cells. In various embodiments, the target molecules are tumor-reactive lymphocyte antigens, tumor or viral lymphocyte cell surface molecules, 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 aforementioned categories of target molecules are not mutually exclusive, and therefore, a given target molecule may be classified into two or more of the aforementioned categories of target molecules. For example, some molecules may be considered both TCAs and checkpoint inhibitors.

[0131] Exemplary types of cancer that can be targeted include acute lymphoblastic leukemia, acute myeloid leukemia, biliary tract cancer, B-cell leukemia, B-cell lymphoma, 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, stomach cancer, gastrointestinal cancer, glioma, hairy cell leukemia, head and neck cancer, Hodgkin lymphoma, liver cancer, lung cancer, medullary thyroid carcinoma, melanoma, multiple myeloma, ovarian cancer, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, pulmonary duct 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 related to the tumor microenvironment are known for substantially all types of cancer.

[0132] Other target molecules include 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.

[0133] 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, or CHK2. In some embodiments, the target molecule is PDL1.

[0134] Examples of immune cells that may be targeted include, but are not limited to, T cells (e.g., cytotoxic T cells, regulatory T cells), B cells, dendritic cells, natural killer (NK) cells, NKT cells, macrophages, and other antigen-presenting cells.

[0135] In certain embodiments, the target molecule is located on the surface of dendritic cells or other antigen-presenting cells, such as XCR1, Clec9a, CD1c, CD11c, CD14, PDL1, macrophage mannose receptor (CD206), and DEC-205.

[0136] In further embodiments, the target molecule is present on the surface of natural killer (NK) cells, such as CD335, CD38, CD2, NKG2D, NKp44, NKp30, CD16, LFA-1, CD27, KIR, NKH1A, and NKp46.

[0137] A preferred format for the targeting portion is described in Section 6.6.1.1. The targeting portion is preferably an antigen-binding portion, for example, an antibody or an antigen-binding portion of an antibody, for example, an scFv as described in Section 6.6.1.1.2, or a Fab as described in Section 6.6.1.1.1.

[0138] In some embodiments, the targeting portion targets exemplary target molecules listed in Table F below, along with references to exemplary antibodies or antibody sequences on which the targeting portion may be based. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8] [Table 6-9] [Table 6-10] [Table 6-11] [Table 6-12] [Table 6-13] [Table 6-14]

[0139] In some embodiments, the targeting moiety competes with the antibodies listed in Table F for binding to the target molecule. In further embodiments, the targeting moiety includes a CDR having the CDR sequence of the antibody listed in Table F. In some embodiments, the targeting moiety includes all six CDR sequences of the antibody listed in Table F. In other embodiments, the targeting moiety includes 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 further embodiments, the targeting moiety includes a VH having the amino acid sequence of the VH of the antibody listed in Table F. In some embodiments, the targeting moiety further includes a VL having the amino acid sequence of the VL of the antibody listed in Table F. In other embodiments, the targeting moiety further includes the universal light chain VL sequence.

[0140] In some embodiments, the targeting moiety targets PDL1 as described in Table F-1 below, along with references to exemplary antibodies or antibody sequences on which the targeting moiety may be based. [Table 7-1] [Table 7-2]

[0141] In some embodiments, the targeting moiety competes with the antibodies listed in Table F-1 for binding to the target molecule. In further embodiments, the targeting moiety includes a CDR having the CDR sequence of the antibody listed in Table F-1. In some embodiments, the targeting moiety includes all six CDR sequences of the antibody listed in Table F. In other embodiments, the targeting moiety includes 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 further embodiments, the targeting moiety includes a VH having the amino acid sequence of the VH of the antibody listed in Table F-1. In some embodiments, the targeting moiety further includes a VL having the amino acid sequence of the VL of the antibody listed in Table F-1. In other embodiments, the targeting moiety further includes the universal light chain VL sequence.

[0142] In some embodiments, the targeting moiety targets PD1 as described in Table F-2 below, along with references to exemplary antibodies or antibody sequences on which the targeting moiety may be based. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4]

[0143] In some embodiments, the targeting moiety competes with the antibodies listed in Table F-2 for binding to the target molecule. In further embodiments, the targeting moiety includes a CDR having the CDR sequence of the antibody listed in Table F-2. In some embodiments, the targeting moiety includes all six CDR sequences of the antibody listed in Table F. In other embodiments, the targeting moiety includes 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 further embodiments, the targeting moiety includes a VH having the amino acid sequence of the VH of the antibody listed in Table F-2. In some embodiments, the targeting moiety further includes a VL having the amino acid sequence of the VL of the antibody listed in Table F-2. In other embodiments, the targeting moiety further includes the universal light chain VL sequence.

[0144] Additional target molecules that can be targeted by IFN receptor antagonists are disclosed in Table I below, and in particular in Table 1, e.g., Hafeez et al., 2020, Molecules 25:4764, doi:10.3390 / molecules25204764. Table 1 of Hafeez et al. is incorporated herein by reference in its entirety.

[0145] 6.6.1.1. Format of the Targeted Section In certain embodiments, the targeting moiety of the IFN receptor antagonist of this disclosure may be any type of antibody or fragment thereof that possesses specific binding to an antigenic determinant. In one embodiment, the targeting moiety is an immunoglobulin molecule or fragment thereof, specifically an IgG class immunoglobulin molecule, more specifically an IgG1 or IgG4 immunoglobulin molecule. The antibody fragment may be VH (or V H ) Fragment, VL (or V L Examples include, but are not limited to, ) fragments, Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, minibodies, diabodies, triabodies, and tetrabodies.

[0146] 6.6.1.1.1.Fab Fab domains have traditionally been produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. Fab domains can contain constant domain and variable region sequences from any suitable species and are therefore mouse, chimeric, human, or humanized.

[0147] 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 immunoglobulin, the VH domain pairs with the VL domain to form the Fv region, and the CH1 domain pairs with the CL domain to further stabilize the binding site. Disulfide bonds between the two constant domains can further stabilize the Fab domain.

[0148] With respect to the IFN receptor antagonists of this disclosure, particularly when the light chains of the targeting regions are not common or universal light chains, it is advantageous to use Fab heterodimerization strategies to enable the correct association of Fab domains belonging to the same targeting region and minimize abnormal pairing of Fab domains belonging to different targeting regions. For example, the Fab heterodimerization strategies shown in Table G below can be used. [Table 9]

[0149] Therefore, in certain embodiments, the correct association between two polypeptides of Fab is facilitated, for example, by exchanging the VL and VH domains of Fab with each other, or by exchanging the CH1 and CL domains with each other, as described in WO2009 / 080251.

[0150] Correct Fab pairing can also be facilitated by introducing one or more amino acid modifications to the CH1 domain and one or more amino acid modifications to the CL domain of the Fab, and / or by introducing one or more amino acid modifications to the VH domain and one or more amino acid modifications to the VL domain. The amino acids to be modified are typically those that are part of the VH:VL and CH1:CL interfaces so that the Fab components pair with each other preferentially rather than with other Fab components.

[0151] In one embodiment, one or more amino acid modifications are limited to conserved framework residues in variable (VH, VL) and constant (CH1, CL) domains, as indicated by the Kabat numbering of the residues. Almagro, 2008, Frontiers in Bioscience 13:1619-1633 provides definitions of framework residues based on Kabat, Chothia, and IMGT numbering schemes.

[0152] In one embodiment, modifications introduced in the VH and CH1 domains and / or VL and CL domains are complementary to each other. Complementarity at the heavy-light chain interface can be achieved based on steric and hydrophobic contacts, electrostatic / charge interactions, or various combinations of interactions. Complementarity between protein surfaces has been widely described in the literature in terms of lock and key fit, knob into hole, protrusion and cavity, donor and acceptor, all of which suggest a structural and chemical correspondence between two interacting surfaces.

[0153] In one embodiment, one or more introduced modifications introduce new hydrogen bonds across the interfaces of Fab components. In one embodiment, one or more introduced modifications introduce new salt bridges across the interfaces of Fab components. Exemplary substitutions are described in WO2014 / 150973 and WO2014 / 082179, the contents of which are incorporated herein by reference.

[0154] In some embodiments, the Fab domain includes a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain, thereby introducing a salt bridge between the CH1 and CL domains (see, for example, Golay et al., 2016, J Immunol 196:3199-211).

[0155] In some embodiments, the Fab domain includes 143Q and 188V substitutions within the CH1 domain, as well as 113T and 176V substitutions within the CL domain, which helps to exchange hydrophobic and polar contact regions between the CH1 and CL domains (see, for example, Golay et al., 2016, J Immunol 196:3199-211).

[0156] In some embodiments, the Fab domain may include modifications in some or all of the VH, CH1, VL, and CL domains to introduce orthogonal Fab interfaces that facilitate the correct assembly of the Fab domain (Lewis et al., 2014 Nature Biotechnology 32:191-198). In one embodiment, the 39K, 62E modification is introduced into the VH domain, the H172A, F174G modification into the CH1 domain, the 1R, 38D, (36F) modification into the VL domain, and the L135Y, S176W modification into the CL domain. In another embodiment, the 39Y modification is introduced into the VH domain and the 38R modification into the VL domain.

[0157] The Fab domain can also be modified to replace the natural CH1:CL disulfide bond with an engineered disulfide bond, thereby increasing the efficiency of pairing of Fab components. For example, the engineered disulfide bond 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).

[0158] Fab domains can also be modified by replacing the CH1 and CL domains with alternative domains that facilitate correct assembly. For example, Wu et al., 2015, MAbs 7:364-76 describe replacing the CH1 domain with the constant domain of the T cell receptor and the CL domain with the b domain of the T cell receptor, and pairing these domain substitutions with additional charge-charge interactions between the VL and VH domains by introducing 38D modification to the VL domain and 39K modification to the VH domain.

[0159] Instead of, or in addition to, the use of Fab heterodimerization strategies to promote correct VH-VL pairing, a VL of a common light chain (also referred to as a universal light chain) can be used for each specific ABD in the IFN receptor antagonists of this disclosure. In various embodiments, using a common light chain as described herein reduces the number of inappropriate species in the IFN receptor antagonist compared to using the original congeneral VL. In various embodiments, the VL domain of the ABD is identified from a monospecific antibody containing a common light chain. In various embodiments, the VH region of the ABD in the IFN receptor antagonist comprises a human heavy chain variable gene segment that is rearranged in vivo in mouse B cells pre-engineered to express a limited human light chain repertoire or a single human light chain congeneral to a human heavy chain, and in response to exposure to the antigen of interest, generates an antibody repertoire containing multiple human VHs congeneral to one of one or two possible human VLs, the antibody repertoire being specific to the antigen of interest. The common light chain is derived from a reconstituted human Vκ1-39Jκ5 sequence or a reconstituted human Vκ3-20Jκ1 sequence, including somatic mutants (e.g., affinity matured mutants). See, for example, U.S. Patent No. 10,412,940.

[0160] 6.6.1.1.2. scFv Single-chain Fv or "scFv" antibody fragments contain the VH and VL domains of the antibody within a single polypeptide chain and can be expressed as single-chain polypeptides, retaining the specificity of the intact antibody from which they originate. Generally, scFv polypeptides further include a polypeptide linker between the VH and VL domains, which allows the scFv to form a desired structure for target binding. Examples of suitable linkers for connecting the VH and VL chains of scFv are the linkers identified in Section 6.5.

[0161] As used herein, unless otherwise specified, an scFv may have a VL variable region and a VH variable region in either order with respect to the N-terminus and C-terminus of the polypeptide, for example, and may contain a VL-linker-VH or a VH-linker-VL.

[0162] scFv may include VH and VL sequences from any suitable species, such as mouse-derived, human-derived, or humanized VH and VL sequences.

[0163] To create nucleic acids encoding scFv, DNA fragments encoding VH and VL are operably linked to another fragment encoding a linker, for example, one of the linkers described in Section 6.5 (typically a repeat of a sequence containing amino acids glycine and serine, such as the amino acid sequence (Gly4~Ser)3 (SEQ ID NO: 50)), thereby enabling the expression of a continuous single-stranded protein having VL and VH regions linked 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).

[0164] 6.7. Separator section The IFN receptor antagonists of this disclosure comprise one or more separator moieties. As components of an IFN receptor antagonist, the separator moiety may be any molecule that enables the simultaneous binding of the anchor moiety and the IFN moiety. Thus, the separator moieties have specific characteristics (e.g., size, shape, steric properties, etc.) such that each provides sufficient separation between the anchor moiety and the IFN moiety for each to bind to its respective target. The separator moieties may be, for example, organic polymers or polypeptides.

[0165] In addition to enabling simultaneous binding to the anchor portion and the IFN portion, the separator portion may further have one or more additional properties. For example, in certain embodiments, the separator portion is a multimerization portion. A "multimerization portion" refers to any polypeptide or other molecule or portion thereof that enables multimerization (e.g., dimerization). Such multimerization includes, for example, non-covalent association of two or more multimerization portions. Various multimerization portions are recognized in the art and contemplated herein.

[0166] Exemplary separator portions of the present disclosure are further described below.

[0167] 6.7.1. Fc Domain and Fc Region The IFN receptor antagonists of the present disclosure typically include a pair of Fc domains that associate to form an Fc region. In native antibodies, the Fc region includes a hinge region at their N-terminus to form the constant domain. Throughout the present disclosure, unless otherwise specified, reference to an Fc domain encompasses an Fc domain having a hinge domain at its N-terminus.

[0168] The Fc domain can be derived from any suitable species operably linked to the ABD or its components. In one embodiment, the Fc domain is derived from a human Fc domain. In a preferred embodiment, the targeting portion or its components are fused to an IgG Fc molecule. The targeting portion or its components can be fused to the N-terminus or C-terminus, or both, of the IgG Fc domain.

[0169] 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.

[0170] The two Fc domains within the Fc region may be identical or different from each other. In natural antibodies, the Fc domains are typically identical, but for the purpose of producing multispecific binding molecules, such as the IFN receptor antagonists of the present disclosure and the MBMs produced by their activation, the Fc domains may advantageously be different to allow for heterodimerization as described in Section 6.7.1.2 below.

[0171] In natural antibodies, the heavy chain Fc domains of IgA, IgD, and IgG are composed of two heavy chain constant domains (CH2 and CH3), and the domains of IgE and IgM are composed of three heavy chain constant domains (CH2, CH3, and CH4). These dimerize to create the Fc region.

[0172] In the IFN receptor antagonists of the present disclosure, the Fc region, and / or the Fc domains thereof, can include heavy chain constant domains from one or more different classes of antibodies, such as from 1, 2, or 3 different classes.

[0173] In one embodiment, the Fc region includes a CH2 domain and a CH3 domain derived from IgG1.

[0174] In one embodiment, the Fc region includes a CH2 domain and a CH3 domain derived from IgG2.

[0175] In one embodiment, the Fc region includes a CH2 domain and a CH3 domain derived from IgG3.

[0176] In one embodiment, the Fc region includes a CH2 domain and a CH3 domain derived from IgG4.

[0177] In one embodiment, the Fc region includes a CH4 domain derived from IgM. The IgM CH4 domain is typically located at the C-terminus of the CH3 domain.

[0178] In one embodiment, the Fc region includes CH2 and CH3 domains derived from IgG, as well as a CH4 domain derived from IgM.

[0179] It will be understood that the heavy chain constant domain for use in producing the Fc region for the IFN receptor antagonist of this disclosure may include variants of the naturally occurring constant domain described above. Such variants may include one or more amino acid mutations compared to the wild-type constant domain. In one example, the Fc region of this disclosure includes at least one constant domain whose sequence differs from that of the wild-type constant domain. It will be understood that the variant constant domain may be longer or shorter than the wild-type constant domain. Preferably, the variant constant domain is at least 60% identical or similar to the wild-type constant domain. In another example, the variant constant domain is at least 70% identical or similar. In yet another example, the variant constant domain is at least 80% identical or similar. In yet another example, the variant constant domain is at least 90% identical or similar. In yet another example, the variant constant domain is at least 95% identical or similar.

[0180] IgM and IgA are naturally present in humans as covalent multimers of a common H2L2 antibody unit. IgM exists as a pentamer when a J chain is incorporated, and as a hexamer when the J chain is absent. IgA exists in monomeric and dimeric forms. The heavy chains of IgM and IgA have an 18-amino acid extension to a C-terminal constant domain known as a tail. The tail contains cysteine ​​residues that form disulfide bonds between heavy chains in the polymer and is thought to play a crucial role in polymerization. The tail also contains glycosylation sites. In certain embodiments, the IFN receptor antagonists of this disclosure do not contain a tail.

[0181] 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, such as FcRn or leukocyte receptors, binding to complement, modified disulfide bond structures, or altered glycosylation patterns. Exemplary Fc modifications that alter effector function are described in Section 6.7.1.1.

[0182] The Fc domain can also be modified to include modifications that improve the manufacturability of asymmetric IFN receptor antagonists, for example, by enabling heterodimerization, which is the preferential pairing of non-identical Fc domains with identical Fc domains. Heterodimerization allows for the production of IFN receptor antagonists in which different polypeptide components are linked together by Fc regions containing Fc domains with different sequences. An example of a heterodimerization strategy is illustrated in Section 6.7.1.2.

[0183] It will be understood that any of the above modifications can be combined in any suitable manner to achieve desired functional properties, and / or combined with other modifications to alter the properties of an IFN receptor antagonist.

[0184] Exemplary Fc domain sequences are provided in Table C below. [Table 10-1] [Table 10-2]

[0185] 6.7.1.1. Changes in Effector Function in Fc Domain In some embodiments, the Fc domain includes one or more amino acid substitutions that reduce binding to the Fc receptor and / or effector function.

[0186] In certain embodiments, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In certain embodiments, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and cytokine secretion. In certain embodiments, the effector function is ADCC.

[0187] In one embodiment, the Fc domain (e.g., the Fc domain of an IFN receptor antagonist half antibody) or Fc region (e.g., one or both Fc domains of an IFN receptor antagonist that can associate to form an Fc region) comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331, and P329 (numbering according to the Kabat EU index). In a more particular embodiment, the Fc domain or Fc region comprises an amino acid substitution at a position selected from the group of L234, L235, and P329 (numbering according to the Kabat EU index). In some embodiments, the Fc domain or Fc region comprises the amino acid substitutions L234A and L235A (numbering according to the Kabat EU index). In such an embodiment, the Fc domain or region is an Igd Fc domain or region, particularly, a human Igd Fc domain or region. In one embodiment, the Fc domain or Fc region comprises an amino acid substitution at position P329. In a more particular embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numbering according to the Kabat EU index). In one embodiment, the Fc domain or Fc region comprises an amino acid substitution at position P329, as well as a further amino acid substitution at a position selected from E233, L234, L235, N297, and P331 (numbering according to the Kabat EU index). In a more particular embodiment, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a particular embodiment, the Fc domain or Fc region comprises amino acid substitutions at positions P329, L234, and L235 (numbering according to the Kabat EU index). In a more particular embodiment, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G (“P329G LALA”, “PGLALA”, or “LALAPG”).

[0188] Typically, the same one or more amino acid substitutions are present in each of the two Fc domains of the Fc region. Thus, in certain embodiments, each Fc domain of the Fc region contains the amino acid substitutions L234A, L235A, and P329G (numbered according to the Kabat EU index), namely, in each of the first and second Fc domains of the Fc region, the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A), and the proline residue at position 329 is replaced with a glycine residue (P329G) (numbered according to the Kabat EU index).

[0189] In one embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In some embodiments, the IgG1 Fc domain is a variant IgG1 containing the D265A, N297A mutation (EU numbered) to reduce effector function.

[0190] In another embodiment, the Fc domain is an IgG4 Fc domain with reduced binding to the Fc receptor. An exemplary IgG4 Fc domain with reduced binding to the Fc receptor may include 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: [Table 11-1] [Table 11-2] [Table 11-3]

[0191] In certain embodiments, IgG4 having reduced effector function comprises the bolded portion of the amino acid sequence of Sequence ID No. 31 of WO2014 / 121087, and may be referred to herein as IgG4s or hIgG4s.

[0192] Regarding the heterodimer Fc region, it is possible to incorporate the above-mentioned combinations of variant IgG4 Fc sequences, for example, an Fc region containing an Fc domain containing the amino acid sequence (or its bolded portion) of sequence number 30 of WO2014 / 121087 and an Fc domain containing the amino acid sequence (or its bolded portion) of sequence number 37 of WO2014 / 121087, or an Fc region containing an Fc domain containing the amino acid sequence (or its bolded portion) of sequence number 31 of WO2014 / 121087 and an Fc domain containing the amino acid sequence (or its bolded portion) of sequence number 38 of WO2014 / 121087.

[0193] 6.7.1.2. Fc Heterodimerized Variants Certain IFN receptor antagonists, unlike innate immunoglobulins, involve dimerization between two Fc domains operably linked to non-identical N-terminal or C-terminal regions. Insufficient heterodimerization of the two Fc domains to form the Fc domain can hinder increasing the yield of the desired heterodimer molecule and present a purification challenge. For example, as disclosed in EP1870459A1; U.S. Patent Nos. 5,582,996; 5,731,168; 5,910,573; 5,932,448; 6,833,441; 7,183,076; U.S. Patent Application Publication No. 2006 / 204493A1; and PCT Publication No. 2009 / 089004A1, various techniques 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.

[0194] In some embodiments, the present disclosure provides IFN receptor antagonists comprising Fc heterodimers, i.e., Fc regions comprising heterogeneous and non-identical Fc domains. Typically, each Fc domain in the Fc heterodimer comprises a CH3 domain of an antibody. The CH3 domains are derived from the constant region of an antibody of any isotype, class, or subclass, preferably an IgG (IgG1, IgG2, IgG3, and IgG4) class, as described in the preceding section.

[0195] Heterodimerization of two different heavy chains at the CH3 domain yields a desired IFN receptor antagonist, while homodimerization of the same heavy chain reduces the yield of the desired IFN receptor antagonist. Therefore, in preferred embodiments, the polypeptides that associate to form the IFN receptor antagonist of this disclosure contain a CH3 domain having modifications that are favorable for heterodimerization compared to an unmodified Fc domain.

[0196] In certain embodiments, the modification that promotes the formation of an Fc heterodimer is a so-called "knob-into-hole" or "knob-in-hole" modification, which includes a "knob" modification in one Fc domain and a "hole" modification in the other Fc domain. Knob-into-hole techniques are described, for example, in U.S. Patent No. 5,731,168, U.S. Patent No. 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 ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide to allow the protrusion to be positioned within the cavity, in order to promote heterodimer formation and inhibit homodimer formation. The protrusions are constructed by replacing smaller amino acid side chains (e.g., tyrosine or tryptophan) from the interface of the first polypeptide. Compensatory cavities of the same or similar size as the protrusions are created at the interface of the second polypeptide by replacing larger amino acid side chains (e.g., alanine or threonine) with smaller amino acid side chains.

[0197] Accordingly, in some embodiments, an amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced with an amino acid residue having a larger side-chain volume, thereby creating a protrusion in the CH3 domain of the first subunit that can be placed in a cavity in the CH3 domain of the second subunit, and an amino acid residue in the CH3 domain of the second subunit of the Fc domain is replaced with an amino acid residue having a smaller side-chain volume, thereby creating a cavity in the CH3 domain of the second subunit that can be placed therein. Preferably, the amino acid residue having a larger side-chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residue having a smaller side-chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protrusion and cavity can be produced by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. An exemplary substitution is Y470T.

[0198] In certain such embodiments, in the first Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), in the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V), optionally, the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbered according to the Kabat EU index). In further embodiments, the first Fc domain may be further modified by replacing the serine residue at position 354 with a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 with a cysteine ​​residue (E356C) (in particular, the serine residue at position 354 with a cysteine ​​residue), and the second Fc domain may be further modified by replacing the tyrosine residue at position 349 with a cysteine ​​residue (Y349C) (numbered according to the Kabat EU index). In certain embodiments, the first Fc domain may include amino acid substitutions S354C and T366W, and the second Fc domain may include amino acid substitutions Y349C, T366S, L368A, and Y407V (numbered according to the Kabat EU index).

[0199] In some embodiments, electrostatic steering (e.g., Gunasekaran et al., 2010, J Biol Chem 285(25):19637-46) can be used to facilitate the association of the first and second Fc domains of the Fc region.

[0200] As an alternative to, or in addition to, the use of Fc domains modified to promote heterodimerization, Fc domains can be modified to enable purification strategies that allow for the selection of Fc heterodimers. In one such embodiment, a polypeptide comprises a modified Fc domain that inhibits its binding to protein A, thus enabling a purification method that yields a heterodimeric protein. See, for example, U.S. Patent No. 8,586,713. Thus, the IFN receptor antagonist comprises a first CH3 domain and a second Ig CH3 domain, where the first Ig CH3 domain and the second Ig CH3 domain differ from each other by at least one amino acid, and this difference of at least one amino acid 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 includes a mutation / modification that reduces or eliminates protein A binding, such as the H95R modification (H435R according to IMGT exon numbering and EU numbering). The second CH3 may further include the Y96F modification (Y436F according to IMGT and EU). This class of modifications is referred to herein as “star” mutations.

[0201] In some embodiments, Fc may contain one or more mutations (e.g., knob and hole mutations) to promote heterodimerization, as well as star mutations to promote purification.

[0202] 6.7.1.3. Hinged Domain The IFN receptor antagonists of this disclosure may include an Fc domain having a hinge domain at its N-terminus. The hinge region may be a native or modified hinge region. The hinge region is typically found at the N-terminus of an Fc region. Unless otherwise indicated by context, the term “hinge domain” in the context of a single or monomeric polypeptide chain refers to a naturally occurring or unnatural hinge sequence that is a monomeric hinge domain, and in the context of a dimeric polypeptide (e.g., a homodimer or heterodimer IFN receptor antagonist formed by the association of two Fc domains), it may include two associated hinge sequences on separate polypeptide chains. Sometimes, two associated hinge sequences are referred to as a “hinge region.” In certain embodiments of IFN receptor antagonists, additional repeats of the hinge region may be incorporated into the polypeptide sequence.

[0203] A native hinge region is typically the hinge region found between the Fab domain and the Fc domain in naturally occurring antibodies. A modified hinge region is any hinge that differs from a native hinge region in length and / or composition. Such hinges may include hinge regions from other species, such as those from humans, mice, rats, rabbits, sharks, pigs, hamsters, camels, llamas, or goats. Other modified hinge regions may include complete hinge regions derived from antibodies of a different class or subclass than those of the heavy chain Fc domain or Fc region. Alternatively, a modified hinge region may include a portion or repeat unit of a native hinge, where each repeat unit is derived from a native hinge region. Further alternatives include modifying a native hinge region by converting one or more cysteine ​​or other residues to neutral residues such as serine or alanine, or by converting suitably positioned residues to cysteine ​​residues. Such means can increase or decrease the number of cysteine ​​residues within the hinge region. Other modified hinge regions may be entirely synthetic and can be designed to have desired properties such as length, cysteine ​​composition, and flexibility.

[0204] Several modified hinge regions are already described, for example, in U.S. Patent Nos. 5,677,425, WO99 / 15549, WO2005 / 003170, WO2005 / 003169, WO2005 / 003170, WO98 / 25971, and WO2005 / 003171, which are incorporated herein by reference.

[0205] In one embodiment, the IFN receptor antagonist of the present disclosure comprises an Fc region in which one or both Fc domains have an intact hinge domain at its N-terminus.

[0206] In various embodiments, positions 233–236 within the hinge region may be G, G, G, and empty; G, G, empty, and empty; G, empty, empty, and empty; or all empty, and the positions are numbered by EU numbering.

[0207] In some embodiments, the IFN receptor antagonists of the present disclosure include a modified hinge region that reduces binding affinity to the Fcγ receptor compared to the wild-type hinge region of the same isotype (e.g., human IgG1 or human IgG4).

[0208] In one embodiment, the IFN receptor antagonist of this disclosure comprises an Fc region in which each Fc domain has an intact hinge domain at its N-terminus, each Fc domain and hinge domain is derived from IgG4, and each hinge domain contains the modified sequence CPPC (SEQ ID NO: 121). The core hinge region of human IgG4 contains the sequence CPSC (SEQ ID NO: 122) compared to IgG1 which contains the sequence CPPC (SEQ ID NO: 121). The serine residues present in the IgG4 sequence result in increased flexibility in this region, and therefore a certain percentage of molecules form disulfide bonds (intrachain disulfides) within the same protein chain rather than crosslinking to other heavy chains within the IgG molecule to form interchain disulfides. (Angel et al., 1993, Mol Immunol 30(1):105-108). By replacing serine residues with proline residues, the same core sequence as IgG1 is obtained, allowing for the complete formation of interchain disulfides within the IgG4 hinge region, thus reducing heterogeneity in the purified product. This altered isotype is referred to as IgG4P.

[0209] 6.7.1.3.1. Chimera Hinge Array A hinged domain can be a chimeric hinged domain.

[0210] For example, a chimeric hinge may be constructed by combining an "upper hinge" sequence derived from the human IgG1, human IgG2, or human IgG4 hinge region with a "lower hinge" sequence derived from the human IgG1, human IgG2, or human IgG4 hinge region.

[0211] In certain embodiments, the chimeric hinge region comprises the amino acid sequence EPKSCDKTHTCPPCPAPPVA (sequence number 123, previously disclosed as sequence number 8 in WO2014 / 121087, which is incorporated herein by reference in its entirety) or ESKYGPPCPPCPAPPVA (sequence number 124, previously disclosed as sequence number 9 in WO2014 / 121087). Such a chimeric hinge sequence can be suitably linked to the IgG4 CH2 region (for example, by incorporating it into the IgG4 Fc domain, e.g., a human Fc domain or a mouse Fc domain, and can be further modified with CH2 and / or CH3 domains to reduce effector function, as described in Section 6.7.1.1, for example).

[0212] 6.7.1.3.2. Hinge arrangement with reduced effector functionality In further embodiments, the hinge region may be modified to reduce the effector function, for example, as described in WO2016161010A2, which is incorporated herein in its entirety by reference. In various embodiments, positions 233–236 of the modified hinge region are G, G, G, and empty; G, G, empty, and empty; G, empty, empty, and empty; or all empty, with the positions numbered by EU numbering (as shown in Figure 1 of WO2016161010A2). These segments can be represented as GGG-, GG--, G---, or ----, where "-" indicates an empty position.

[0213] Position 236 is empty in canonical human IgG2 but is occupied in other canonical human IgG isotypes. Positions 233–235 are occupied by non-G residues in all four human isotypes (as shown in Figure 1 of WO2016161010A2).

[0214] Hinge modifications within positions 233-236 can be combined with position 228, which is occupied by P. Position 228 is originally occupied by P in human IgG1 and IgG2, but by S in human IgG4 and by R in human IgG3. The S228P mutation in IgG4 antibodies is advantageous for stabilizing IgG4 antibodies and reducing heavy-light chain pair exchange between exogenous and endogenous antibodies. Preferably, positions 226-229 are occupied by C, P, P, and C, respectively.

[0215] The exemplary hinge region has residues 226–236, sometimes referred to as the intermediate (or core) and lower hinge, and is occupied by modified hinge sequences designated as GGG-(233–236), GG--(233–236), G---(233–236), and G-less(233–236). Optionally, the hinge domain amino acid sequence may include CPPPAPGGG-GPSVF (SEQ ID NO: 125, previously disclosed as SEQ ID NO: 1 in WO2016161010A2), CPPPPAPGG--GPSVF (SEQ ID NO: 126, previously disclosed as SEQ ID NO: 2 in WO2016161010A2), CPPPPAP---GPSVF (SEQ ID NO: 127, previously disclosed as SEQ ID NO: 3 in WO2016161010A2), or CPPPPAP----GPSVF (SEQ ID NO: 128, previously disclosed as SEQ ID NO: 4 in WO2016161010A2).

[0216] The modified hinge region described above can be incorporated into a heavy chain constant region, which typically includes CH2 and CH3 domains and may have additional hinge segments (e.g., upper hinges) adjacent to the specified region. Such additional constant region segments are typically of the same isotype, preferably human isotypes, but can be hybrids of different isotypes. The isotype of such additional human constant region segments is preferably human IgG4, but can be human IgG1, IgG2, or IgG3, or hybrids thereof, where the domains are of different isotypes. Exemplary sequences of human IgG1, IgG2, and IgG4 are shown in Figures 2–4 of WO2016161010A2.

[0217] In certain embodiments, the modified hinge sequence can be linked to the IgG4 CH2 region (for example, by incorporating it into the IgG4 Fc domain, e.g., a human Fc domain or a mouse Fc domain, and further modifying it with the CH2 and / or CH3 domains to reduce effector function, as described in Section 6.7.1.1).

[0218] 6.8. Nucleic acids and host cells In another embodiment, the Disclosure provides nucleic acids encoding the IFN receptor antagonists of the 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.

[0219] A single nucleic acid can 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 three or more polypeptide chains (for example, a single nucleic acid can 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). To control expression separately, open reading frames encoding two or more polypeptide chains can be under the control of separate transcriptional regulatory elements (e.g., promoters and / or enhancers). Open reading frames encoding two or more polypeptides can also be controlled by the same transcriptional regulatory element, separated by an internal ribosome entry site (IRES) sequence, and allowed to be translated into separate polypeptides.

[0220] 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 less than or equal to the number of polypeptide chains in the IFN receptor antagonist (for example, if two or more polypeptide chains are encoded by a single nucleic acid).

[0221] The nucleic acids in this disclosure may be DNA or RNA (e.g., mRNA).

[0222] In another aspect, the Disclosure provides host cells and vectors containing nucleic acids of the Disclosure. The nucleic acids may be present in a single vector or in separate vectors present in the same host cell or in separate host cells, as described more in detail below herein.

[0223] 6.8.1. Vectors This disclosure provides vectors comprising nucleotide sequences encoding one or two polypeptide chains of IFN receptor antagonists or their components as described herein, for example, half-antibodies of IFN receptor antagonists. The vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs).

[0224] A variety of vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rous sarcoma virus, MMTV, or MOMLV), or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses such as Semlik Forest virus, Eastern equine encephalitis virus, and flavivirus.

[0225] Additionally, cells in which DNA has been stably incorporated into the chromosome can be selected by introducing one or more markers that enable selection of the transfected host cell. These markers may provide, for example, prototropy to a trophic host, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be either directly ligated to the expressed DNA sequence or introduced into the same cell by co-transformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.

[0226] Once a DNA sequence containing an expression vector or construct is prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. To achieve this, various techniques may be used, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene guns, lipid-based transfection, or other conventional techniques. Methods and conditions for culturing the resulting transfected cells and recovering the expressed polypeptide are known to those skilled in the art and may be modified or optimized according to the specific expression vector and mammalian host cell used, as described herein.

[0227] 6.8.2.Cells This disclosure also provides host cells containing the nucleic acids of this disclosure.

[0228] In one embodiment, a host cell is genetically engineered to contain one or more nucleic acids as described herein.

[0229] In one embodiment, host cells are genetically engineered using an expression cassette. The term “expression cassette” refers to a nucleotide sequence that can influence 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 bringing about expression, such as an inducible promoter, may also be used.

[0230] This disclosure also provides host cells containing the vectors described herein.

[0231] The cells may be, but are not limited to, eukaryotic cells, bacterial cells, insect cells, or human cells. Preferred eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Preferred insect cells include, but are not limited to, Sf9 cells.

[0232] 6.9. Pharmaceutical Compositions The IFN receptor antagonists of this disclosure 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 specific uses, such as veterinary use or pharmaceutical use in humans. The form of the composition (e.g., dry powder, liquid formulation, etc.), as well as the excipients, diluents, and / or carriers used, depends on the intended use of the IFN receptor antagonist and, in the case of therapeutic use, the mode of administration.

[0233] For therapeutic use, the composition may be supplied as part of a sterile pharmaceutical composition containing a pharmaceutically acceptable carrier. This composition may be in any preferred form (depending on the desired method of administration to the patient). The pharmaceutical composition may be administered to the patient by various routes, such as orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intratumorally, intrathecally, topically, or topically. The most preferred route of administration in any given case 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.

[0234] Pharmaceutical compositions can be conveniently presented 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 may be in the form of a lyophilized dry powder or liquid containing an amount of IFN receptor antagonist suitable for a single dose. Dry powder unit dosage forms may be packaged in a kit with a syringe, a suitable amount of diluent, and / or other components useful for administration. Unit doses in liquid form may be conveniently supplied in the form of a syringe pre-filled with an amount of IFN receptor antagonist suitable for a single dose.

[0235] The pharmaceutical composition may also be supplied in bulk formulation form, as it contains an amount of IFN receptor antagonist suitable for multiple doses.

[0236] Pharmaceutical compositions may be prepared for storage as lyophilized formulations or aqueous solutions by mixing an IFN receptor antagonist of desired purity with any choice of pharmaceutically acceptable carriers, excipients, or stabilizers (all referred to herein as “carriers”) typically used in the art, namely buffers, stabilizers, preservatives, isotonic agents, nonionic cleaning agents, antioxidants, and various other additives. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives should be non-toxic to the recipient at the dosage and concentration used.

[0237] Buffers help maintain pH within a range close to physiological conditions. They can exist at a wide variety of concentrations, but typically they will be present in a range of about 2 mM to about 50 mM. Suitable buffers for use in this disclosure include both organic and inorganic acids and their salts, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citrate-trisodium citrate mixture, citrate-monosodium citrate mixture, etc.), succinate buffers (e.g., succinate-monosodium succinate mixture, succinate-sodium hydroxide mixture, succinate-disodium succinate mixture, etc.), tartaric acid buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumarate-monosodium fumarate mixture, disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconate-sodium glyconate mixture, gluconate-sodium hydroxide mixture, gluconate-potassium glyconate mixture, etc.), and Examples include (glucate) mixtures, oxalate buffers (e.g., oxalate-sodium oxalate mixture, oxalate-sodium hydroxide mixture, oxalate-potassium oxalate mixture, etc.), lactate buffers (e.g., lactate-sodium lactate mixture, lactate-sodium hydroxide mixture, lactate-potassium lactate mixture, etc.), and acetate buffers (e.g., acetate-sodium acetate mixture, acetate-sodium hydroxide mixture, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts (e.g., Tris) can be used.

[0238] Preservatives may be added to delay microbial growth and may be added in amounts ranging from about 0.2% to 1% (w / v). Suitable preservatives for use in this disclosure include phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halide (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkylparabens (e.g., methyl or propylparaben), catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonic agents, sometimes known as “stabilizers,” may be added to ensure the isotonicity of the liquid compositions of this disclosure and include polyhydric sugar alcohols, such as trihydric or higher sugar alcohols (e.g., glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol). Stabilizers refer to a broad category of excipients that can function as volume extenders or additives, helping to solubilize therapeutic drugs or prevent them from denatured or adhering to the container walls.Typical stabilizers include polyhydric sugar alcohols (listed above), amino acids (e.g., arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.), organic sugars or sugar alcohols (e.g., lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, etc., including cyclitols such as inositol), polyethylene glycol, amino acid polymers, and sulfur-containing reducing agents (e.g., urea, glutathione, thio). These can be saturates (e.g., suconic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate), low molecular weight polypeptides (e.g., peptides with 10 or fewer residues), proteins (e.g., human serum albumin, bovine serum albumin, gelatin, or immunoglobulins), hydrophilic polymers (e.g., polyvinylpyrrolidone), monosaccharides (e.g., xylose, mannose, fructose, glucose), disaccharides (e.g., lactose, maltose, sucrose, and trehalose), trisaccharides (e.g., raffinose), and polysaccharides (e.g., dextran). The stabilizer may be present in an amount ranging from 0.5% to 10% by weight of the IFN receptor antagonist.

[0239] Nonionic surfactants or detergents (also known as "wetting agents") can be added to aid in the solubilization of glycoproteins and protect them from agitation-induced aggregation, thereby allowing the formulation to be exposed to a stressful shear surface without causing protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), polyoxomers (184, 188, etc.), and pluronic polyols. Nonionic surfactants may be present in concentrations ranging from approximately 0.05 mg / mL to approximately 1.0 mg / mL (e.g., approximately 0.07 mg / mL to approximately 0.2 mg / mL).

[0240] Various additional excipients include bulking agents (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and cosolvents.

[0241] The IFN receptor antagonists of this disclosure can be formulated, for example, as a pharmaceutical composition containing the IFN receptor antagonist, which contains one or more pharmaceutically acceptable excipients or carriers. To prepare a pharmaceutical composition or sterile composition containing the IFN receptor antagonists of this disclosure, the IFN receptor antagonist preparation can be combined with one or more pharmaceutically acceptable excipients or carriers.

[0242] For example, IFN receptor antagonist formulations can be prepared by mixing the IFN receptor antagonist with a physiologically acceptable carrier, excipient, or stabilizer, for example, in the form of a lyophilized powder, slurry, aqueous solution, lotion, or suspension (e.g., Hardman et al., 2001, Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro, 2000, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.), 1993, Pharmaceutical Dosage Forms: General Medications, Marcel Dekker, NY; Lieberman, et al. (eds.), 1990, Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. See also: al. (eds.), 1990, Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie, 2000, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY.

[0243] The effective dose for a particular subject may vary depending on factors such as the condition being treated, the subject's overall health, the method and method of administration and dosage, 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., and Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).

[0244] The compositions of this disclosure may also be administered via one or more routes of administration using one or more of the various methods known in the art. As will be understood by those skilled in the art, the route and / or method of administration will vary depending on the desired outcome. Routes of administration selected for IFN receptor antagonists include, for example, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other common routes of administration by injection or infusion. Common administration may usually represent modes of administration other than enteral and topical administration by injection, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, subarachnoid, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, intraarachnoid, intraspine, epidural, and intrasternal injections and infusions. Alternatively, the compositions of this disclosure may be administered via topical, epithelial, or mucosal routes of administration, such as non-common routes such as intranasal, oral, vaginal, rectal, sublingual, or topical. In one embodiment, the IFN receptor antagonist is administered by infusion. In another embodiment, the IFN receptor antagonist of this disclosure is administered subcutaneously.

[0245] 6.9.1. Pharmaceutical compositions for the delivery of nucleic acids encoding IFN receptor antagonists The IFN receptor antagonists of this disclosure can be delivered by nucleic acids encoding the IFN receptor antagonist, for example, as plasmids, DNA, mRNA, or via viral vectors encoding the IFN receptor antagonist under the control of a suitable promoter.

[0246] In one embodiment, the delivery vector is a virus including retroviruses, adenoviruses, herpes simplex viruses, poxviruses, vaccinia viruses, lentiviruses, or adeno-associated viruses. In one embodiment, the delivery vector is adeno-associated viruses (AAVs) including serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, or engineered or naturally selected variants thereof.

[0247] Exemplary viral vectors include recombinant adenovirus and adeno-associated virus vectors (rAAV). rAAV vectors are based on defective non-pathogenic parvovirus adeno-associated virus type 2. Most such vectors are derived from plasmids containing only the AAV inverted terminal repeat adjacent to the transgene expression cassette. Efficient gene transport and stable transgene delivery through integration into the genome of transduced cells are key features of this vector system. Useful AAV serotypes for delivering IL27 transgenes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV 8.2, AAV9, and AAV rh10, as well as pseudotyped AAVs such as AAV2 / 8, AAV2 / 5, and AAV2 / 6.

[0248] In one embodiment, the nucleic acid encoding the IFN receptor antagonist (or its components) also contains an adeno-associated virus (AAV) nucleic acid sequence. In one embodiment, the vector is a chimeric adeno-associated virus containing gene elements from two or more serotypes. For example, an AAV vector (designated as AAV1 / 2 or AAV RC1 / 2) having a rep gene from AAV1 and a cap gene from AAV2 can be used as a delivery vector to deliver the IFN receptor antagonist expressing the nucleic acid to cells in need or to the cells of a patient.In one embodiment, the delivery vectors are 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, A AV5 / 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, A AV6 / 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 virus vectors, or derivatives thereof.Gao et al., “Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy,” PNAS 99(18):11854-11859, Sep.3, 2002 is incorporated herein by reference to AAV vectors and chimeric virus vectors useful as delivery vectors, as well as their construction and use.

[0249] AAV can be manufactured on a clinical scale by several different processes. Examples of systems that can be used include (1) plasmid DNA transfection in mammalian cells, (2) Ad infection of stable mammalian cell lines, (3) infection of mammalian cells with recombinant herpes simplex virus (rHSV), and (4) infection of insect cells (Sf9 cells) with recombinant baculovirus (as outlined by Penaud-Budloo et al., 2018, Mol Ther Methods Clin Dev. 8: 166-180).

[0250] Replication-deficient recombinant adenovirus vectors (Ad) are produced with high titers and can readily infect several different cell types. Most adenovirus vectors are engineered so that the transgene replaces the Ad Ela, Elb, and / or E3 genes, and the replication-deficient vector then grows in human 293 cells that supply the gene function deleted in trans. Ad vectors can transduce multiple types of tissues in vivo, including non-dividing differentiated cells found in the liver, kidney, and muscle. Conventional Ad vectors have high transport capacity.

[0251] Packaging cells are used to form viral particles capable of infecting host cells. Examples of such cells include 293 cells for packaging adenoviruses, and w2 or PA317 cells for packaging retroviruses. Viral vectors used in gene therapy are typically produced by producer cell lines that package nucleic acid vectors within viral particles. The vectors typically contain (where applicable) the minimum viral sequences necessary for packaging and subsequent integration into the host, with other viral sequences replaced by expression cassettes encoding the proteins to be expressed. Missing viral functions are supplied trans by the packaging cell line. For example, AAV vectors used in gene therapy typically contain only the inverted end sequence (ITR) sequence from the AAV genome, necessary for packaging and integration into the host genome. The viral DNA is packaged into a cell line containing helper plasmids that encode other AAV genes, namely rep and cap, but lacking the ITR sequence. The cell line is also infected with adenovirus as a helper. The helper virus facilitates the replication of the AAV vector and the expression of AAV genes from the helper plasmids. Helper plasmids are not packaged in sufficient quantities due to the lack of ITR sequences. Adenovirus contamination can be reduced, for example, by heat treatment, which makes the adenovirus more susceptible than AAV.

[0252] Nucleic acid molecules (e.g., mRNA) or viruses can be formulated as the sole pharmaceutically active ingredient in a pharmaceutical composition, or in combination with other activators for a specific disorder to be treated. Optionally, other drugs, pharmaceuticals, carriers, adjuvants, and diluents may be included in the compositions provided herein. For example, wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as one or more of colorants, release agents, coating agents, sweeteners, flavorings, and fragrances, preservatives, antioxidants, chelating agents, and inert gases may also be present in the composition. Other exemplary agents and excipients that may be included in the composition include, for example, water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, sodium sulfite; oil-soluble antioxidants such as ascorbyl 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.

[0253] 6.10. Indications and Method of Use This disclosure provides methods and applications for using the IFN receptor antagonists described herein.

[0254] The IFN receptor antagonists disclosed herein can be used to modulate immune responses in a variety of applications.

[0255] In certain embodiments, the Disclosure provides a method for treating cancer or an inflammatory or immune (e.g., autoimmune) disease, comprising administering an IFN receptor antagonist or pharmaceutical composition described herein to a subject in need thereof, wherein the IFN receptor antagonist expresses a type I interferon receptor and comprises an anchoring moiety (e.g., a targeting moiety) that binds to a target molecule present on the surface of disease-associated target cells.

[0256] This disclosure further provides a method for locally modulating an immune response in a target tissue, comprising administering an IFN receptor antagonist or pharmaceutical composition described herein, having one or more anchoring moieties (e.g., targeting moieties) capable of binding to a target molecule expressed in the target tissue.

[0257] In some embodiments, the administration is not local to the tissue. For example, if the target tissue is cancerous tissue, the administration may be systemic or subcutaneous.

[0258] The IFN receptor antagonists of this disclosure 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.

[0259] The IFN receptor antagonists of this disclosure can be used to treat any proliferative disorder (e.g., cancer) that expresses a target molecule (either on tumor cells or in the tumor microenvironment, e.g., in the extracellular matrix or tumor lymphocytes). In certain embodiments, the IFN antagonists of this disclosure are administered to subjects receiving, previously receiving, or likely to receive oncolytic viral therapy.

[0260] In some embodiments, cancers include 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 glandular tubule cancer. Cancer, germ cell tumors, endometrial cancer, ependymoma, esophageal cancer, sensory neuroblastoma, fibrous histiocytoma, Ewing's sarcoma, eye cancer, germ cell tumors, gallbladder cancer, stomach cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, glioma, head and neck cancer, hairy cell leukemia, hepatocellular carcinoma, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumors, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cancer, liver cancer, intraepithelial lobules Lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cervical squamous cell carcinoma of unknown primary origin, midline cancer involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, Parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureteral cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sézary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, gastric cancer, T-cell lymphoma, teratoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, or Wilms' tumor.

[0261] Table I below shows exemplary indications for which IFN receptor antagonists targeting specific target molecules can be used. [Table 12-1] [Table 12-2] [Table 12-3]

[0262] Additional target molecules and corresponding indications are disclosed, for example, in Hafeez et al., 2020, Molecules 25:4764, doi:10.3390 / molecules25204764, particularly in Table 1. Table 1 is incorporated in its entirety by reference.

[0263] In further embodiments, IFN receptor antagonists can be used to modulate immune responses induced by other agents. Thus, in some embodiments, the IFN receptor antagonists of this disclosure are administered as adjuvant therapy with immunogenic agents. In some embodiments, the immunogenic agent is an adjuvant vaccine or a non-adjuvant vaccine. Thus, IFN receptor antagonists can enhance antigen-specific immune responses induced by vaccines. In various embodiments, the vaccine is a prophylactic or therapeutic cancer vaccine, or a prophylactic or therapeutic vaccine against an infectious agent, such as a virus, bacteria, or parasite.

[0264] 6.10.1. Combination Therapy The IFN receptor antagonists of this disclosure may be administered in combination with one or more other agents in therapy. For example, the IFN receptor antagonists of this disclosure may be administered concurrently with at least one additional therapeutic agent. The term “therapeutic agent” encompasses any agent administered to a subject in need of treatment for symptoms or disease. Such additional therapeutic agents may include any active ingredients suitable for the specific indication being treated, preferably having complementary activities that do not adversely affect each other.

[0265] Without being constrained by theory, administering the IFN receptor antagonists of this disclosure in combination with oncolytic virus therapy is thought to enable enhanced efficacy and reduced side effects of oncolytic virus therapy by inhibiting IFN signaling, which is caused by the upregulation of type I IFN in response to oncolytic virus administration. Therefore, in some embodiments, the additional therapeutic agent is an oncolytic virus.

[0266] In certain embodiments, additional therapeutic agents are immunosuppressants, such as mycophenolate mofetil (MMF), mycophenolic acid (MPA), cyclosporine A, FK506-like compounds (e.g., FK506, FK506 derivatives, and FK506 analogs), rapamycin compounds (rapamycin, rapamycin derivatives, and rapamycin analogs), and corticosteroids (e.g., hydrocortisone, hydroxyltriamcinolone, alpha-methyldexamethasone, dexamethasone phosphate, beclomethasone). Dipropionate, clobetasol valerate, desonide, desoxymethasone, desoxycorticosterone acetate, dexamethasone, dichlorizone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluchlorolone acetonide, fludrocortisone, flumetasone pivalate, fluocinolone acetonide, fluocinonide, flucortin butyl ester, fluocortolone, fluprednilidene acetate, fludrenolone, halcinonide, hi Drocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolon, fludrocortisone, diflurosone diacetate, fluradrenolon acetonide, medrizone, amsinafel, amsinafid, betamethasone and its ester balance, chloroprednisone, chloroprednisone acetate, crocorterone, cresinolone , dichlorizone, diflurprednate, fluchloronide, flunizolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocoltamate, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate, triamcinolone, and mixtures thereof), nonsteroidal anti-inflammatory drugs (e.g., oxicams such as piroxicam, isoxicam, tenoxicam, sudoxicam;Salicylates such as aspirin, disalside, benolilate, trilysate, sapphin, sorprin, diflunisal, fendosal, diclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxepac, flofenac, thiopinac, didomethacin, acematacin, fenthiazac, zomepirac, clindanac, oxepinac, felbinac, and ketorolac; fenametes such as mefenamic acid, meclofenamic acid, flufenamic acid, diflumic acid, and tolfenamic acid; ibuprofen, naproxen, benoxaprofen This includes, but is not limited to, propionic acid derivatives such as flurbiprofen, ketoprofen, fenoprofen, fenbufen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, thioxaprofen, suprofen, aluminoprofen, tiaprofenic acid, pyrazoles such as phenylbutazone, oxyfenbutazone, feprazone, azapropazone, and trimethazone, and anti-inflammatory cytokines or chemokines (e.g., IL-4, IL-6, IL-10, IL-11, and IL-13).

[0267] Such other agents are preferably present in combination in amounts effective for the intended purpose. The effective amount of such other agents depends on the amount of IFN receptor antagonist used, the type of disorder or treatment, and other factors discussed above. IFN receptor antagonists are generally used in the same dosages and routes of administration as described herein, or in about 1–99% of the dosages described herein, or in any dosage and route deemed empirically / clinically appropriate.

[0268] Such combination therapies described above include combined administrations (where two or more therapeutic agents are contained in the same or separate compositions) and separate administrations, in which case the administration of the IFN receptor antagonist of this disclosure may be performed before, concurrently with, and / or after the administration of additional therapeutic agents and / or adjuvants.

[0269] 7. Array Certain sequences in this disclosure are provided in Table S below. [Table 13-1] [Table 13-2] [Table 13-3]

[0270] 8. Numbered Embodiments While various specific embodiments have been illustrated and described, it will be understood that various modifications can be made without departing from the spirit and scope of this disclosure. This disclosure is illustrated by the numbered embodiments described below. 1. A type I interferon (IFN) receptor antagonist, (a) Anchor region for cells expressing type I interferon receptor, (b) The type I interferon alpha receptor 1 (IFNAR1) portion, (c) Type I interferon (IFN) portion, (d) A separator portion that connects the anchor portion to the IFFANAR1 portion or the IFN portion, (e) an IFN receptor antagonist comprising, optionally, a linker connecting the IFNAR1 portion and the IFN portion. 2. The IFN receptor antagonist according to Embodiment 1, wherein the anchor portion can bind to extracellular matrix (ECM) antigens, tumor-reactive lymphocyte antigens, cell surface molecules of tumors 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. 3. The IFN receptor antagonist according to Embodiment 1 or 2, wherein the anchor portion is an anchor portion for immune cells. 4. The IFN receptor antagonist according to Embodiment 2, wherein the anchor portion is an anchor portion for B cells. 5. The IFN receptor antagonist according to Embodiment 1 or 2, wherein the anchor portion is an anchor portion for cancer cells. 6. An IFN receptor antagonist according to any one of embodiments 1 to 5, wherein the anchor portion is the targeting portion. 7. The IFN receptor antagonist according to Embodiment 6, wherein the targeting portion is scFv or Fab. 8. An IFN receptor antagonist according to Embodiment 6 or 7, wherein the targeting portion can bind to a target molecule identified in Section 6.6.1. 9. An IFN receptor antagonist according to any one of Embodiments 6 to 8, wherein the targeting portion (a) comprises (i) the CDR sequence or (ii) the VH sequence and VL sequence of the antibody listed in Table F, or (b) competes with the antibody listed in Table F for binding to the target molecule. 10. An IFN receptor antagonist according to any one of embodiments 6 to 9, wherein the targeting portion can bind to a checkpoint inhibitor. 11. The IFN receptor antagonist according to Embodiment 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. 12. The IFN receptor antagonist according to Embodiment 11, wherein the checkpoint inhibitor is PDL1. 13. The IFN receptor antagonist according to Embodiment 12, wherein the targeting portion is an antigen-binding fragment of an anti-PDL1 antibody. 14. An IFN receptor antagonist according to any one of Embodiments 1 to 5, wherein the anchor portion is a cell surface protein binding molecule. 15. An IFN receptor antagonist of embodiment 14, wherein the anchor portion is PD1 or its PDL1 binding portion. 16. An IFN receptor antagonist according to any one of Embodiments 1 to 15, wherein the separator portion is an Fc domain. 17. An IFN receptor antagonist of Embodiment 16, wherein the Fc domain includes a hinge domain. 18. An IFN receptor antagonist according to any one of embodiments 1 to 17, wherein the linker is a non-cleavable linker (NCL). 19. The IFN receptor antagonist according to Embodiment 18, wherein the NCL is the NCL listed in Table E. 20. An IFN receptor antagonist according to any one of embodiments 1 to 19, further comprising an additional linker connecting the separator portion to the IFNAR1 portion. 21. An IFN receptor antagonist according to any one of embodiments 1 to 19, further comprising an additional linker connecting the separator portion to the IFN portion. 22. The IFN receptor antagonist according to embodiment 20 or 21, wherein the additional linker is an NCL. 23. The IFN receptor antagonist according to Embodiment 22, wherein the additional linker is an NCL listed in Table E. 24. An IFN receptor antagonist according to any one of embodiments 1 to 23, further comprising an additional anchor portion. 25. The IFN receptor antagonist according to Embodiment 24, wherein the additional anchor portion is an additional targeting portion. 26. The IFN receptor antagonist according to Embodiment 25, wherein the additional targeting portion is scFv or Fab. 27. An IFN receptor antagonist of embodiment 26, wherein the additional targeting portion specifically binds to the same target as the anchor portion. 28. An IFN receptor antagonist according to any one of embodiments 25 to 27, wherein the additional targeting portion can bind to a target molecule identified in Section 6.6.1. 29. An IFN receptor antagonist according to any one of Embodiments 25 to 28, wherein the additional targeting portion comprises (a) the (i) CDR sequence or (ii) the VH and VL sequences of the antibody listed in Table F, or (b) competes with the antibody listed in Table F for binding to the target molecule. 30. An IFN receptor antagonist according to any one of embodiments 25 to 29, wherein the additional targeting portion can bind to a checkpoint inhibitor. 31. The IFN receptor antagonist according to Embodiment 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. 32. The IFN receptor antagonist according to Embodiment 31, wherein the checkpoint inhibitor is PDL1. 33. The IFN receptor antagonist according to Embodiment 32, wherein the additional targeting portion is an antigen-binding fragment of an anti-PD1 antibody. 34. The IFN receptor antagonist according to Embodiment 24, wherein the anchor portion is a cell surface protein binding molecule. 35. An IFN receptor antagonist of embodiment 34, wherein the anchor portion is PD1 or its PDL1 binding portion. 36. An IFN receptor antagonist according to any one of embodiments 24 to 35, further comprising an additional separator portion operably connected to the additional anchor portion. 37. The IFN receptor antagonist according to Embodiment 36, wherein the additional separator portion is an additional Fc domain. 38. The IFN receptor antagonist according to Embodiment 37, wherein the additional Fc domain includes a hinge domain. 39. An IFN receptor antagonist according to any one of embodiments 1 to 38, wherein the IFN portion is masked by the IFNAR1 portion. 40. The IFN receptor antagonist according to any one of Embodiments 1 to 39, wherein the IFN receptor antagonist comprises a first polypeptide chain and a second polypeptide chain. 41. The first polypeptide chain described above, (a) The anchor portion or its components, (b) The separator portion and, (c) The IFN receptor antagonist according to Embodiment 40, comprising the IFNAR1 portion. 42. The second polypeptide chain described above, (a) Additional anchor portion or component thereof, (b) The IFN receptor antagonist according to Embodiment 41, comprising an additional separator portion. 43. The IFN receptor antagonist according to embodiment 41 or 42, wherein the first polypeptide chain further comprises the IFN moiety. 44. The IFN receptor antagonist according to Embodiment 43, wherein the IFN portion is located at the C-terminus of the IFNAR1 portion. 45. The IFN receptor antagonist according to Embodiment 43, wherein the IFN portion is located at the N-terminus of the IFNAR1 portion. 46. ​​An IFN receptor antagonist according to any one of embodiments 41 to 45, wherein the IFNAR1 portion is located at the C-terminus of the separator portion. 47. The first polypeptide chain is oriented from the N-terminus to the C-terminus. (a) The anchor portion or its components, (b) The separator portion and, (c) The IFNAR1 portion mentioned above, (d) An IFN receptor antagonist according to any one of embodiments 40 to 43, comprising the IFN portion. 48. The first polypeptide chain is oriented from the N-terminus to the C-terminus. (a) The anchor portion or its components, (b) The separator portion and, (c) The IFN portion mentioned above, (d) An IFN receptor antagonist according to any one of embodiments 40 to 43, comprising the IFNAR1 portion. 49. The IFN receptor antagonist according to Embodiment 42, wherein the second polypeptide chain further comprises the IFN moiety. 50. The second polypeptide chain is oriented from the N-terminus to the C-terminus. (a) Additional anchor portion or component thereof, (b) Additional separator section, (c) The IFN receptor antagonist according to Embodiment 49, comprising the IFN portion. 51. An IFN receptor antagonist according to any one of embodiments 40 to 48, wherein the second polypeptide chain further comprises an additional IFNAR1 moiety and an additional IFN moiety. 52. An IFN receptor antagonist according to any one of Embodiments 1 to 51, wherein the IFN portion comprises an amino acid sequence having at least about 90% sequence identity with (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 up to 15 amino acid cleavages at its N-terminus and / or C-terminus. 53. The IFN receptor antagonist according to Embodiment 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 up to 15 amino acid cleavages at its N-terminus and / or C-terminus. 54. An IFN receptor antagonist according to any one of Embodiments 1 to 51, wherein the IFN portion comprises (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 up to 15 amino acid cleavages at its N-terminus and / or C-terminus. 55. An IFN receptor antagonist according to Embodiment 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 up to 15 amino acid cleavages at its N-terminus and / or C-terminus. 56. An IFN receptor antagonist according to any one of Embodiments 1 to 51, wherein the IFN portion comprises (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 up to 15 amino acid cleavages at its N-terminus and / or C-terminus. 57. An IFN receptor antagonist according to Embodiment 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 up to 15 amino acid cleavages at its N-terminus and / or C-terminus. 58. An IFN receptor antagonist according to any one of Embodiments 1 to 51, wherein the IFN portion is (a) a fully mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ, or (b) a mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ having up to 15 amino acid cleavages at its N-terminus and / or C-terminus. 59. An IFN receptor antagonist according to Embodiment 58, wherein the IFN portion is (a) full-length mature human IFNα2b or (b) mature human IFNα2b having up to 15 amino acid cleavages at its N-terminus and / or C-terminus. 60. An IFN receptor antagonist according to any one of Embodiments 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. 61. An IFN receptor antagonist according to any one of Embodiments 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. 62. An IFN receptor antagonist according to any one of Embodiments 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. 63. An IFN receptor antagonist according to any one of Embodiments 1 to 59, wherein the IFNAR1 portion is (i) the SD2 domain and SD3 domain of human IFNAR1, (ii) the SD1 domain, SD2 domain and SD3 domain of human IFNAR1, or (iii) the SD1 domain, SD2 domain, SD3 domain and SD4 domain of human IFNAR1. 64. An IFN receptor antagonist according to Embodiment 51, wherein the additional IFN portion comprises (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 up to 15 amino acid cleavages at its N-terminus and / or C-terminus, having at least about 90% sequence identity. 65. An IFN receptor antagonist according to Embodiment 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 up to 15 amino acid cleavages at its N-terminus and / or C-terminus. 66. An IFN receptor antagonist according to Embodiment 51, wherein the additional IFN portion comprises (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 up to 15 amino acid cleavages at its N-terminus and / or C-terminus, having at least about 95% sequence identity. 67. An IFN receptor antagonist according to Embodiment 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 up to 15 amino acid cleavages at its N-terminus and / or C-terminus. 68. An IFN receptor antagonist according to Embodiment 51, wherein the additional IFN portion comprises (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 up to 15 amino acid cleavages at its N-terminus and / or C-terminus, having at least about 98% sequence identity. 69. The IFN receptor antagonist according to Embodiment 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 up to 15 amino acid cleavages at its N-terminus and / or C-terminus. 70. An IFN receptor antagonist according to Embodiment 51, wherein the additional IFN portion is (a) a fully mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ, or (b) a mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ having up to 15 amino acid cleavages at its N-terminus and / or C-terminus. 71. The IFN receptor antagonist according to Embodiment 70, wherein the additional IFN portion is (a) full-length mature human IFNα2b or (b) mature human IFNα2b having up to 15 amino acid cleavages at its N-terminus and / or C-terminus. 72. The IFN receptor antagonist according to Embodiment 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. 73. The IFN receptor antagonist according to Embodiment 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. 74. The IFN receptor antagonist according to Embodiment 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. 75. The IFN receptor antagonist according to Embodiment 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. 76. The IFN receptor antagonist according to any one of Embodiments 1 to 75, wherein the IFN receptor antagonist does not contain the IFNAR2 portion. 77. The IFN receptor antagonist according to Embodiment 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. 78. The IFN receptor antagonist according to Embodiment 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. 79. The IFN receptor antagonist according to Embodiment 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. 80. The IFN receptor antagonist according to Embodiment 76, wherein the IFNAR2 portion is (i) the D1 domain of human IFNAR2, or (ii) the D1 and D2 domains of human IFNAR2. 81. An IFN receptor antagonist according to any one of Embodiments 1 to 80, comprising any half-antibody pair as specified in Table 2. 82. An IFN receptor antagonist, optionally an IFN receptor antagonist according to any one of embodiments 1 to 80, comprising a polypeptide chain having the configuration of two half-antibodies illustrated in Figure 1B. 83. An IFN receptor antagonist, optionally an IFN receptor antagonist according to any one of embodiments 1 to 80, comprising a polypeptide chain having the configuration of two half-antibodies illustrated in Figure 1C. 84. An IFN receptor antagonist, optionally an IFN receptor antagonist according to any one of embodiments 1 to 80, comprising a polypeptide chain having the configuration of two half-antibodies illustrated in Figure 1D. 85. An IFN receptor antagonist, optionally an IFN receptor antagonist according to any one of embodiments 1 to 80, comprising a polypeptide chain having the configuration of two half-antibodies illustrated in Figure 1E. 86. An IFN receptor antagonist, optionally an IFN receptor antagonist according to any one of embodiments 1 to 80, comprising a polypeptide chain having the configuration of two half-antibodies illustrated in Figure 1F. 87. A type I interferon (IFN) receptor antagonist, (a) A first polypeptide, oriented from the N-terminus to the C-terminus, (i) First targeting portion, (ii) First Fc domain, (iii) First linker, (iv) IFNAR1 portion, (v) Second linker, and (vi) A first polypeptide including the IFN portion, (b) A second polypeptide, oriented from the N-terminus to the C-terminus, (i) Second targeting portion, and (ii) an IFN receptor antagonist which is optionally an IFN receptor antagonist according to any one of Embodiments 1 to 86, comprising a second polypeptide comprising a second Fc domain associated with the first Fc domain. 88. A type I interferon (IFN) receptor antagonist, (a) A first polypeptide, oriented from the N-terminus to the C-terminus, (i) First targeting portion, (ii) First Fc domain, (iii) First linker, (iv) an IFN moiety; (v) Second linker, and (vi) A first polypeptide containing the IFNAR1 portion, (b) A second polypeptide, oriented from the N-terminus to the C-terminus, (i) Second targeting portion, and (ii) an IFN receptor antagonist which is optionally an IFN receptor antagonist according to any one of Embodiments 1 to 86, comprising a second polypeptide comprising a second Fc domain associated with the first Fc domain. 89. A type I interferon (IFN) receptor antagonist, (a) A first polypeptide, oriented from the N-terminus to the C-terminus, (i) First targeting portion, (ii) First Fc domain, (iii) First linker, (iv) The first IFNAR1 portion, (v) Second linker, and (vi) A first polypeptide comprising the first IFN portion, (b) A second polypeptide, oriented from the N-terminus to the C-terminus, (i) Second targeting portion, (ii) A second Fc domain associated with the first Fc domain, (iii) The third linker, (iv) The second IFNAR1 portion, (v) The fourth linker, and (vi) an IFN receptor antagonist comprising a second polypeptide comprising a second IFN moiety, which is optionally an IFN receptor antagonist according to any one of embodiments 1 to 86. 90. A type I interferon (IFN) receptor antagonist, (a) A first polypeptide, oriented from the N-terminus to the C-terminus, (i) First targeting portion, (ii) First Fc domain, (iii) First linker, (iv) The first IFN portion, (v) Second linker, and (vi) A first polypeptide comprising the first IFNAR1 portion, (b) A second polypeptide, oriented from the N-terminus to the C-terminus, (i) Second targeting portion, (ii) A second Fc domain associated with the first Fc domain, (iii) The third linker, (iv) The second IFN section, (v) The fourth linker, and (vi) an IFN receptor antagonist which is optionally an IFN receptor antagonist according to any one of embodiments 1 to 86, comprising a second polypeptide comprising a second IFNAR1 portion. 91. A nucleic acid or a group of nucleic acids encoding an IFN receptor antagonist according to any one of Embodiments 1 to 90. 92. A host cell that has been modified to express an IFN receptor antagonist according to any one of Embodiments 1 to 90 or a nucleic acid(s) according to Embodiment 70. 93. A method for producing an IFN receptor antagonist according to any one of Embodiments 1 to 90, comprising culturing a host cell according to Embodiment 92 and recovering the IFN receptor antagonist expressed thereby. 94. A pharmaceutical composition comprising an IFN receptor antagonist according to any one of Embodiments 1 to 90 and an excipient. 95. The pharmaceutical composition according to Embodiment 94, further comprising an oncolytic virus. 96. A method for inhibiting IFN signaling in cells expressing (a) an IFN receptor and (b) a molecule to which the anchor portion specifically binds, comprising contacting the cells with an IFN receptor antagonist according to any one of Embodiments 1 to 90 or a pharmaceutical composition according to Embodiment 94. 97. The method according to Embodiment 96, wherein the cells are immune cells. 98. The method according to embodiment 96 or 97, wherein the cells are B cells. 99. The method according to Embodiment 96, wherein the cells are cancer cells. 100. The method according to any one of embodiments 96 to 99, wherein the method is an in vitro method. 101. The method according to any one of embodiments 96 to 99, comprising administering the IFN receptor antagonist to a subject requiring such administration. 102. The method according to Embodiment 101, wherein the subject is a patient with cancer. 103. The method according to Embodiment 102, further comprising administering an oncolytic virus to the subject. 104. The method according to Embodiment 103, wherein the oncolytic virus is oncolytic bullous stomatitis virus (VSV). 105. The method according to Embodiment 101, wherein the subject is a patient having an immune disorder or condition. 106. The method according to Embodiment 105, wherein the immune disorder or condition is systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSc), or Sjögren's syndrome (SS). 107. A method for reducing inflammation, comprising administering to a subject in need thereof an IFN receptor antagonist according to any one of Embodiments 1 to 90 or a pharmaceutical composition according to Embodiment 94. 108. A method for treating a subject suffering from cancer, comprising administering to the subject in need of such treatment an IFN receptor antagonist according to any one of Embodiments 1 to 90 or a pharmaceutical composition according to Embodiment 94. 109. The method according to Embodiment 108, further comprising administering an oncolytic virus to the subject. 110. The method according to Embodiment 109, wherein the oncolytic virus is oncolytic vesicular stomatitis virus (VSV). 111. A method for treating a subject suffering from an immune disorder or condition, comprising administering to the subject an IFN receptor antagonist according to any one of Embodiments 1 to 90 or a pharmaceutical composition according to Embodiment 94. 112. The method according to Embodiment 111, wherein the immune disorder or condition is systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSc), or Sjögren's syndrome (SS). [Examples]

[0271] 9. Examples 9.1. Materials and Methods 9.1.1. Design of IFN receptor antagonist constructs The IFN receptor antagonist constructs depicted in Figure 1 were designed as dimers (e.g., homodimers or heterodimers) containing two polypeptide chains. Table E-1 below provides the core polypeptide sequences used in the studies described herein. Using the core polypeptide sequences in Table E-1, IFN receptor antagonists and control constructs can be generated, thereby allowing the addition of a targeting moiety, e.g., Fab or scFv, to the N-terminus of each polypeptide chain. [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4]

[0272] 9.1.2. Production of IFN receptor antagonist constructs Constructs encoding IFN receptor antagonists were generated in standard mammalian protein expression DNA vectors (pcDNA3.4 or similar) suitable for high-yield protein production and containing standard elements such as promoter sequences, poly(A) sequences, regulatory elements, and resistance genes. Where applicable, sequences were codon-optimized. A 29-amino acid signal sequence from mouse inactive tyrosine-protein kinase transmembrane receptor ROR1 (mROR1) was added to the N-terminus of the construct to function as a signal for secretion. All IFN receptor antagonists were expressed as preproteins containing signal sequences cleaved by intracellular processing to produce mature proteins. Constructs were expressed in Expi293FTM cells by transient transfection (Thermo Fisher Scientific). Proteins from the Expi293F supernatant were purified using a ProteinMaker system (Protein BioSolutions, Gaithersburg, MD) equipped with either a HiTrap® Protein G HP or a MabSelect SuRe pcc column (Cytiva). After single-step elution, the proteins were neutralized, dialyzed to a final buffer of phosphate-buffered saline (PBS) containing 5% glycerol, aliquoted, and stored at -80°C. The samples were further analyzed by SE-UPLC to determine the presence of high or low molecular weight species compared to the target species.

[0273] 9.1.3. Manipulation of KG-1a reporter cells The premyeloblastic macrophage cell line KG-1a was transduced with an interferon-stimulated response element (ISRE)-driven luciferase reporter construct and maintained in Iskoff-modified Dulbecco's 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 was knocked out in this clone using CRISPR-Cas9 technology, and the resulting cell line KG-1a / ISRE-Luc / PDL1 KO was validated by flow cytometry. KG-1a / ISRE-Luc was modified to overexpress PDL1 (amino acids M1-T290, accession number NP_054862.1), and the resulting cell line, KG-1a / ISRE-Luc / hPDL1, was validated by flow cytometry.

[0274] 9.1.4. Staining and Flow Cytometry KG-1a / ISRE-Luc cells, 2.5 × 10⁶ per well 5 Cells were seeded at a density and incubated on ice for 30 minutes with serial dilutions of an IFN receptor antagonist. Cells were washed twice and counter-stained with goat anti-human IgG F(ab')2AF647 on ice for a further 30 minutes. After staining was complete, cells were washed, fixed, and data were acquired using iQue Plus (Sartorius).

[0275] 9.1.5. Luciferase assay setup for IFN signaling evaluation Cell suspensions and dilutions of IFN receptor antagonists or control constructs were prepared using RPMI1640 medium supplemented with 2 mM L-glutamine / penicillin / streptomycin + 10% FBS as the assay medium.

[0276] On assay day, KG-1a / ISRE-Luc reporter cells were centrifuged and 5 × 10⁶ cells were added to assay medium. 5were resuspended at a density of / mL. IFNα2b, IFN receptor antagonist, and control constructs were diluted 1:5 according to an 11-point dilution range, and the 12th point contained no recombinant protein. 2.5×10 4 Reporter cells were added to 96-well white flat-bottom plates and incubated with serially diluted IFNα2b, IFN receptor antagonist, and control constructs in the presence or absence of 200 pM IFNα2b, 45 pM IFNα2b, or 90 pM IFNβ. After incubating the plates at 37 °C and 5% CO2 for 5 hours, 100 μL of ONE-Glo™ (Promega) reagent was added to the lysed cells to detect luciferase activity. The emitted light was captured in relative light units (RLU) on a multi-label plate reader Envision (PerkinElmer). All serial dilutions were tested in duplicate.

[0277] 9.1.6. Evaluation of cell viability by luminescence assay Iscove's modified Dulbecco's medium supplemented with 2 mM L-glutamine / penicillin / streptomycin and 20% fetal bovine serum (FBS) was used as assay medium to prepare cell suspensions and dilutions of recombinant IFNα2b, IFNβ, or control constructs.

[0278] On the day of the assay, KG-1a / ISRE-Luc reporter cells were centrifuged and resuspended in assay medium at a density of 1×10 5 / mL and 5×10 3Reporter cells were seeded in wells and incubated with a fixed amount of IFNα2b (900 pM) or IFNβ (30 pM) along with titration of recombinant IFNα2b, IFNβ, or a control construct. All constructs were serially diluted (1:5) over a 9-point titration range (50 nM to 5.12 fM), with the 10th point containing no recombinant protein or fixed amount of IFNα2b or IFNβ. 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 incubated at 37°C and 5% CO2 for 1 hour, followed by detection of NanoLuc® luciferase activity. The emitted light was measured in relative light units (RLU) on a multi-label plate reader Envision (PerkinElmer). EC of the tested constructs was measured. 50 The values ​​were determined using GraphPad Prism™ software from a four-parameter logistic equation over a 10-point dose-response curve. The percentage inhibition of the cytotoxic response for each tested construct was calculated using the following formula: % inhibition = ((maximum luminescence (construct) - minimum luminescence (construct)) / (maximum IFN luminescence - minimum IFN luminescence)) × 100. All serial dilutions were tested in double series.

[0279] 9.2. Example 1: In vitro binding affinity of IFN receptor antagonists Targeted monovalent and bivalent IFN receptor antagonists and control constructs were designed and produced as described in sections 9.1.1 and 9.1.2. The binding affinity of the IFN receptor antagonists and control constructs was evaluated as described in section 9.1.4, and the data obtained from viable cells were used as a measure of binding affinity. The strongest binding affinity was observed with cis-masked bivalent Fc-R1-IFNα2b and monovalent Fc-R1-IFNα2b constructs (Figure 2A).

[0280] 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. The potency of both monovalent and bivalent Fc-IFNa2b was reduced compared to IFNa2b. Compared to Fc-IFNα2b, cis-masked bivalent Fc-R1-IFNα2b (bivalent Fc-R1-IFNα2b) and monovalent Fc-R1-IFNα2b (Fc-R1-IFNα2b), as well as the lesser-trans-masked monovalent Fc-IFNα2b×R1 (Fc-IFNα2b×R1) constructs, exhibited reduced potency.

[0281] In summary, these results indicate that cis-masked constructs containing an IFNAR1 masking moiety occupy IFNAR2 receptors on the cell membrane, preventing free IFN binding to these receptors and attenuating the downstream intracellular cascade.

[0282] 9.3. Example 2: In vitro signaling activity of targeted IFN receptor antagonists Targeted monovalent and bivalent IFN receptor antagonists and control constructs were designed and produced as described in sections 9.1.1 and 9.1.2. The signaling activity of the targeted 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.

[0283] In the first evaluation, the activity of PDL1-targeted 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 exhibited lower potency (Figure 3A). Compared to untargeted Ab-IFNα2b, masked untargeted constructs exhibited lower potency regardless of their masking moieties, with monovalent cis-masked (monovalent Iso-R1-IFNα2b) and bivalent masked (bivalent Iso-R1-IFNα2b) exhibiting the lowest potency (Figures 3B and 3C). The construct aPDL1-IFNα2b exhibited the strongest activity, surpassing that shown by the IFNα2b control (Figure 3A), highlighting the effect of PDL1 targeting. Other targeting-dependent activations of IFNAR were observed using trans-masked IFNAR1-masking subconstructs, with monovalent aPDL1-R1×IFNα2b exhibiting stronger potency than iso-R1×IFNα2b. No targeting-dependent increase in activity was observed in cis-masked monovalent and divalent R1-masked constructs.

[0284] Next, the activity of PDL1-targeted IFN receptor antagonists and isotype control constructs was evaluated in the presence of 200 pM IFNα2b in PDL1 KO cells (Figures 3D-3F). All PDL1-targeted monovalent and bivalent IFN receptor antagonists and isotype control constructs were associated with a relatively constant level of reporter activity due to the presence of 200 pM IFNα2b, and none of the constructs showed concentration-dependent changes in activity, demonstrating that IFN receptor antagonists do not possess target-independent antagonist activity.

[0285] In the following evaluation set, the activity of PDL1-targeted IFN receptor antagonists and isotype control constructs was assessed in the presence of 200 pM IFNα2b in PDL1 overexpression (OE) cells (Figures 3G-3I). Titration with IFNα2b yielded comparable concentration-response curves (Figures 3D and 3F). When PDL1-targeted monovalent IFN receptor antagonists and isotype control constructs were evaluated using PDL1 OE cells, the PDL1-targeted construct with the IFNAR1 masking moiety, aPDL1-R1-IFNα2b, showed a concentration-dependent reduction in activity and targeted-dependent IFNAR blockade (Figure 3H). Furthermore, the structure of aPDL1-R1-IFNα2b appeared to be associated with potent reduction in activity. The masking and IFN moieties of aPDL1-R1-IFNα2b are located on the same polypeptide chain and are linked by a linker as depicted in Figure 1B. As depicted in Figure 1D, the similar but trans-masked monovalent construct, PDL1-IFNα2b×R1, in which the masking and IFN moieties are located on different polypeptide chains, was associated with incomplete IFNAR blockade.

[0286] The divalent construct aPDL1-R1-IFNα2b was associated with similar activity reduction as the monovalent cis-masked construct (Figure 3I). Both monovalent and divalent aPDL1-R1-IFNα2b achieved complete blockade of IFNAR activity, while the trans-masked aPDL1-IFNα2b×PDL1-R1 achieved partial blockade of IFNAR activity (Figures 3H and 3I).

[0287] Next, the activity of PDL1-targeted IFN receptor antagonists and isotype control constructs was evaluated against exemplary anti-IFNAR1 and anti-IFNAR2 antibodies in the presence of 45 pM IFNα2b (Figures 4A-4F) or 95 pM IFNβ (Figures 4G-4L) in PDL1 KO (Figures 4A-4C and 4G-4I) or PDL1 OE cells (Figures 4D-4F and 4J-4L). Titration with masked monovalent or bivalent PDL1-R1-IFN yielded dose-dependent PDL1-dependent inhibition response curves for IFNα2b (Figures 4A-4F) and IFNβ (Figures 4G-4L). In contrast, anti-IFNAR1 and IFNAR2 antibodies yielded lower blocking power against either IFNα2b or IFNβ, which was not PDL1-dependent. In summary, these data demonstrate that masked formats of Ab-R1-IFNa2b monovalent and bivalent enable potent targeted IFN receptor blockade.

[0288] 9.4. Example 3: In vitro cytoprotective activity of targeted IFN receptor antagonists Targeted monovalent and bivalent IFN receptor antagonists and control constructs were designed and produced as described in sections 9.1.1 and 9.1.2. The cytoprotective activity of the targeted 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.

[0289] In the initial evaluation set, the cytoprotective activity of IFN receptor antagonists and control constructs was assessed in PDL1-overexpressing (OE) and PDL1 KO KG-1a / ISRE-Luc reporter cells co-treated with 900 pM IFNα2b for 5 days. This IFNα2b treatment resulted in either a cell proliferation inhibitory response, detected as a flat luminescence curve due to a lack of cell division, or cytotoxicity, detected as a decrease in luminescence signaling due to a reduction in cell number. Neither the PDL1-targeted unmasked construct nor the isotype control unmasked construct blocked the cell proliferation inhibitory / cytotoxic response to IFNα2b treatment (Figures 5A and 6A, and Table E-2). In contrast, PDL1-targeted monovalent and bivalent masked IFN receptor antagonists were associated with a concentration-dependent increase in luminescence in PDL1 OE KG-1a / ISRE-Luc reporter cells, while isotype controls were associated with relatively constant levels of luminescence (Figures 5B-5C) and percentage inhibition of the IFNα2b treatment-induced cell proliferation inhibitory / cytotoxic response (Table E-2). In PDL1 KO KG-1a / ISRE-Luc reporter cells, neither monovalent nor bivalent IFN receptor antagonists were associated with increased luminescence (Figures 6B-6C) or increased percentage inhibition (Table E-2), suggesting that the cytoprotective activity of IFN receptor antagonists is target-dependent. Anti-IFNAR1 and anti-IFNAR2 antibodies yielded relatively moderate cytoprotective activity that was not PDL1-dependent (Figures 5D and 6D, and Table E-2).

[0290] In the second evaluation set, IFNβ was used to trigger a cell proliferation inhibitory / cytotoxic response, and the cytoprotective activity of 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 evaluation set, PDL1-targeted monovalent and bivalent masked IFN receptor antagonists were associated with a concentration-dependent increase in luminescence in PDL1 OE KG-1a / ISRE-Luc reporter cells (Figures 7A–7C and 8A–8C), as well as a percentage inhibition of the IFNα2b treatment-induced cell proliferation inhibitory / cytotoxic response, rather than with the control construct (Table E-2). Again, anti-IFNAR1 and anti-IFNAR2 antibodies resulted in relatively moderate increases in luminescence that were not PDL1-dependent (Figures 7D and 8D, and Table E-2). In summary, these data demonstrate that monovalent and bivalent masked IFN receptor antagonists enable potent target-dependent cytoprotective activity. [Table 15]

[0291] 9.5. Example 4: IFN-induced cell proliferation inhibitory blockade activity by targeted IFN receptor antagonists Monovalent and bivalent IFNAR1 or IRNAR2 masked constructs were designed and produced as described in Sections 9.1.1 and 9.1.2. Cell viability in the presence of IFNa2b or IFNβ was evaluated using IFN receptor antagonists and control constructs in PDL1 KO and PDL1 overexpression (OE) KG-1a / ISRE-Luc reporter cells, as described in Section 9.1.6.

[0292] In PDL1-KO cells, IFNα2b exhibited a dose-dependent inhibitory effect on cell proliferation (Figure 9A). In the presence of IFNα2b, all isotypes or PDL1-targeted monovalent IFN receptor antagonists and control constructs showed relatively consistent levels of luminescence (Figure 9B). Targeted and isotype bivalent IFN receptor antagonists were associated with weak blockade of cell proliferation inhibitory effects in the absence of PD-L1 expression (Figure 9C).

[0293] Next, cell viability was evaluated in PD-L1 OE KG-1a / ISRE-Luc cells using IFN receptor antagonists and control constructs in the presence of 900 pM IFNα2b. All control molecules were associated with relatively constant levels of luminescence (Figure 9D). However, PD-L1-targeted monovalent and bivalent IFN receptor antagonists were associated with concentration-dependent blockade of the IFNα2b cell proliferation inhibitory effect (Figures 9E and 9F). Both monovalent and bivalent cis-masked molecules (aPDL1-R1-IFNα2b) achieved significant blockade of IFNα2b cell proliferation inhibitory activity (Figures 9E and 9F). The trans-masked molecule (aPDL1-IFNα2b×R1) also achieved significant blockade, albeit at a lower level compared to the cis-masked monovalent aPDL1-R1-IFNα2b (Figure 9E).

[0294] In the next evaluation, cell viability was assessed in PDL1 KO KG-1a / ISRE-Luc cells in the presence of IFNβ. Again, IFNβ itself also exhibited a dose-dependent inhibitory effect on cell proliferation (Figure 9G). In the presence of IFNβ, all targeted monovalent IFN receptor antagonists and control constructs showed relatively consistent levels of luminescence (Figure 9H). Targeted and isotyped bivalent IFN receptor antagonists were associated with weak blockade of the inhibitory effect on cell proliferation (Figure 9I).

[0295] Next, cell viability was evaluated in PDL1 OE KG-1a / ISRE-Luc cells in the presence of 30 pM IFNβ. All control molecules were associated with relatively constant levels of luminescence (Figures 9J-9L). However, PD-L1-targeted monovalent and bivalent IFN receptor antagonists were associated with concentration-dependent blockade of IFNβ activity. Both monovalent and bivalent cis-masked molecules (aPDL1-R1-IFNα2b) achieved significant blockade of IFNβ cell proliferation inhibitory activity (Figures 9K and 9L). The trans-masked molecule (aPDL1-IFNα2b×R1) also achieved significant blockade, albeit at a lower level compared to the cis-masked monovalent aPDL1-R1-IFNα2b (Figure 9K).

[0296] 9.6. Example 5: Effect of targeted IFN receptor antagonist on IP10 release Monovalent and bivalent IFNAR1 or IRNAR2-masked constructs were designed and produced as described in sections 9.1.1 and 9.1.2. The effects of the IFNAR1-masked and IFNAR2-masked constructs were evaluated in PDL1-expressing monocyte-derived dendritic cells (MoDcCs). These were incubated with the individual constructs for 3 days, during which the supernatant was collected and assayed for IP10 using AlphaLISA.

[0297] PDL1 expression was confirmed in MoDCs (Figure 10B). The IFNAR1-masked construct showed targeting-dependent blocking activity of IFNα2b-mediated IP10 release in MoDCs (Figure 10A). In contrast, the IFNAR2-masked construct did not block IP10 release (Figure 10A). Furthermore, the monovalent IFNAR1-masked PDL1-targeted construct was associated with higher IP10 release blocking ability compared to the divalent IFNAR1-masked PDL1-targeted construct (Figure 10A).

[0298] 9.7. Example 6: In vitro signaling activity of targeted IFN receptor antagonists Isotyped or PDL1-targeted IFN receptor antagonists and control constructs were designed and produced as described in sections 9.1.1 and 9.1.2. The signaling activity of the isotyped or PDL1-targeted IFN receptor antagonists and control constructs was evaluated using PDL1-KO and PDL1-overexpressing (OE) KG-1a / ISRE-Luc reporter cells as described in sections 9.1.3 and 9.1.5.

[0299] In the first evaluation, the activity of isotypes or PDL1-targeted 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 isotypes and PDL1-targeted bivalent IFN receptor antagonists showed relatively low levels of ISRE-Luc activity (Figure 11A). In the presence of 20 pM hIFNα2b, all bivalent IFN receptor antagonists were associated with low levels of antagonism (Figure 11B). In the presence of 10 pM hIFNβ, antagonism of IFNβ-containing constructs was observed in a targeting-independent manner (Figure 11C).

[0300] Next, the activity of isotypes or PDL1-targeted bivalent IFN receptor antagonists and control constructs was evaluated in PDL1 OE KG-1a / ISRE-Luc reporter cells. Again, in the absence of hIFNα2b or hIFNβ, all isotypes and PDL1-targeted bivalent IFN receptor antagonists showed relatively low levels of ISRE-Luc activity (Figure 11D). In the presence of 20 pM hIFNα2b, both bivalent PDL1-targeted IFN receptor antagonists containing IFNα2b and IFNβ were associated with significant antagonism (Figure 11E). In the presence of 10 pM hIFNβ, both bivalent PDL1-targeted IFN receptor antagonists containing IFNα2b and IFNβ were associated with significant antagonism (Figure 11F).

[0301] In all experiments, the aPDL1-R1 construct without any IFN molecules showed no antagonism (Figures 11A-11F).

[0302] 9.1. Example 7: In vitro signaling activity of PDL1 and EGFR-targeted uIFN-containing IFN receptor antagonists Isotypes containing a universal type I interferon (uIFN) moiety, PDL1, or EGFR-targeted IFN receptor antagonists and control constructs were designed and produced as described in sections 9.1.1 and 9.1.2. The signaling activity of the isotypes, or PDL1-targeted IFN receptor antagonists and control constructs was evaluated using PDL1-KO and PDL1-OE KG-1a / ISRE-Luc reporter cells or EGFR OE and EGFR-free cells as described in sections 9.1.3 and 9.1.5.

[0303] In the absence of hIFNα2b or hIFNβ, both the isotype and PDL1-targeted IFN antagonist constructs showed similar ISRE-Luc activity in PDL1 KO cells (Figure 12A). The isotype control construct showed similar activity in PDL1 OE cells, while the PDL1-targeted IFN antagonist did not show activity in these cells (Figure 12B). In the presence of 20 pM hIFNα2b, both constructs showed stable levels of ISRE-Luc activity in PDL1 KO cells (Figure 12C). The PDL1-targeted IFN antagonist inhibited this activity in PDL1 OE cells (Figure 12D), while the isotype control did not. Similarly, in the presence of 10 pM hIFNβ, both constructs showed stable levels of ISRE-Luc activity in PDL1 KO cells (Figure 12E). However, in PDL1 OE cells, PDL1-targeted IFN antagonists showed strong antagonistism.

[0304] Next, EGFR-targeted and isotype constructs were evaluated. In the absence of hIFNα2b or hIFNβ, both the isotype and EGFR-targeted IFN antagonist constructs showed similar ISRE-Luc activity in EGFR-free cells (Figure 13A). The isotype control construct showed similar activity in PDL1 OE cells, while the PDL1-targeted IFN antagonist showed reduced activity in these cells (Figure 13B). In the presence of 20 pM hIFNα2b, both constructs showed stable levels of ISRE-Luc activity in "EGFR-free" cells (Figure 13C). In EGFR OE cells, the EGFR-targeted IFN antagonist showed significant antagonism (Figure 13D), while the isotype control did not. Similarly, in the presence of 10 pM hIFNβ, both constructs exhibited stable levels of ISRE-Luc activity in "EGFR-free" cells (Figure 13E), and again, EGFR-targeted IFN antagonists showed significant antagonism in EGFR-OE cells.

[0305] 10. Citation of References All publications, patents, patent applications, and other documents referenced herein are incorporated herein by reference in whole for all purposes to the same extent as if each individual publication, patent, patent application, or other document were individually indicated as being incorporated herein by reference for all purposes. In the event of any conflict between one or more teachings of the references incorporated herein and the present disclosure, the teachings herein shall prevail.

Claims

1. It is a type I interferon (IFN) receptor antagonist, (a) An anchor portion for cells expressing type I interferon receptor, (b) The type I interferon alpha receptor 1 (IFNAR1) portion, (c) Type I interferon (IFN) portion, (d) A separator portion connecting the anchor portion to the IFNAR1 portion or the IFN portion, (e) an IFN receptor antagonist comprising, optionally, a linker connecting the IFNAR1 portion and the IFN portion.

2. The IFN receptor antagonist according to claim 1, wherein the anchor portion can bind to extracellular matrix (ECM) antigens, tumor-reactive lymphocyte antigens, cell surface molecules of tumors 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.

3. The IFN receptor antagonist according to claim 1, wherein the anchor portion is the targeting portion.

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

5. The IFN receptor antagonist according to claim 3, wherein the targeting portion can bind to a checkpoint inhibitor, and optionally 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 according to claim 5, wherein the checkpoint inhibitor is PDL1.

7. The IFN receptor antagonist according to 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 separator portion 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 according to claim 9, wherein the NCL is the NCL listed in Table E.

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

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

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

14. The IFN receptor antagonist according to claim 13, wherein the additional anchor portion is an additional targeting portion.

15. The IFN receptor antagonist according to claim 14, wherein the additional targeting portion is scFv or Fab.

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

17. An IFN receptor antagonist according to any one of claims 14 to 16, wherein the additional targeting portion can bind to a checkpoint inhibitor, optionally 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 according to claim 17, wherein the checkpoint inhibitor is PDL1.

19. The IFN receptor antagonist according to 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 separator portion operably connected to the additional anchor 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 first polypeptide chain is (a) The anchor portion or its components, (b) The separator portion and, (c) The IFN receptor antagonist according to claim 22, comprising the IFNAR1 portion.

24. The second polypeptide chain is (a) Additional anchor portion or component thereof, (b) The IFN receptor antagonist according to claim 23, comprising an additional separator portion.

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

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

27. The second polypeptide chain is oriented from the N-terminus to the C-terminus. (a) Additional anchor portion or component thereof, (b) Additional separator section, (c) The IFN receptor antagonist according to claim 26, comprising 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. An 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) a full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε, or IFNκ having up to 15 amino acid cleavages at its N-terminus and / or C-terminus.

30. The IFN receptor antagonist according to 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 up to 15 amino acid cleavages at its N-terminus and / or C-terminus.

31. The IFN receptor antagonist according to claim 30, wherein the IFN portion is (a) fully mature human IFNα2b or (b) mature human IFNα2b having up to 15 amino acid cleavages at its N-terminus and / or C-terminus.

32. An IFN receptor antagonist according to any one of claims 1 to 31, wherein the IFNAR1 portion comprises or consists of an amino acid sequence having at least 90%, at least 95%, at least 98%, or 100% sequence identity with (i) the SD2 domain and SD3 domain of human IFNAR1, (ii) the SD1 domain, SD2 domain and SD3 domain of human IFNAR1, or (iii) the SD1 domain, SD2 domain, SD3 domain and SD4 domain of human IFNAR1.

33. The IFN receptor antagonist according to claim 28, wherein the additional IFN portion comprises (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 up to 15 amino acid cleavages at its N-terminus and / or C-terminus, having at least about 90%, at least about 95%, at least about 98%, or 100% sequence identity.

34. The IFN receptor antagonist according to 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 up to 15 amino acid cleavages at its N-terminus and / or C-terminus.

35. The IFN receptor antagonist according to claim 28, wherein the additional IFNAR1 portion comprises or consists of an amino acid sequence having at least 90%, at least 95%, or 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.

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

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 according to claim 37, wherein the IFNAR2 portion comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 98% sequence identity with (i) the D1 domain of human IFNAR2, or (ii) the D1 and D2 domains of human IFNAR2.

39. An IFN receptor antagonist is optionally an IFN receptor antagonist according to any one of claims 1 to 38, comprising a polypeptide chain having (a) the configuration of two half-antibodies illustrated in Figure 1B, (b) the configuration of two half-antibodies illustrated in Figure 1C, (c) the configuration of two half-antibodies illustrated in Figure 1D, (d) the configuration of two half-antibodies illustrated in Figure 1E, and (e) the configuration of two half-antibodies illustrated in Figure 1F.

40. A nucleic acid or a plurality of nucleic acids encoding an IFN receptor antagonist according to any one of claims 1 to 39.

41. A host cell that has been manipulated to express an IFN receptor antagonist according to any one of claims 1 to 39 or a nucleic acid(s) according to claim 40.

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

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

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

45. The method according to claim 44, wherein the cells are (a) immune cells, optionally B cells, or (b) cancer cells.

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

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

48. The method according to claim 47, further comprising administering an oncolytic virus to the subject.

49. A method for treating a subject suffering from an immune disorder or condition, 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, wherein the immune disorder or condition is optionally systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSc), or Sjögren's syndrome (SS).

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