KR20250048583A - Interferon precursor protein and uses thereof - Google Patents

Abstract

The present disclosure provides a protease-activated IFN proprotein. The IFN proprotein comprises an IFN moiety that is attached to an Fc moiety via a protease-cleavable linker such that binding to a receptor is sterically hindered. When a protease acts, for example , at a tumor site, the protease-cleavable linker is cleaved to release and activate the IFN moiety. Optionally, the IFN proprotein further comprises a targeting moiety that recognizes a tumor-associated antigen and directs the proprotein to the tumor site. The present disclosure also provides pharmaceutical compositions comprising the IFN proprotein, methods of using the IFN proprotein for therapy, and nucleic acids encoding the IFN proprotein, recombinant cells expressing the IFN proprotein, and methods of producing the IFN proprotein.

Description

Interferon precursor protein and uses thereof

1. Cross-reference of related applications

This application claims the benefit of U.S. Provisional Application No. 63/399,040, filed August 18, 2022, U.S. Provisional Application No. 63/383,804, filed November 15, 2022, and U.S. Provisional Application No. 63/481,303, filed January 24, 2023, the contents of each of which are incorporated herein by reference in their entirety.

2. Sequence List

This application contains an electronically submitted sequence listing, which is incorporated herein by reference in its entirety. The copy created on August 15, 2023 is named RGN-020WO_SL.xml and is 361,113 bytes in size.

3. Background Technology

Type I interferon (IFN) is known to directly inhibit tumor cell proliferation. Type I IFN is useful in the treatment of several types of cancer, including hematological malignancies (chronic myeloid leukemia, hairy cell leukemia, multiple myeloma, non-Hodgkin lymphoma) and solid tumors (melanoma, renal carcinoma, Kaposi sarcoma). See , for example, Zitvogel et al ., 2015, Nat Rev Immunol 15:405-414 and Antonelli et al. , 2015, Cytokine Growth Factor Rev 26:121-131.

A particular advantage of type I IFN therapy is that it can intervene at multiple points in the generation of antitumor immune responses, including stimulation of innate and adaptive cytotoxic lymphocyte populations, negative regulation of suppressor cell types, and effects on tumor cells by modulating proliferation, apoptosis, differentiation, migration, and cell surface antigen expression (Parker et al. , 2016, Nature Reviews Cancer 16:131-144).

One of the major obstacles to the use of type I IFNs in the clinic is the serious side effects associated with this treatment. The most common side effects are flu-like symptoms, hematologic toxicity, transaminase elevations, nausea, fatigue, and psychiatric sequelae. These side effects can interfere with reaching and maintaining doses necessary to maximize therapeutic effects and can completely offset the clinical benefits of type I IFN therapy (Lotrich, 2009; Dialogues Clin Neurosci 11:417-425). Type I IFNs signal through the IFNAR1/IFNAR2 complex, which is expressed on most cells and tissues of the body. Thus, they are targeted to tumor-reactive immune cells ( e.g., Diamond et al. , 2011; J Exp Med. 208(10):1989-2003; The ability to preferentially or specifically deliver active type I IFNs to the tumor microenvironment is essential for the continued clinical use of type I IFNs. Strategies to modify type I IFNs are needed to obtain novel forms of the drug that are preferentially active in tumor-reactive immune cells and/or tumors and to reduce side effects on normal IFNAR-expressing cells.

Therefore, the industry needs new type I IFN therapies with improved therapeutic efficacy and safety profiles.

4. Contents of the invention

The present disclosure relates to IFN precursor proteins that are activated by a protease ( e.g. , a protease expressed in a tumor environment).

An IFN proprotein comprises an IFN moiety that is sterically hindered from binding to a receptor, and is configured such that the linker of the IFN proprotein is cleaved by a protease to activate the IFN moiety, thereby alleviating the steric hindrance of the IFN moiety. The IFN proprotein may further comprise a targeting moiety that directs the IFN proprotein to a specific tissue or cell type.

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

The IFN precursor protein can further comprise, for example , an N-terminal, targeting moiety (or a component thereof, e.g. , a chain of a Fab) to one or both Fc domains. The targeting moiety comprises, for example, an antigen binding domain ("ABD") capable of binding to a target molecule present on the tumor surface ( e.g., a tumor-associated antigen) or to another component of the tumor microenvironment ( e.g., the extracellular matrix ("ECM") or tumor lymphocytes).

Exemplary IFN moieties that may be used in the IFN precursor proteins of the present disclosure are described in Section 6.3.

Protease-cleavable linkers that may be used in the IFN precursor proteins of the present disclosure are described in Section 6.4.

Non-cleavable linkers that can be used in the IFN precursor proteins of the present disclosure are described in Section 6.5.

Targeting moieties that may be used in the IFN precursor proteins of the present disclosure are described in Section 6.6, and targeting moiety formats are disclosed in Section 6.7.

Fc domains that can be incorporated into the IFN precursor proteins of the present disclosure are described in Section 6.8.

Exemplary IFN precursor proteins of the present disclosure are described in Section 6.2 and numbered Embodiments 1 to 146.

The present disclosure also provides a nucleic acid encoding an IFN proprotein of the present disclosure. The nucleic acid encoding the IFN proprotein can be a single nucleic acid ( e.g. , a vector encoding all of the polypeptide chains of the IFN proprotein) or a plurality of nucleic acids ( e.g. , two or more vectors encoding different polypeptide chains of the IFN proprotein). The present disclosure also provides host cells and cell lines engineered to express the nucleic acids and IFN proproteins of the present disclosure. The present disclosure also provides methods for producing the IFN proproteins of the present disclosure. Exemplary nucleic acids, host cells, cell lines, and methods for producing IFN proproteins are described in paragraph 6.9 and embodiments 147-149.

The present disclosure also provides pharmaceutical compositions comprising the IFN precursor protein of the present disclosure. Exemplary pharmaceutical compositions are described in Section 6.10 and numbered embodiment 150.

The present invention further provides methods of using the IFN precursor protein and the pharmaceutical compositions of the present disclosure for purposes such as treating cancer. Exemplary methods are described in Section 6.11 and numbered Examples 151 to 201.

5. Brief description of the drawing
Figures 1a-1c are cartoons showing the configurations of three IFN proproteins of the present disclosure having a protease-cleavable linker next to the interferon moiety. Figure 1a shows an IFN proprotein having the overall configuration antibody-PCL-IFN-PCL-Fc. Figure 1b shows a single hinge IFN proprotein having the Fab-PCL-IFN-PCL-hinge-Fc configuration. Figure 1c shows a double hinge IFN proprotein having the Fab-hinge-PCL-IFN-PCL-hinge-Fc configuration. Although shown with targeting moieties in the form of a Fab, the VH, VL and CL domains of the Fab are optional for non-targeting IFN proproteins or may be replaced with other targeting moieties, such as a scFv.
Figures 2a-2f are cartoons showing the configurations of six IFN precursor proteins of the present disclosure having a protease-cleavable linker (PCL) on only one side of the interferon moiety and a non-cleavable linker (NCL) on the other side of the interferon moiety. Figure 2a shows an IFN precursor protein having the overall configuration antibody-NCL-IFN-PCL-Fc. Figure 2b shows a single hinge IFN precursor protein having the configuration Fab-NCL-IFN-PCL-hinge-Fc. Figure 2c shows a double hinge IFN precursor protein having the configuration Fab-hinge-NCL-IFN-PCL-hinge-Fc. Figure 2d shows an IFN precursor protein having the overall configuration antibody-PCL-IFN-NCL-Fc. Figure 2e shows a single hinge IFN precursor protein having a Fab-PCL-IFN-NCL-hinge-Fc configuration. Figure 2f shows a double hinge IFN precursor protein having a Fab-hinge-PCL-IFN-NCL-hinge-Fc configuration. Although shown to have targeting moieties in the form of a Fab, the VH, VL and CL domains of the Fab are optional for non-targeting IFN precursor proteins or can be replaced by other targeting moieties such as scFv.
Figure 3 is a table of exemplary target IFN precursor proteins and their constituent polypeptide chains according to Figure 1. TM represents a targeting moiety, HC represents an antibody heavy chain, LC represents an antibody light chain, IFN represents an interferon (IFN) moiety, where denotes the N- and C-terminal truncations in the IFN sequence of the IFN moiety, respectively ( e.g. , as described in Section 6.3), PCL denotes a protease cleavable linker ( e.g. , as described in Section 6.4), Fc denotes an Fc domain ( e.g. , as described in Section 6.8), Hinge refers to the hinge sequence of an antibody, and LongHinge and ShortHinge denote full-length or truncated versions of the immunoglobulin hinge sequence ( e.g. , as described in Section 6.8.3). Additionally, although the IFN moiety is shown as being flanked by a protease cleavable linker (PCL), one of the PCLs of each of Chain 1 and Chain 2 may be replaced with a non-cleavable linker (NCL) , for example, as described in Section 6.5.
Figures 4a-4c are size exclusion ultra-performance liquid chromatography (SE-UPLC) profiles of exemplary IFN molecules that can be incorporated into the IFN precursor protein structure of the present disclosure. Figure 4a shows the SEC profile of an IFN molecule having an N-terminal Fc domain and a C-terminal IFN moiety, Fc-IFNα1. Figure 4b shows the SEC profile of an IFN molecule having an N-terminal Fc domain and a C-terminal IFN moiety, Fc-IFNα2b. Figure 4c shows the SEC profile of an IFN molecule having an N-terminal IFN domain and a C-terminal Fc domain, IFNα2b-Fc.
Figures 5a-5c are In vitro activity of exemplary IFN molecules linked to an Fc molecule at the N-terminus or C-terminus is shown. Cartoon images in Figure 5a represent N-terminal and C-terminal Fc fusions of IFN. Fc-IFN is a full representation of an IFN molecule having an N-terminal Fc domain and a C-terminal IFN moiety, and IFN-Fc is a full representation of an IFN molecule having an N-terminal IFN moiety and a C-terminal Fc domain. Figure 5b is Graph showing the in vitro activity of exemplary IFN molecules, Fc-IFNα2b and IFNα2b-Fc, compared to unlinked IFNα2b. Figure 5c is a graph showing the activity of Fc-IFN molecules compared to other unlinked IFNs.
Figures 6a-6d are SE-UPLC profiles of exemplary mutant IFN molecules that can be incorporated into the IFN precursor protein structure of the present disclosure. Figure 6a shows the SE-UPLC profile of the mutant IFN molecule Fc-IFNα2bR33A. Figure 6b shows the SE-UPLC profile of the mutant IFN molecule Fc-IFNα2bR149A. Figure 6c shows the SE-UPLC profile of the mutant IFN molecule Fc-IFNα2bR120A. Figure 6d shows the SE-UPLC profile of the mutant IFN molecule Fc-IFNα2bS152A.
Figures 7a-7b are The in vitro activity of exemplary mutant IFN molecules that can be incorporated into the IFN precursor protein structure of the present disclosure is illustrated. The cartoon image in FIG. 7a shows the overall structure of a wild-type (WT) or mutant (Mut) Fc-IFN molecule. FIG. 7b is a graph showing the in vitro activity of Fc-IFNα2b molecules having mutations affecting the IFNAR1 or IFNAR2 interface.
Figures 8a-8c are SE-UPLC profiles of exemplary IFN precursor protein structures. Figure 8a shows the SE-UPLC profile of aPD1-singleHinge-FLIFN, a single hinge full-length IFN precursor protein. Figure 8b shows the SE-UPLC profile of aPD1-doubleHingeLong-IFN, a double hinge IFN precursor protein. Figure 8c shows the SEC profile of an IFN precursor protein with two Fc regions.
Figures 9a-9b are In vitro enzymatic digestion of single- and double-hinge IFN proproteins is shown. Figure 9a is a gel image showing three single-hinge proproteins, two double-hinge IFN proproteins, and a positive control cleaved by uPA. Similarly, Figure 9b is a gel image showing the same single- and double-hinge IFN proproteins cleaved by MMP2 and MMP9.
Figures 10a-10b illustrate the in vitro activity of exemplary IFN precursor proteins compared to IFNα2b and Fc-IFNα2b. Figure 10a is a graph showing the in vitro activity of two single-hinge IFN precursor proteins (full-length and truncated) and one double-hinge IFN precursor protein. Figure 10b is a graph showing how the activity of the same IFN precursor proteins of Figure 10a changes when MMP buffer alone is added (dotted line) or when MMP buffer and enzyme mixture are added together (dotted line).

6. Specific details for carrying out the invention

6.1. Definition

As used herein, the following terms have the following meanings:

ABD chain, targeting moiety chain : The targeting moiety and the antigen binding site (ABD) therein may be present as a single polypeptide chain ( e.g. , in the case of scFv or scFab) or may be formed by the joining of two or more polypeptide chains ( e.g. , in the case of Fab or Fv). The terms "ABD chain" and "targeting moiety chain" as used herein mean all or a portion of the ABD or targeting moiety present in a single polypeptide chain. The use of the terms "ABD chain" or "targeting moiety chain" is for convenience and descriptive purposes only and does not imply any particular composition or method of production. Furthermore, references to the ABD or targeting moiety in describing the IFN precursor protein encompass the ABD chain or the targeting moiety chain unless the context clearly indicates otherwise. Thus, when describing an IFN proprotein in which an Fc domain is operatively linked to a targeting moiety, the Fc domain can be covalently linked, directly or indirectly ( e.g. , via a linker), via a peptide bond to (1) a first ABD or targeting moiety chain of a Fab or Fv (to another component of the Fab or Fv and a second related ABD or targeting moiety chain), or (2) an ABD or targeting moiety chain comprising a scFv or scFab.

About, Approximately : The terms "about,""approximately," and the like are used throughout the specification before a number to indicate that the number is not necessarily exact ( e.g. , to describe ratios, variations in measurement accuracy and/or precision, timing , etc. ). It should be understood that disclosure of "about X" or "approximately X" (wherein X is a number) is also disclosure of "X." Thus, for example, disclosure of an embodiment where one sequence has "about X% sequence identity" to another sequence is also disclosure of an embodiment where the other sequence has "X% sequence identity."

Activate, Activate : The terms “activate,” “activate,” and the like, in relation to the IFN precursor proteins of the present disclosure, refer to the release of an IFN moiety from a sterically blocked constant domain due to protease-mediated enzymatic cleavage of a protease-cleavable linker.

AND, OR : Unless otherwise indicated, the conjunction "or" is intended to be used in its correct sense as a Boolean logical operator, encompassing both feature selection in alternatives (either A or B, where the choice of A is mutually exclusive with B) and feature selection in conjunctions (either A or B, where both A and B are chosen). In some places in the text, the term "and/or" is used for the same purpose, and this should not be construed to imply that "or" is being used to refer to mutually exclusive alternatives.

Antibody : The term "antibody" as used herein refers to a polypeptide (or set of polypeptides) of the immunoglobulin family that can noncovalently, reversibly, and specifically bind to an antigen. For example, naturally occurring "antibodies" of the IgG type are tetramers comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, namely CH1, CH2, and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigen. The constant regions of antibodies can mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system ( e.g. , effector cells) and the first component (Clq) of the classical complement system. The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, bispecific or multispecific antibodies, and anti-idiotypic (anti-id) antibodies. Antibodies can have any isotype/class ( e.g. , IgG, IgE, IgM, IgD, IgA, and IgY) or subclass ( e.g. , IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. In contrast, the constant domains of the light (CL) and heavy (CH1, CH2, or CH3) chains impart important biological properties such as secretion, placental mobility, Fc receptor binding, complement binding, etc. By convention, the numbering of constant region domains increases as they move away from the antigen binding domain or amino terminus of the antibody. The N-terminus is a variable region, the C-terminus is a constant region, and the CH3 and CL domains represent the carboxy-terminus of the heavy and light chains of a native antibody, respectively. Unless the context indicates otherwise, for convenience, references to antibodies also refer to engineered antibodies as well as antibody fragments comprising a non-native antigen-binding domain and/or an antigen-binding domain having a non-native configuration.

Antigen Binding Domain : The term "antigen binding domain" or "ABD" as used herein refers to a portion of an antibody or antibody fragment ( e.g. , a targeting moiety) that has the ability to noncovalently, reversibly, and specifically bind to an antigen. Examples of antibody fragments that may include an ABD include, but are not limited to, a single chain Fv (scFv), a Fab fragment (a monovalent fragment consisting of the VL, VH, CL, and CH1 domains), a F(ab)2 fragment (a bivalent fragment consisting of two Fab fragments joined by a disulfide bridge at the hinge region), a Fd fragment consisting of the VH and CH1 domains, a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a dAb fragment consisting of a VH domain (Ward et al. , 1989, Nature 341:544-546), and an isolated complementarity determining region (CDR). Thus, the term "antibody fragment" encompasses both proteolytic fragments of antibodies ( e.g. , Fab and F(ab) ₂ fragments) and engineered proteins ( e.g. , scFv) comprising one or more portions of an antibody. Antibody fragments may also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs and bis-scFvs (see, e.g. , Hollinger and Hudson, 2005, Nature Biotechnology 23: 1126-1136).

Associated : In the context of an IFN precursor protein, the term "associated" means a functional relationship between two or more polypeptide chains. In particular, the term "associated" means that two or more polypeptides are associated with each other, for example , noncovalently, via molecular interactions, or covalently, via one or more disulfide bridges or chemical cross-links, to form a functional IFN precursor protein. Examples of associations that may be present in the IFN precursor proteins of the present disclosure include, but are not limited to, association between Fc domains to form an Fc region (either a homodimer or a heterodimer, as described in Section 6.8), association between the VH and VL regions in a Fab or Fv, association between CH1 and CL in a Fab.

Cancer : The term "cancer" refers to a disease characterized by the uncontrolled (and often rapid) growth of abnormal cells. Cancer cells may spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, adrenal cancer, ganglion cancer, biliary tract cancer, bone cancer, endometrial cancer, eye cancer, fallopian tube cancer, genital cancer, colon cancer, meninges cancer, esophagus cancer, peritoneum cancer, pituitary cancer, penile cancer, placenta cancer, pleura cancer, salivary gland cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, upper aerodigestive cancer, urinary tract cancer, vaginal cancer, vulva cancer, lymphoma, leukemia, lung cancer, etc. ( e.g. , any TAA-positive cancer of any of the foregoing types).

Complementarity Determining Region : The term "complementarity determining region" or "CDR" as used herein refers to a sequence of amino acids within an antibody variable region that confers antigen specificity and binding affinity. For example, typically each heavy chain variable region has three CDRs ( e.g. , CDR-H1, CDR-H2, and CDR-H3), and each light chain variable region has three CDRs (CDR-L1, CDR-L2, and CDR-L3). The precise amino acid sequence boundaries of a given CDR can be determined using any of several well-known systems, including those described in: Kabat et al ., 1991, "Sequences of Proteins of Immunological Interest," 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering system), Al-Lazikani et al ., 1997, JMB 273:927-948 (“Chothia” numbering system) and ImMunoGenTics (IMGT) numbering (Lefranc, 1999, The Immunologist 7:132-136; Lefranc et al. , 2003, Dev. Comp. Immunol. 27:55-77 (“IMGT” numbering system). For example, in the classical format, according to Kabat, the CDR amino acid residues of the heavy chain variable domain (VH) are 31-35 (CDR-H1), 50-65 (CDR-H2), 95-102 (CDR-H3), and the CDR amino acid residues of the light chain variable domain (VL) are 24-34 (CDR-L1), According to Chothia, the CDR amino acids of VH are numbered 26-32 (CDR-H1), 52-56 (CDR-H2), 95-102 (CDR-H3), and those of VL are numbered 26-32 (CDR-L1), 50-52 (CDR-L2), 91-96 (CDR-L3). Combining the CDR definitions of Kabat and Chothia, the CDRs are numbered 26-35 (CDR-H1), 50-65 (CDR-H2), 95-102 (CDR-H3) of human VH and 24-34 (CDR-L1), 50-56 (CDR-L2), 91-96 (CDR-L3) of human VL. According to IMGT, the CDR amino acid residues of VH are numbered as approximately 26-35 (CDR-H1), 51-57 (CDR-H2), 93-102 (CDR-H3), and the CDR amino acid residues of VL are numbered as approximately 27-32 (CDR-L1), 50-52 (CDR-L2), 89-97 (CDR-L3) ("Kabat" criteria numbering). According to IMGT, the CDR regions of an antibody can be determined using the IMGT/DomainGap Align program.

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

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

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

The term "CH3 domain" refers to the heavy chain constant region that is C-terminal to the CH2 domain in a heavy chain constant domain. In some embodiments, the term "CH3 domain" refers to the region of an immunoglobulin molecule spanning amino acids 341 to 447 (EU numbers). The term "CH3 domain" includes wild-type CH3 domains and variants thereof ( e.g. , non-naturally occurring CH3 domains or altered CH3 domains). For example, the term "CH3 domain" includes wild-type IgG1, IgG2, IgG3, and IgG4 CH3 domains and variants thereof having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5, and/or at most 5, 4, 3, 2 or 1 mutations ( e.g. , substitutions, deletions and/or additions). Exemplary CH3 domains include CH3 domains with mutations that alter biological activities of the antibody, such as ADCC, CDC, purification, dimerization, and half-life.

The term “CL domain” refers to the constant region of an immunoglobulin light chain. The term “CL domain” encompasses both wild-type CL domains ( e.g. , kappa or lambda light chain constant regions) and variants thereof ( e.g. , non-naturally occurring CL domains or altered CL domains). For example, the term “CL domain” encompasses wild-type kappa and lambda constant domains and variants thereof having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and/or at most 5, 4, 3, 2 or 1 mutations ( e.g. , substitutions, deletions and/or additions).

Effector functions: The term "effector functions" refers to activities of an antibody molecule that are mediated by binding through antibody domains other than the antigen binding domain, and are typically mediated by binding of an effector molecule. Effector functions include complement-mediated effector functions, which are mediated, for example, by binding of the C1 component of complement to an antibody. Activation of complement is important for opsonization and lysis of cellular pathogens. Activation of complement may also stimulate inflammatory responses and be involved in autoimmune hypersensitivity. Effector functions also include Fc receptor (FcR)-mediated effector functions, which may be triggered upon 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, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production. The effector function of antibodies can be altered by altering, for example , enhancing or decreasing, the affinity of the antibody for the effector molecule, such as an Fc receptor or a complement component. The binding affinity is generally varied by altering the binding site of the effector molecule. In this case, it is desirable to identify the site of interest and alter at least part of the site in an appropriate manner. It is also expected that altering the binding site of the antibody for the effector molecule will not significantly alter the overall binding affinity, but may alter the geometry of the interaction, rendering the effector mechanism ineffective, such as in unproductive binding. Additionally, it is thought that effector function can also be altered by modifying sites that are not directly involved in binding of effector molecules, but are otherwise involved in the performance of effector function.

Epitope : An epitope or antigenic determinant is a portion of an antigen that is recognized by an antibody or other antigen-binding moiety, as described herein. An epitope may be linear or conformational.

Fab : The term "Fab" refers to a pair of polypeptide chains, the first polypeptide chain comprising a variable heavy (VH) domain of an antibody operably linked, typically at its N-terminus, to a first constant domain (referred to herein as C1), and the second polypeptide chain comprising a variable light (VL) domain of an antibody operably linked, typically at its N-terminus, to a second constant domain (referred to herein as C2) that can pair with the first constant domain. In a native antibody, the VH is at the N-terminus of the first constant domain (C1) of the heavy chain and the VL is at the N-terminus of the constant domain of the light chain (CL). The Fabs of the present disclosure may be arranged in their native orientation or may include domain substitutions or exchanges that facilitate correct VH or VL pairing. For example, it is possible to replace the CH1 and CL domain pairs of a Fab with CH3 domain pairs to facilitate correct altered Fab-chain pairing in the heterodimeric molecule. It is also possible to invert CH1 and CL so that CH1 is attached to VL and CL is attached to VH (a configuration commonly known as Crossmab). The term "Fab" includes single-chain Fab.

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

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

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

The IFN precursor protein of the present disclosure generally comprises two half antibodies comprising an IFN moiety flanked by a protease cleavable linker, each comprising an Fc domain C-terminal to a C-terminal protease cleavable linker and a constant domain N-terminal to an N-terminal protease cleavable linker. One or both half antibodies of the IFN precursor protein may additionally comprise a targeting moiety.

The term "half-antibody" is used for descriptive purposes only and does not imply any particular composition or method of production. The designation of half-antibodies as "first" half-antibodies, "second" half-antibodies, "left" half-antibodies, "right" half-antibodies, etc., is for convenience and descriptive purposes only.

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

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

Linker : The term “linker” as used herein refers to a protease-cleavable linker or a non-cleavable linker.

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

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

Polypeptides, Peptides and Proteins : The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acid residues.

Preprotein : A "preprotein" is a protein precursor that is inactive and can be activated by proteolysis by a protease. A precursor protein is therefore "protease-activatable."

Protease : The term “protease” as used herein refers to an enzyme that catalyzes the hydrolysis of a peptide bond. Generally, a protease useful in the present disclosure, e.g. , a protease described in Section 6.4.1, recognizes and cleaves a substrate having a particular sequence motif, e.g. , as described in Section 6.4.2. Preferably, the protease is expressed at a higher level in cancer tissue as compared to normal tissue.

Protease-cleavable linker : As used herein, the term "protease-cleavable linker" or "PCL" means a peptide whose amino acid sequence comprises one or more ( e.g. , two, three or more) substrate sequences for one or more proteases. Exemplary protease-cleavable linkers are described in Section 6.4 and exemplary protease-cleavable linker sequences are disclosed in Section 6.4.4.

Recognize : As used herein, the term "recognize" means that an antibody or antibody fragment ( e.g. , a targeting moiety) locates and interacts with ( e.g. , binds to) an epitope of interest.

Single Chain Fab or scFab : The term "single chain Fab" or "scFab" as used herein refers to an ABD comprising a VH domain, a CH1 domain, a VL domain, a CL domain and a linker. In some embodiments, the domains and linker are arranged from N-terminus to C-terminus 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 suitably a non-cleavable linker of at least 30 amino acids, preferably 32 to 50 amino acids. Single chain Fab fragments are typically stabilized via native disulfide bonds between the CL domain and the CH1 domain. Additionally, these single-chain Fab molecules can be further stabilized by creating an interchain disulfide bond through insertion of a cysteine residue ( e.g. , at position 44 in the VH domain and position 100 in the VL domain according to Kabat numbering).

Single chain Fv or scFv : The term "single chain Fv" or "scFv" as used herein refers to an ABD comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore (1994), Springer-Verlag, New York, pp. 269-315. The VH and VL are arranged in N-terminal to C-terminal order, VH-VL or VL-VH, and are typically separated by a linker, such as a linker as set forth in Table 105.

Spacer: The term "spacer" as used herein refers to a peptide having an amino acid sequence that is incorporated into a linker that comprises a substrate, but is not a substrate for a protease. The spacer can be used to separate the substrate from other domains in the molecule, such as the ABD. In some embodiments, the residues in the spacer minimize aminopeptidase and/or exopeptidase action, thereby preventing cleavage of the N-terminal amino acid.

Binds specifically (or selectively) : The term "binds specifically (or selectively)" to an antigen or epitope refers to a binding reaction that determines the presence of a cognate antigen or epitope in a heterogeneous population of proteins and other molecules. The binding reaction may, but need not, be mediated by an antibody or antibody fragment. The term "specific binding" does not exclude cross-species reactivity. For example, an antigen-binding domain ( e.g. , an antigen-binding fragment of an antibody) that "binds specifically" to an antigen of one species may also "bind specifically" to antigens of one or more other species. Thus, such cross-species reactivity does not, in and of itself, alter the classification of the antigen-binding domain as a "specific" binder. In certain embodiments, an antigen binding domain of the present disclosure that specifically binds a human antigen has cross-species reactivity with one or more non-human mammalian species, e.g. , a primate species (including but not limited to one or more of Macaca fasciculis , Macaca mulatta , and Macaca nemestrina ) or a rodent species, e.g. , Mus musculus .

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

Substrate : The term "substrate" refers to the peptide sequence on which a protease acts and on which the protease will cleave peptide bonds.

Target Molecule : The term “target molecule” as used herein refers to any biological molecule ( e.g. , a protein, carbohydrate, lipid, or a combination thereof) expressed on the cell surface or extracellular matrix, which is capable of specifically binding to the target moiety of the disclosed IFN proprotein.

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

T-Cell Antigen, TCA : The term "T-cell antigen" or "TCA" refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) expressed on the surface of T lymphocytes and is useful for preferentially targeting a pharmacological agent to a specific site. In some embodiments, the site is cancer tissue and/or the T cell antigen is a tumor reactive lymphocyte antigen, a cell surface molecule of a tumor or viral lymphocyte, or an immune checkpoint inhibitor expressed on T lymphocytes.

Tumor : The term "tumor" is used interchangeably herein with the term "cancer", and, for example , both terms include solid and liquid, e.g. , malignant or circulating, tumors. The terms "cancer" or "tumor" as used herein include premalignant and malignant cancers and tumors.

Tumor-Associated Antigen, TAA : The term "tumor-associated antigen" or "TAA" refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) expressed in whole or in fragment ( e.g. , MHC/peptide) on the surface of a cancer cell, that is useful for preferentially targeting a pharmacological agent to the cancer cell. In some embodiments, the TAA is a marker expressed by both normal cells and cancer cells, e.g. , a lineage marker. In some embodiments, the TAA is a cell surface molecule that is overexpressed in cancer cells as compared to normal cells, e.g., 1-fold overexpressed, 2-fold overexpressed, 3-fold overexpressed, or more as compared to normal cells. In some embodiments, the TAA is a cell surface molecule that is inappropriately synthesized in a cancer cell, e.g., a molecule that comprises a deletion, addition, or mutation as compared to the molecule expressed in a normal cell. In some embodiments, TAAs are expressed entirely or as fragments ( e.g. , MHC/peptides) exclusively on the cell surface of cancer cells, and will not be synthesized or expressed on the surface of normal cells. Thus, the term "TAA" encompasses antigens specific for cancer cells, sometimes known in the art as tumor specific antigens ("TSAs").

Treat, Treatment, Treating : As used herein, the terms "treat,""treatment," and "treating" refer to a reduction or improvement in the progression, severity, and/or duration of a disorder, or an improvement in one or more symptoms (preferably one or more discernible symptoms) of a disorder, resulting from the administration of one or more IFN receptor agonists of the invention (e.g., IFN proproteins that are capable of agonizing an IFN receptor after activation). In some embodiments, the disorder is a proliferative disorder and the terms "treat,""treatment," and "treating" refer to an improvement in at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which is not necessarily discernible to the patient. In other embodiments, the terms "treat,""treatment," and "treating" refer to inhibiting the progression of a proliferative disorder physically, e.g. , by stabilizing an discernible symptom, physiologically, e.g. , by stabilizing a physical parameter, or both. In other embodiments, the terms “treat,” “treatment,” and “treating” refer to a reduction or stabilization of tumor size or number of cancer cells.

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

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

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

6.2. IFN precursor protein

The present disclosure relates to IFN proproteins comprising an IFN moiety that is attenuated by steric hindrance of flanking constant domains. The IFN proprotein is configured such that upon encountering a protease, such as a protease overexpressed in a tumor environment, the protease cleavable linker is cleaved, releasing the IFN. This is accomplished by incorporating an IFN moiety flanked by a protease cleavable linker (PCL) between the constant/FC domains of an antibody. Such proteins are therefore sometimes referred to as "internal" IFN structures. For example, an IFN proprotein that can act on an IFN receptor after activation is also referred to herein as an "IFN receptor agonist."

Typically, an IFN precursor protein comprises two half antibodies, each comprising a pair of Fc domains that join to form an Fc region (typically comprising a hinge sequence), an N-terminus that is a cleavable or non-cleavable linker, an IFN moiety, and an additional linker that is either cleavable or non-cleavable. In some embodiments, each half antibody comprises two protease cleavable linkers flanking the IFN moiety. Exemplary IFN precursor proteins having two protease cleavable linkers on each half antibody are illustrated in FIGS. 1A-1C . In other embodiments, each half antibody comprises a single protease cleavable linker on one side of the IFN moiety, and the other linker on the IFN moiety is a non-cleavable linker. Exemplary IFN precursor proteins having two protease cleavable linkers on each half antibody are illustrated in FIGS. 2A-2F . At the N-terminus of the protease-cleavable linker is an antibody constant domain, which may be joined by the entire constant domain, including the Fc domain, to form another Fc region, or by only a portion of the constant domain (e.g., the CH1 domain).

Exemplary configurations of IFN precursor proteins of the present disclosure are illustrated in FIGS. 1A-1C and 2A-2F. As illustrated, the IFN moiety and surrounding linker can comprise a hinge domain only at the C-terminus (“single hinge”, e.g. , as in FIGS. 1A , 1B , 2A , 2B , 2D and 2E ) or at both the N- and C-terminals (“dual hinge”, e.g. , as in FIGS. 1C , 2C and 2F ).

In general, IFN precursor proteins are

a) In the first polypeptide chain:

i) a first immunoglobulin constant domain;

ii) first linker;

iii) type I interferon (IFN) moiety;

iv) second linker and

v) comprising a first Fc domain; and

b) In the second polypeptide chain:

i) a second immunoglobulin constant domain;

ii) third linker;

iii) type I interferon (IFN) moiety;

iv) a fourth linker; and

v) a second Fc domain capable of combining with the first Fc domain to form an Fc region.

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

Preferably, the IFN moiety of the IFN precursor protein is sterically hindered from binding to the IFN receptor by the Fc domain and/or the constant domain.

An exemplary IFN precursor protein is illustrated in FIG. 1A, wherein the targeting moiety (represented as a Fab domain paired with VH-CH1 and VL-CL) is optional. The IFN precursor protein of FIG. 1A comprises a first polypeptide chain, a second polypeptide chain, an optional third polypeptide chain, and an optional fourth polypeptide chain, wherein the first polypeptide chain comprises the first polypeptide chain.

a) The first polypeptide chain consists of:

i) 1st VH1 domain (optional);

ii) the first CH1 domain;

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

iv) a first protease cleavable linker (PCL);

v) type I interferon (IFN) moiety;

vi) a second protease cleavable linker (PCL); and

vii) second Fc domain;

b) The second polypeptide chain consists of:

i) Second VH1 domain (optional);

ii) the second CH1 domain;

iii) a third Fc domain comprising a hinge domain, a CH2 domain and a CH3 domain (the CH3 domain is a second immunoglobulin constant domain);

iv) a third protease cleavable linker (PCL);

v) type I interferon (IFN) moiety;

vi) a fourth protease cleavable linker (PCL); and

vii) 4th Fc domain;

c) The optional third polypeptide chain comprises:

i) 1st VL domain;

ii) the first CL domain; and

d) The fourth polypeptide chain consists of:

i) a second VL domain; and

ii) 2nd CL domain;

The first polypeptide chain binds to the second polypeptide chain, thereby forming an Fc region by binding the first Fc domain and the third Fc domain, and the second Fc domain and the fourth Fc domain form another Fc region.

If present, the third polypeptide chain binds to the first polypeptide chain, thereby forming a first VH, CH1, VL and CL to form a first targeting moiety, and the fourth polypeptide chain binds to the second polypeptide chain, thereby forming a second VH, CH1, VL and CL to form a second targeting moiety. Alternatively, the first and second polypeptide chains may comprise a scFv at their N-terminus.

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

Another exemplary IFN precursor protein is depicted in Figure 1B, wherein the targeting moiety (represented as a Fab domain paired with VH-CH1 and VL-CL) is optional. The IFN precursor protein of Figure 1B comprises a first polypeptide chain, a second polypeptide chain, an optional third polypeptide chain, and an optional fourth polypeptide chain, wherein the first polypeptide chain comprises the first polypeptide chain.

a) The first polypeptide chain consists of:

i) 1st VH1 domain (optional);

ii) the first CH1 domain;

iii) a first protease cleavable linker (PCL);

iv) type I interferon (IFN) moiety;

v) a second protease cleavable linker (PCL); and

vi) first Fc domain;

b) The second polypeptide chain consists of:

i) Second VH1 domain (optional);

ii) the second CH1 domain;

iii) a third protease cleavable linker (PCL);

iv) type I interferon (IFN) moiety;

v) a fourth protease cleavable linker (PCL); and

vi) second Fc domain;

c) The optional third polypeptide chain comprises:

i) 1st VL domain;

ii) the first CL domain; and

d) The fourth polypeptide chain consists of:

i) a second VL domain; and

ii) 2nd CL domain;

The first polypeptide chain is linked to a second polypeptide chain such that the first Fc domain and the second Fc domain form an Fc region.

If present, the third polypeptide chain binds to the first polypeptide chain, thereby forming a first VH, CH1, VL and CL to form a first targeting moiety, and the fourth polypeptide chain binds to the second polypeptide chain, thereby forming a second VH, CH1, VL and CL to form a second targeting moiety. Alternatively, the first and second polypeptide chains may comprise a scFv at their N-terminus.

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

A variant of the IFN precursor protein of FIG. 1b is depicted in FIG. 1c. The IFN precursor protein of FIG. 1c further comprises a first hinge domain between the first CH1 domain and the first protease cleavable linker and a second hinge domain between the second CH1 domain and the third protease cleavable linker.

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

a) The first polypeptide chain:

i) 1st VH1 domain (optional);

ii) the first CH1 domain;

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

iv) first non-cleavable linker (NCL);

v) type I interferon (IFN) moiety;

vi) a first protease cleavable linker (PCL); and

vii) comprising a second Fc domain,

b) The second polypeptide chain:

i) Second VH1 domain (optional);

ii) the second CH1 domain;

iii) a third Fc domain comprising a hinge domain, a CH2 domain and a CH3 domain (the CH3 domain is a second immunoglobulin constant domain);

iv) a second non-cleavable linker (NCL);

v) type I interferon (IFN) moiety;

vi) a second protease cleavable linker (PCL); and

vii) comprising a fourth Fc domain,

c) an optional third polypeptide chain:

i) 1st VL domain;

ii) comprising a first CL domain, and

d) The fourth polypeptide chain is:

i) a second VL domain; and

ii) 2nd CL domain;

The first polypeptide chain binds to the second polypeptide chain, thereby forming an Fc region by binding the first Fc domain and the third Fc domain, and the second Fc domain and the fourth Fc domain form another Fc region.

If present, the third polypeptide chain binds to the first polypeptide chain, thereby forming a first VH, CH1, VL and CL to form a first targeting moiety, and the fourth polypeptide chain binds to the second polypeptide chain, thereby forming a second VH, CH1, VL and CL to form a second targeting moiety. Alternatively, the first and second polypeptide chains may comprise a scFv at their N-terminus.

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

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

a) The first polypeptide chain consists of:

i) 1st VH1 domain (optional);

ii) the first CH1 domain;

iii) a first non-cleavable linker (NCL);

iv) type I interferon (IFN) moiety;

v) a first protease cleavable linker (PCL); and

vi) first Fc domain;

b) The second polypeptide chain consists of:

i) Second VH1 domain (optional);

ii) the second CH1 domain;

iii) a second non-cleavable linker (NCL);

iv) type I interferon (IFN) moiety;

v) a second protease cleavable linker (PCL); and

vi) comprising a second Fc domain,

c) an optional third polypeptide chain:

i) 1st VL domain;

ii) comprising a first CL domain, and

d) The fourth polypeptide chain is:

i) a second VL domain; and

ii) comprising a second CL domain,

Here, the first polypeptide chain is linked to the second polypeptide chain such that the first Fc domain and the second Fc domain form an Fc region.

If present, the third polypeptide chain binds to the first polypeptide chain, thereby forming a first VH, CH1, VL and CL to form a first targeting moiety, and the fourth polypeptide chain binds to the second polypeptide chain, thereby forming a second VH, CH1, VL and CL to form a second targeting moiety. Alternatively, the first and second polypeptide chains may comprise a scFv at their N-terminus.

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

A variant of the IFN precursor protein of FIG. 2b is depicted in FIG. 2c. The IFN precursor protein of FIG. 2c further comprises a first hinge domain between the first CH1 domain and the first protease non-cleavable linker and a second hinge domain between the second CH1 domain and the second protease non-cleavable linker.

Another exemplary IFN precursor protein is shown in Figure 2d, wherein the targeting moiety (represented as a Fab domain paired with VH-CH1 and VL-CL) is optional. The IFN precursor protein of Figure 2d comprises a first polypeptide chain, a second polypeptide chain, an optional third polypeptide chain, and an optional fourth polypeptide chain, wherein the first polypeptide chain comprises the first polypeptide chain.

a) The first polypeptide chain:

i) 1st VH1 domain (optional);

ii) the first CH1 domain;

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

iv) a first protease cleavable linker (PCL);

v) type I interferon (IFN) moiety;

vi) a first protease non-cleavable linker (NCL); and

vii) comprising a second Fc domain,

b) The second polypeptide chain:

i) Second VH1 domain (optional);

ii) the second CH1 domain;

iii) a third Fc domain comprising a hinge domain, a CH2 domain and a CH3 domain (the CH3 domain is a second immunoglobulin constant domain);

iv) a second protease cleavable linker (PCL);

v) type I interferon (IFN) moiety;

vi) a second protease non-cleavable linker (NCL); and

vii) comprising a fourth Fc domain,

c) an optional third polypeptide chain:

i) 1st VL domain;

ii) comprising a first CL domain,

d) The fourth polypeptide chain is:

i) a second VL domain; and

ii) comprising a second CL domain,

The first polypeptide chain binds to the second polypeptide chain, thereby forming an Fc region by binding the first Fc domain and the third Fc domain, and the second Fc domain and the fourth Fc domain form another Fc region.

If present, the third polypeptide chain binds to the first polypeptide chain, thereby forming a first VH, CH1, VL and CL to form a first targeting moiety, and the fourth polypeptide chain binds to the second polypeptide chain, thereby forming a second VH, CH1, VL and CL to form a second targeting moiety. Alternatively, the first and second polypeptide chains may comprise a scFv at their N-terminus.

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

Another exemplary IFN precursor protein is shown in Figure 2e, wherein the targeting moiety (represented as a Fab domain paired with VH-CH1 and VL-CL) is optional. The IFN precursor protein of Figure 2e comprises a first polypeptide chain, a second polypeptide chain, an optional third polypeptide chain, and an optional fourth polypeptide chain, wherein the first polypeptide chain comprises the first polypeptide chain.

a) The first polypeptide chain:

i) 1st VH1 domain (optional);

ii) the first CH1 domain;

iii) a first protease cleavable linker (PCL);

iv) type I interferon (IFN) moiety;

v) a first protease non-cleavable linker (NCL); and

vi) first Fc domain;

b) The second polypeptide chain:

i) Second VH1 domain (optional);

ii) the second CH1 domain;

iii) a second protease cleavable linker (PCL);

iv) type I interferon (IFN) moiety;

v) a second protease non-cleavable linker (NCL); and

vi) comprising a second Fc domain,

c) an optional third polypeptide chain:

i) 1st VL domain;

ii) comprising a first CL domain, and

d) The fourth polypeptide chain is:

i) a second VL domain; and

ii) comprising a second CL domain,

Here, the first polypeptide chain is linked to the second polypeptide chain such that the first Fc domain and the second Fc domain form an Fc region.

If present, the third polypeptide chain binds to the first polypeptide chain such that the first VH, CH1, VL and CL form a first targeting moiety, and the fourth polypeptide chain binds to the second polypeptide chain such that the second VH, CH1, VL and CL form a second targeting moiety. Alternatively, the first and second polypeptide chains may comprise a scFv at their N-terminus.

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

A variant of the IFN precursor protein of FIG. 2e is depicted in FIG. 2f. The IFN precursor protein of FIG. 2e further comprises a first hinge domain between the first CH1 domain and the first protease cleavable linker and a second hinge domain between the second CH1 domain and the second protease cleavable linker.

Typically, the Fc domain of the IFN precursor protein is depicted in Figures 1a to 1c, and the hinge domain is depicted in Figures 2a to 2f.

Therefore, IFN precursor proteins typically contain two to four protease-cleavable linkers. Cleavage of all protease-cleavable linkers of an IFN precursor protein into four protease-cleavable linkers releases an activated IFN precursor protein containing an IFN moiety and lacking a C-terminal Fc moiety, an N-terminal constant domain, and a targeting moiety (if present). Cleavage of all protease-cleavable linkers of an IFN precursor protein into two protease-cleavable linkers removes one of the sterically blocking moieties, resulting in an IFN molecule consisting of either a targeting moiety (antibody-IFN) or an Fc domain (IFN-Fc).

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

The sequence and length of the hinge and linker sequences can also vary, as can the sequence of the IFN moiety (including full-length or N- and/or C-terminally truncated IFN sequences). Exemplary IFN moieties are described in Section 6.3, including IFNα and IFNβ based moieties described in Sections 6.3.1 and 6.3.2, below. Exemplary protease cleavable linker sequences are set forth in Section 6.4. Exemplary non-cleavable linker and hinge sequences are set forth in Sections 6.5 and 6.8.3, respectively. Exemplary targeting moieties are set forth in Section 6.6.

6.3. IFN moiety

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

The IFN moiety of the present disclosure may comprise 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 cell membrane receptor IFNAR, composed of IFNAR1 and IFNAR2, which is ubiquitously expressed in all nucleated cells. Ligand binding is initiated by the high-affinity receptor subunit IFNAR2 (Piehler et al ., 2012, Immunological Reviews, doi.org/10.1111/imr.12001). Type I IFNs can therefore act on virtually every cell in the body. Sixteen type I interferon subtypes have been identified, and they vary widely in their affinity and activity for IFNAR2.

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

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

The type I IFN moiety can comprise a sequence that differs from the wild-type IFN sequence by one or more mutations, such as substitutions, deletions or insertions. Substitutions that reduce receptor binding and thereby attenuate IFN activity can be suitably used. Amino acids with N- or C-terminal deletions (or truncations) can also be used ( e.g. , amino acids truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids from the N- or C-terminus of a mature type I IFN). Without being bound by theory, the inventors believe that the terminal truncations impose additional steric constraints on the IFN moiety and reduce IFN activity until the protease-cleavable linker in the IFN proprotein is cleaved.

Further details on exemplary type I IFN moieties are provided below.

6.3.1. Interferon-α moiety

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

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

MASPFALLMV LVVLSCKSSC SLG CDLPETH SLDNRRTLML LAQMSRISPS

SCLMDRHDFG FPQEEFDGNQ FQKAPAISVL HELIQQIFNL FTTKDSSAAW

DEDLLDKFCT ELYQQLNDLE ACVMQEERVG ETPLMNADSI LAVKKYFRRI

TLYLTEKKYS PCAWEVVRAE IMRSLSLSTN LQERLRRKE (SEQ ID NO: 1)

The human IFNα1b gene differs from the IFNα1a allelic variants by a single nucleotide change in the coding region that results in a single change in the amino acid sequence (Val114 instead of Ala114 in the mature protein, and Val137 instead of Ala137 in the full-length polypeptide).

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

MALTFALLVA LLVLSCKSSC SVG CDLPQTH SLGSRRTLML LAQMRRISLF

SCLKDRHDFG FPQEEFGNQF QKAETIPVLH EMIQQIFNLF STKDSSAAWD

ETLLDKFYTE LYQQLNDLEA CVIQGVGVTE TPLMKEDSIL AVRKYFQRIT

LYLKEKKYSP CAWEVVRAEI MRSFSLSTNL QESLRSKE (SEQ ID NO: 2)

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

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

In some embodiments, the IFNα moiety has one or more amino acid substitutions ( e.g. , substitutions that alter IFNAR binding and/or agonistic activity). Exemplary substitutions are set forth in WO 2013/107791, 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 comprises:

a) L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K or R33A or R33Q, H34A, D35A, Q40A, H57Y, E58N, Q61S, F64A, N65A, T69A, L80A, D82E, Y85A, T86I, Y89A, D114R or D114A, L117A, R120A or R120E or R120K, K121E, R125A, K133A, K134A, R144A, A145G or A145M, M148A, R149A, R149K, S152A, One or more substituents selected from L153A, N156A; and/or

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

c) Wherein one or more amino acids in 9, 17, 47, 65, 66, 117, 123, 128, 147 and 157 are replaced with alanine, glycine or threonine as described in WO2021126929A1.

The amino acid positions of the substituents described above are provided based on mature IFNα2b.

In other embodiments, the IFNα moiety comprises one or more amino acid substitutions as set forth in Table 1. Table 1 shows IFNα substitutions identified based on amino acid positions within the base sequence of IFNα2.

Table 1
Exemplary IFNα mutants (see sequence of mature IFNα2) IFNα sequence mutations Source/Effect of Substituent L15A WO2018014068A9 R22A WO2018014068A9 R23A WO2018014068A9 S25A WO2018014068A9 L26A Thomas et al. , 2011, Cell, 146(4):621-632 F27A Thomas et al. , 2011, Cell, 146(4):621-632 L30A Thomas et al. , 2011, Cell, 146(4):621-632 L30V WO2018014068A9 K31A WO2018014068A9 D32A WO2018014068A9 R33K WO2018014068A9 R33Q WO2018014068A9 R33A WO2013059885A2 H34A WO2018014068A9 D35E WO2016201337A1 Q40A WO2018014068A9 H57A Thomas et al. , 2011, Cell, 146(4):621-632 H57S WO2013134138A1 E58A Thomas et al. , 2011, Cell, 146(4):621-632 Q61A WO2016201337A1 Q90A Piehler et al ., 2012, Immunological Reviews, doi.org/10.1111/imr.12001 E96A Piehler et al ., 2012, Immunological Reviews, doi.org/10.1111/imr.12001 D114R WO2018014068A9 L117A WO2018014068A9 R120A WO2018014068A9 R120E WO2018014068A9 Q124R WO2022015711A1 R125A WO2018014068A9 R125E WO2018014068A9 K131A WO2018014068A9 E132A WO2018014068A9 K133A WO2018014068A9 K134A WO2018014068A9 L135A WO2013134138A1 R144A WO2022015711A1 R144D WO2018014068A9 R144E WO2018014068A9 R144G WO2018014068A9 R144H WO2018014068A9 R144I WO2018014068A9 R144K WO2018014068A9 R144L WO2013059885A2 R144N WO2018014068A9 R144Q WO2018014068A9 R144S WO2013059885A2 R144T WO2013059885A2 R144V WO2018014068A9 R144Y WO2013059885A2 A145D WO2013059885A2 A145E WO2018014068A9 A145G WO2018014068A9, WO2022015711A1 A145H WO2013059885A2 A145I WO2018014068A9 A145K WO2013059885A2 A145L WO2018014068A9 A145M WO2018014068A9 A145N WO2018014068A9 A145Q WO2018014068A9 A145S WO2018014068A9 A145T WO2018014068A9 A145V WO2018014068A9 A145Y WO2013059885A2 M148A WO2018014068A9 R149A WO2022015711A1, Piehler et al ., 2012, Immunological Reviews, doi.org/10.1111/imr.12001 R149K Piehler et al ., 2012, Immunological Reviews, doi.org/10.1111/imr.12001 S152A Piehler et al ., 2012, Immunological Reviews, doi.org/10.1111/imr.12001, WO2022015711A1 L153A WO2018014068A9 N156A WO2018014068A9 R162A WO2016201337A1 E165D WO2016201337A1 L30A, H57Y, E58N, Q61S WO2018014068A9 R33A, H57Y, E58N, Q61S WO2013059885A2 H57S, E58S, Q61S Alteration of IFNα-IFNAR1 binding affinity at site 1 H57Y, E58N, Q61S WO2007000769A2 N65A, L80A, Y85A, Y89A WO2018014068A9 N65A, L80A, Y85A, Y89A, D114A WO2018014068A9 N65A, L80A, Y85A, Y89A, L174A WO2018014068A9 N65A, L80A, Y85A, Y89A, R120A WO2018014068A9 Y85A, Y89A, D114A WO2018014068A9 Q90A, R120A Piehler et al ., 2012, Immunological Reviews, doi.org/10.1111/imr.12001 D114A, R120A WO2018014068A9 L117A, R120A WO2018014068A9 L117A, R120A, K121A WO2018014068A9 R120A, K121A WO2018014068A9 R120E, K121E WO2018014068A9 R144A, H57Y, E58N, Q61S WO2013059885A2 M148A, H57Y, E58N, Q61S WO2018014068A9 R149A, R162A WO2013134138A1 L153A, H57Y, E58N, Q61S WO2018014068A9 Delete residue L161-E165 WO2018014068A9

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

Sequences of exemplary IFNα moieties that may be utilized in the IFN precursor proteins of the present invention are presented in Table 2 below:

Table 2
Exemplary IFNα moieties Structure order SEQ ID NO: IFNα1a CDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFGFPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNADSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE IFNα1b CDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFGFPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNVDSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE 3 IFNα2a CDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE 4 IFNα2b CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE 5 IFNα2b (R33A) CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDAHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE 6 IFNα2b (R33K) CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDKHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE 7 IFNα2b (Q90A) CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYAQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE 8 IFNα2b (E96A) CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLAACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE 9 IFNα2b (R120A) CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVAKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE 10 IFNα2b (A145M) CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRMEIMRSFSLSTNLQESLRSKE 11 IFNα2b (R149A) CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMASFSLSTNLQESLRSKE 12 IFNα2b (R149K) CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMKSFSLSTNLQESLRSKE 13 IFNα2b (S152A) CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFALSTNLQESLRSKE 14 THSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQ 15 CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQ 16

6.3.2. Interferon 1-β moiety

Interferon 1-β (IFN1β or IFN 1-β) is a cytokine naturally produced by the immune system in response to biological and chemical stimuli. IFN1β is a glycosylated monomer with a molecular weight of approximately 22 kDa that is produced in large quantities by fibroblasts and is also known as fibroblast interferon. IFN1β binds to the IFNAR receptor, which consists of a dimer of IFNAR1 and IFNAR2, and induces signals through the JAK/STAT pathway and other pathways. IFN1β can also function by binding to IFNAR1 alone and signal independently of the JAK-STAT pathway (see, e.g. , Stanifer et al. , 2019, Int. J Mol. Sci. 20(6):1445).

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

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

The amino acid sequence of human IFNβ precursor is deposited in GenBank, accession number AAA36040.1, and is reproduced below (signal peptide is underlined).

MTNKCLLQIA LLLCFSTTAL S MSYNLLGFL QRSSNFQCQK LLWQLNGRLE

YCLKDRMNFD IPEEIKQLQQ FQKEDAALTI YEMLQNIFAI FRQDSSSTGW

NETIVENLLA NVYHQINHLK TVLEEKLEKE DFTRGKLMSS LHLKRYYGRI

LHYLKAKEYS HCAWTIVRVE ILRNFYFINR LTGYLRN (SEQ ID NO: 23)

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

In various embodiments, the IFNβ moiety comprises one or more amino acid substitutions and/or deletions compared to IFN1β1. In some embodiments, the substitution is C17S (with respect to mature IFN1β1) and the deletion is one of the C-terminal truncations described in IFN-ΔΙ as US 2009/0025106 A1. IFNA2, IFNA3, IFNA4, IFNA5, IFNA6, IFN-Δ7, IFN-Δδ, IFNA9, and IFN-ΔΙ Ο.

6.3.3. Other type I interferons

In certain embodiments, the type I IFN moiety is another moiety other than an IFNα or IFNβ moiety, such as an interferon-ω (IFNω), interferon-ε (IFNε), or interferon-κ (IFNκ) moiety.

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.

MALLFPLLAALVMTSYSPVGSLGCD LPQNHGLLSRNTLVLLHQMRRISPFLCLKDRRDF

RFPQEMVKGSQLQKAHVMSVLHEMLQQIFSLFHTERSSAAWNMTLLDQLHTGLHQQL

QHLETCLLQVVGEGESAGAISSPALTLRRYFQGIRVYLKEKKYSDCAWEVVRMEIMKSL

FLSTNMQERLRSKDRDLGSS (SEQ ID NO: 24)

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

Human IFNε is identified by UniProt accession number Q86WN2 and has the amino acid sequence described below, with the signal sequence underlined.

MIIKHFFGTVLVLLASTTIFS LDLKLIIFQQRQVNQESLKLLNKLQTLSIQQCLPHRKNFLLP

QKSLSPQQYQKGHTLAILHEMLQQIFSLLFRANISLDGWEENHTEKFLIQLHQQLEYLEAL

MGLEAEKLSGTLGSDNLRLQVKMYFRRIHDYLENQDYSTCAWAIVQVEISRCLFFVFSL

TEKLSKQGRPLNDMKQELTTEFRSPR (SEQ ID NO: 25)

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

Human IFNκ is identified by UniProt accession number Q9P0W0 and has the amino acid sequence described below, with the signal sequence underlined.

MSTKPDMIQKCLWLEILMGIFIAGTLS LDCNLLNVHLRRVTWQNLRHLSSMSNSFPVEC

LRENIAFELPQEFLQYTQPMKRDIKKAFYEMSLQAFNIFSQHTFKYWKERHLKQIQIGLD

QQAEYLNQCLEEDKNENEDMKEMKENEMKPSEARVPQLSSLELRRYFHRIDNFLKEKK

YSDCAWEIVRVEIRRCLYYFYKFTALFRRK (SEQ ID NO: 26)

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

6.4. Protease-cleavable linker

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

The protease cleavable linker can range from 8 amino acids to more than 100 amino acids. In various embodiments, the protease cleavable linker ranges from 8 amino acids to 15 amino acids, from 10 amino acids to 20 amino acids, from 20 amino acids to 80 amino acids, and in certain embodiments, the non-cleavable peptide linker has a length of 20 amino acids to 60 amino acids, from 20 amino acids to 40 amino acids, from 30 amino acids to 50 amino acids, from 20 amino acids to 80 amino acids, or from 30 amino acids to 70 amino acids.

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

In various aspects of the IFN precursor protein comprising four protease cleavable linkers, the first and third protease cleavable linkers are cleavable by the same protease and/or the second and fourth protease cleavable linkers are cleavable by the same protease. In some embodiments, the protease is a protease set forth in Table 101.

In a further aspect of an IFN precursor protein comprising four protease cleavable linkers, the first and third protease cleavable linkers comprise the same substrate sequence and/or the second and fourth protease cleavable linkers comprise the same substrate sequence. In some embodiments, the substrate sequences are set forth in Table 102, and in other embodiments, the first and third protease cleavable linkers also comprise the same spacer sequence and/or the second and fourth protease cleavable linkers also comprise the same spacer sequence. In some embodiments, the spacer sequences are set forth in Table 103.

In an additional aspect, in an IFN precursor protein comprising four protease cleavable linkers, the first and third linkers comprise the same linker sequence and/or the second and fourth linkers comprise the same linker sequence. In some embodiments, the linker sequences are set forth in Table 104.

In some embodiments of an IFN precursor protein comprising four protease cleavable linkers, the first and third protease cleavable linkers are identical to the second and fourth protease cleavable linkers.

In other embodiments, the first and third protease cleavable linkers are different from the second and fourth protease cleavable linkers.

In the aspects and embodiments described above, the different linkers can be cleaved by the same protease, by different proteases, or, if the linker comprises multiple substrate sequences, the different linkers can be cleaved by multiple proteases, at least one of which is common and at least one of which can be cleaved by different proteases.

Exemplary protease-cleavable linker sequences are presented in Section 6.4.4.

6.4.1. Protease

Exemplary protease substrate sequences that may be incorporated into the protease-cleavable linker are provided in Table 101 below.

In certain embodiments, the protease is selected from the group consisting of matrix metalloprotease (MMP)-2, MMP-9, legume asparaginyl endopeptidase, thrombin, fibroblast-activating protease (FAP), MMP-1, MMP-3, MMP-7, MMP-8, MMP-12, MMP-13, MMP-14, membrane type 1 matrix metalloprotease (MT1-MMP), plasmin, transmembrane protease, serine (TMPRSS-3/4), cathepsin A, cathepsin B, cathepsin D, cathepsin E, cathepsin F, cathepsin H, cathepsin K, cathepsin L, cathepsin L2, cathepsin O, cathepsin S, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, Caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, human neutrophil elastase, urokinase/urokinase-type plasminogen activator (uPA), disintegrin and metalloprotease (ADAM) 10, ADAM 12, ADAM 17, ADAM with thrombospondin moiety (ADAMTS), ADAMTS5, beta secretase (BACE), granzyme A, granzyme B, guanidinobenzoatase, hepsin, matriptase, matriptase 2, meprin, neprilysin, prostate-specific membrane antigen (PSMA), tumor necrosis factor-converting enzyme (TACE), kallikrein-related peptidase (KLK) 3, KLK 5, KLK 7, KLK 11, NS3/4 of hepatitis C virus protease (HCV-NS3/4), tissue plasminogen activator (tPA), calpain, calpain 2, glutamate carboxypeptidase II, plasma calcrain, AMSH-like protease, AMSH, γ-secretase component, antiplasmin cleaving enzyme (APCE), desaicin 1, apoptosis-associated cysteine peptidase, or N-acetylated alpha-linked acid dipeptidase-like 1.

6.4.2. Temperament

Exemplary substrate sequences that are cleavable by oncogenic proteases and can be incorporated into protease-cleavable linkers are provided in Table 102 below.

Table 102
Substrate sequences for protease-cleavable linkers Substrate sequence design Protease cleavage SEQ ID NO: (DE) ₈ RPLALWRS(DR) ₈ SU1 MMP7 27 AARGPAIH SU2 28 AAYHLVSQ SU3 Collagenase 29 AGLGISST SU4 Collagenase 30 AGLGVVER SU5 Collagenase 31 ALAL SU6 lysosomal enzyme 32 ALFFSSPP SU7 33 ALFKSSFP SU8 34 ALLLALL SU9 TOP 35 AQFVLTEG SU10 Collagenase 36 AQNLLGMV SU11 37 AVGLLAPP SU12 Serine protease 38 DAFK SU13 Urokinase plasminogen activator (uPA) 39 DEVD SU14 caspase-3 40 DEVDP SU15 caspase-3 41 DPRSFL SU16 Thrombin 42 DVAQFVLT SU17 Collagenase 43 DVLK SU18 Plasmin 44 DWLYWPGI SU19 45 EDDDDKA SU20 Enterokinase 46 EP(Cit)G(Hoof)YL SU21 MMP2, MMP9, MMP14 47 EPQALAMS SU22 Collagenase 48 ESLPVVAV SU23 Collagenase 49 ESPAYYTA SU24 MMP 50 F(Pip)RS SU25 Thrombin FK SU26 lysosomal enzyme FPRPLGITGL SU27 51 FRLLDWQW SU28 52 GFLG SU29 lysosomal enzyme 53 GGAANLVRGG SU30 MMP11 54 GGGRR SU31 Urokinase plasminogen activator (uPA) 55 GGPRGLPG SU32 Cathepsin K 56 GGQPSGMWGW SU33 57 GGSIDGR SU34 Factor Xa 58 GGWHTGRN SU35 59 GIAGQ SU36 Collagenase 60 GKAFRR SU37 Calicrane 2 61 GPAGLYAQ SU38 62 GPAGMKGL SU39 63 GPEGLRVG SU40 Collagenase 64 GPLGIAGI SU41 Collagenase 65 GPLGVRG SU42 66 GPQGIAGQ SU43 Collagenase 67 GPQGLLGA SU44 Collagenase 68 GPRSFG SU45 69 GPRSFGL SU46 70 GPSHLVLT SU47 71 GVSQNYPIVG SU48 HIV protease 72 GVVQASCRLA SU49 CMV protease 73 GWEHDG SU50 interleukin 1β converting enzyme 74 HSSKLQ SU51 Prostate Specific Antigen 75 HSSKLQEDA SU52 Prostate Specific Antigen 76 HSSKLQL SU53 Prostate Specific Antigen 77 HTGRSGAL SU54 78 IDGR SU55 Factor Xa 79 IEGR SU56 Factor Xa 80 ILPRSPAF SU57 81 IPVSLRSG SU58 MMP 82 ISSGL SU59 MMP 83 ISSGLL SU60 MMP 84 ISSGLLS SU61 MMP 85 ISSGLLSS SU62 MMP 86 ISSGLSS SU63 MMP 87 KGSGDVEG SU64 caspase-3 88 KQEQNPGST SU65 FAP 89 KRALGLPG SU66 MMP7 90 LAAPLGLL SU67 91 LAPLGLQRR SU68 92 LAQKLKSS SU69 93 LAQRLRSS SU70 94 LEATA SU71 MMP9 95 LKAAPRWA SU72 96 LLAPSHRA SU73 97 LPGGLSPW SU74 98 LSGRSANI SU75 Serine protease 99 LSGRSANP SU76 Serine protease 100 LSGRSDDH SU77 Serine protease 101 LSGRSDIH SU78 Serine protease 102 LSGRSDNH SU79 Serine protease 103 LSGRSDNI SU80 Serine protease 104 LSGRSDNP SU81 Serine protease 105 LSGRSDQG SU82 Serine protease 106 LSGRSDQH SU83 Serine protease 107 LSGRSDTH SU84 Serine protease 108 LSGRSDYH SU85 Serine protease 109 LSGRSGNH SU86 Serine protease 110 LVLASSSFGY SU87 Herpes simplex virus protease 111 MDAFLESS SU88 Collagenase 112 MGLFSEAG SU89 113 MIAPVAYR SU90 114 MVLGRSLL SU91 115 NLL SU92 Cathepsin B NTLSGRSENHSG SU93 116 NTLSGRSGNHGS SU94 117 PAGLWLDP SU95 118 PGGPAGIG SU96 119 PIC(Et)FF SU97 Cathepsin D 120 PLGC(me)AG SU98 MMP 121 PLGL SU99 122 PLGLAG SU100 MMP 123 PLGLAX SU101 MMP 124 PLGLWA SU102 MMP 125 PLGLWSQ SU103 MMP 126 PLTGRSGG SU104 127 PMAKK SU105 128 PPRSFL SU106 Thrombin 129 PR(S/T)(L/I)(S/T) SU107 MMP9 PRFRIIGG SU108 Plasmin 130 PVGYTSSL SU109 131 PVQPIGPQ SU110 Collagenase 132 QALAMSAI SU111 Collagenase 133 QGRAITFI SU112 134 QNQALRMA SU113 135 RGPA SU114 136 RGPAFNPM SU115 137 RGPATPIM SU116 138 RKSSIIIRMRDVVL SU117 Plasmin 139 RLQLKAC SU118 MMP 140 RLQLKL SU119 MMP 141 RMHLRSLG SU120 142 RPSPMWAY SU121 143 RQARVVNG SU122 Matriptase 144 SAGFSLPA SU123 145 SAPAVESE SU124 Collagenase 146 SARGPSRW SU125 147 SGEPAYYTA SU126 148 SGGPLGVR SU127 149 SGRIGFLRTA SU128 MMP14 150 SGRSA SU129 Urokinase plasminogen activator (uPA) 151 SGRSANPRG SU130 152 SMLRSMPL SU131 153 SPLPLRVP SU132 154 SPLTGRSG SU133 155 SPRSIMLA SU134 156 SSRGPAYL SU135 157 SSRHRRALD SU136 Plasmin 158 SSSFDKGKYKKGDDA SU137 Plasmin 159 SSSFDKGKYKRGDDA SU138 Plasmin 160 STFPFGMF SU139 161 TARGPSFK SU140 162 TGRGPSWV SU141 163 TSGRSANP SU142 164 TSTSGRSANPRG SU143 165 VAGRSMRP SU144 166 VAQFVLTE SU145 Collagenase 167 VHMPLGFLGP SU146 168 VPLSLYSG SU147 MMP9 169 VVPEGRRS SU148 170 WATPRPMR SU149 171 YGAGLGVV SU150 Collagenase 172 HPVGLLAR SU151 173

6.4.3. Spacer

Exemplary spacer sequences that may be incorporated into the protease-cleavable linker are set forth in Table 103 below. In addition to the spacer sequences set forth in Table 103, any of the non-cleavable linker sequences described in Section 6.5, e.g. , any of the non-cleavable linker sequences set forth in Table 105, or a portion thereof, may be used as the spacer sequence. In some embodiments, the spacer sequence is completely absent from the protease-cleavable linker.

Table 103
Spacer sequence of protease-cleavable linker spacer sequence design SEQ ID NO: (GGGGS) _n SP1 174 (GGGS) _n SP2 175 (GGS) _n SP3 176 (GS) _n SP4 177 (GSGGS) _n SP5 178 GGGGSGGGGS SP6 179 GGGGSGGGGSGGGGS SP7 180 GGGGSGGGGSGGGGSGGGGS SP8 181 GGGKSGGGKSGGGKS SP9 182 GGGKSGGKGSGKGGS SP10 183 GGGS SP11 184 GGGSG SP12 185 GGKGSGGKGSGGKGS SP13 186 GGSGGGGSGGGGS SP14 187 GGSGGS SP15 188 GGSGGSGGSGS SP16 189 GSGGG SP17 190 GSGSG SP18 191 GSS SP19 GSSG SP20 192 GSSGGSGGSG SP21 193 GSSGGSGGSGG SP22 194 GSSGGSGGSGGS SP23 195 GSSGGSGGSGGSG SP24 196 GSSGGSGGSGGSGGGS SP25 197 GSSGGSGGSGS SP26 198 GSSGT SP27 199 GSSSG SP28 200 QGQSGQ SP29 201 QGQSGQG SP30 202 QGQSGS SP31 203 QSGQ SP32 204 QSGQG SP33 205 QSGS SP34 206 SGQ SP35 SGQG SP36 207 SGS SP37 (G) _n SP38 208

In some implementations, as used in Table 103 above, n is an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

6.4.4. Exemplary protease-cleavable linkers

Exemplary protease-cleavable linkers comprising one or more substrate sequences as well as a spacer sequence are provided in Table 104 below.

Table 104
Protease-cleavable linker sequence Linker sequence design Protease cleavage SEQ ID NO: GGGISSGLLSGRSDNHGGGISSGLLSGRSDNHGGS PCL1 209 GGGISSGLLSGRSDNHGGGISSGLLSGRSDNHGGS PCL2 209 GGSGGSIPVSLRSGGGISSGLLSGRSDNHGGSGGS PCL3 210 GGSGGSVPLSLYSGGGISSGLLSGRSDNHGGSGGS PCL4 211 GGSHPVGLLARGGGHPVGLLARGGGHPVGLLARGS PCL5 212 GGSHPVGLLARGGGGHPVGLLARGGSGRSAGGSGRSA PCL6 213 AVGLLAPPGGLSGRSANI PCL7 ADAM17_2, FAPa_1, CTSL1_1 214 AVGLLAPPGGLSGRSANP PCL8 FAPa_1, ADAM17_2, CTSL1_1 215 AVGLLAPPGGLSGRSDDH PCL9 MMP14_1, MMP14_1, MMP14_1 216 AVGLLAPPGGLSGRSDIH PCL10 MMP14_1, MMP14_1, MMP14_1 217 AVGLLAPPGGLSGRSDNH PCL11 MMP14_1, MMP14_1 218 AVGLLAPPGGLSGRSDNI PCL12 MMP14_1, CTSL1_1, ADAM17_2 219 AVGLLAPPGGLSGRSDNP PCL13 CTSL1_1, ADAM17_2, FAPa_1 220 AVGLLAPPGGLSGRSDQH PCL14 221 AVGLLAPPGGLSGRSDTH PCL15 FAPa_1, CTSL1_1, ADAM17_2 222 AVGLLAPPGGLSGRSDYH PCL16 223 AVGLLAPPGGTSTSGRSANPRG PCL17 224 AVGLLAPPSGRSANPRG PCL18 225 AVGLLAPPTSGRSANPRG PCL19 226 GGALFKSSFPGPAGLYAQPLAQKLKSSGGK PCL20 CTSL1_1, MMP14_1, ADAM17_2 227 GGGGSGGGGSGGGGSFVGGTGGGGSGGGGSGGS PCL21 228 GGGGSGGGGSGGGGSISSGLLSGRSDDNHGGSGGS PCL22 229 GGGGSGGGGSGGGGSVPLSLYSGGGSGGSGGSGS PCL23 230 GGGGSGGGGSGPLGLWSQGGGGSGGGGSGGGGSGG PCL24 231 GGGGSGGGGSKKAAPGGGGSGGGGSGGGGSGGS PCL25 232 GGGGSGGGGSKKAAPVNGGGGGSGGGGSGGGGS PCL26 233 GGGGSGGGGSPMAKKGGGGSGGGGSGGGGSGGS PCL27 234 GGGGSGGGGSPMAKKVNGGGGGSGGGGSGGGGS PCL28 235 GGGGSGGGGSQARAKGGGGSGGGGSGGGGSGGS PCL29 236 GGGGSGGGGSQARAKVNGGGGSGGGGSGGGGS PCL30 237 GGGGSGGGGSRQARVVNGGGGGSGGGGSGGGGS PCL31 238 GGGGSGGGGSRQARVVNGGGGGSVPLSLYSGGGGGSGGGGS PCL32 239 GGGGSGGGGSRQARVVNSVPLSLYSGGGGGSGGGGS PCL33 240 GGGGSGGGGSVHMPLGFLGPGGGGSGGGGSGGS PCL34 241 GGGGSVHMPLGFLGPGRSRGSFPGGGGS PCL35 242 GGGGSVHMPLFLLGPPMAKKGGGGSGGGGSGGS PCL36 243 GGGGSVHMPLFLLGPRQARVVNGGGGSGGGGS PCL37 244 GGGGSVHMPLFLLGPRQARVVNGGGGSGGGGSGG PCL38 245 GGPLAQKLKSSALFKSSFPGPAGLYAQGGR PCL39 ADAM17_2, CTSL1_1, MMP14_1 246 GLSGRSDNHGGAVGLLAPP PCL40 247 GLSGRSDNHGGVHMPLGFLGP PCL41 248 ISSGLLSGRSANI PCL42 MMP, serine protease 249 ISSGLLSGRSANP PCL43 MMP, serine protease 250 ISSGLLSGRSANPRG PCL44 MMP, serine protease 251 ISSGLLSGRSDDH PCL45 MMP, serine protease 252 ISSGLLSGRSDIH PCL46 MMP, serine protease 253 ISSGLLSGRSDNH PCL47 MMP, serine protease 254 ISSGLLSGRSDNI PCL48 CTSL1_1, MMP14_1 255 ISSGLLSGRSDNP PCL49 MMP, serine protease 256 ISSGLLSGRSDQH PCL50 MMP, serine protease 257 ISSGLLSGRSDTH PCL51 MMP, serine protease 258 ISSGLLSGRSDYH PCL52 MMP, serine protease 259 ISSGLLSGRSGNH PCL53 MMP, serine protease 260 ISSGLLSSGGSGGSLSGRSDNH PCL54 261 ISSGLLSSGGSGGSLSGRSGNH PCL55 262 KGGPGGPAGIGGPLAQRLRSSALFKSSFPGR PCL56 FAPa_1, ADAM17_1, CTSL1_1 263 KSGPGGPAGIGALFSSPPLAQKLKSSGGR PCL57 FAPa_1, CTSL1_2, ADAM17_2 264 LSGRSDNHGGAVGLLAPP PCL58 265 LSGRSDNHGGSGGSISSGLLSS PCL59 266 LSGRSDNHGGSGGSQNQALRMA PCL60 267 LSGRSDNHGGVHMPLGFLGP PCL61 268 LSGRSGNHGGSGGSISSGLLSS PCL62 269 LSGRSGNHGGSGGSQNQALRMA PCL63 270 QNQALRMAGGSGGSLSGRSDNH PCL64 271 QNQALRMAGGSGGSLSGRSGNH PCL65 272 RGGALFKSSFPLAQKLKSSGPAGLYAQGGK PCL66 CTSL1_1, ADAM17_2, MMP14_1 273 RGGGPAGLYAQPLAQKLKSSALFKSSFPGG PCL67 MMP14_1, ADAM17_2, CTSL1_1 274 SGGFPRSGGSFNPRTFGSKRKRRGSRGGGG PCL68 Thrombin, factor Xa, hepsin 275 SGPLAQKLKSSGPAGLYAQALFKSSFPGSK PCL69 ADAM17_2, MMP14_1, CTSL1_1 276 TSTSGRSANPRGGGAVGLLAPP PCL70 277 TSTSGRSANPRGGGVHMPLGFLGP PCL71 278 VHMPLGFLGPGGLSGRSDNH PCL72 279 VHMPLGFLGPGGTSTSGRSANPRG PCL73 280 SGRSA GGG SGRSA GGG SGRSA PCL74 uPA 281 HPVGLLAR GGG HPVGLLAR GGG SGRSA GGG SGRSA PCL75 MPA (MMP-2 and uPA) 282 GPLGVRGK PCL76 MMP-2 283 HPVGLLAR PCL77 MMP-2 173 GPQGIAGQ PCL78 MMP-2, MMP-9, and some MT1-MMPs 67 VPMSMRGG PCL79 MMP-9 and MMP-2 284 IPVSLRSG PCL80 MMP-2 and some MMP-9 or MMP-7 82 RPFSMIMG PCL81 MMP-9 and MMP-7, some MMP-3 285 VPLSLTMG PCL82 MMP-7, some MMP-9, MMP-2, MPT-1 - MMP 286 VPLSLYSG PCL83 MMP-2, MMP-9, MMP-7 169 IPESLRAG PCL84 MMP-2, MMP-7, MMP-9, some MPT-1-MMP 287 VPLSLYSGGGISSGLLSGRSDNH PCL85 288 GGGISSGLLSGRSDNHGGGS PCL86 289 GGGHPVGLLARGGGS PCL87 290 GGGSGGGSGGGGISSGLLSGRSDNHGGGSGGGSGGS PCL88 291 GGGGISSGLLSGRSDNHGGGISSGLLSGRSDNHGGS PCL89 292 GGGSGGSIPVSLRSGGGISSGLLSGRSDNHGGSGGS PCL90 293 GGGSGGSVPLSLYSGGGISSGLLSGRSDNHGGSGGS PCL91 294 GGGSHPVGLLARGGGHPVGLLARGGGHPVGLLARGS PCL92 295 GGGSHPVGLLARGGGGHPVGLLARGGSGRSAGGSGRS PCL93 296 GISSGLLSGRSDNHG PCL94 297 GGGSISSGLLSGRSDNHGGGS PCL95 298

In certain embodiments, the protease-cleavable linker comprises an amino acid sequence having at most 5, at most 4, at most 3, at most 2 or at most 1 amino acid substitutions compared to a sequence set forth in Table 104. Thus, in some embodiments, the protease-cleavable linker comprises or consists of any amino acid sequence of Table 104 having from 1 to 5 amino acid substitutions compared to a sequence set forth in Table 104.

6.5. Non-cleavable linker

In certain aspects, the present disclosure provides an IFN precursor protein wherein two or more components of the IFN precursor protein are linked to each other by a peptide linker. For example, but not limited to, the linker can be used to link an Fc domain and a targeting moiety or other domains within a targeting moiety ( e.g., the VH and VL domains of a scFv).

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

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

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

In some of the embodiments described above, the non-cleavable linker has a length in the range of 5 amino acids to 50 amino acids, for example , a length in the range of 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25 or 5 to 20 amino acids. In other embodiments as described above, the non-cleavable linker has a length in the range of 6 amino acids to 50 amino acids, for example , a length in the range of 6 to 50, 6 to 45, 6 to 40, 6 to 35, 6 to 30, 6 to 25 or 6 to 20 amino acids. In other embodiments as described above, the non-cleavable linker has a length in the range of 7 amino acids to 50 amino acids, for example , a length in the range of 7 to 50, 7 to 45, 7 to 40, 7 to 35, 7 to 30, 7 to 25 or 7 to 20 amino acids.

In particular, charged ( e.g. , charged hydrophilic linkers) and/or flexible non-cleavable linkers are preferred.

Examples of flexible non-cleavable linkers that can be used in the IFN precursor proteins of the present disclosure include those disclosed in Chen et al ., 2013, Adv Drug Deliv Rev. 65(10): 1357-1369 and Klein et al. , 2014, Protein Engineering, Design & Selection 27(10): 325-330. Particularly useful flexible non-cleavable linkers are or comprise repeats of glycine and serine, for example , monomers or multimers of G _n S (SEQ ID NO:299) or SG _n (SEQ ID NO:300), wherein n is an integer from 1 to 10, for example , 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the non-cleavable linker is or comprises a monomer or multimer of _G4S repeats (SEQ ID NO: 301), for example, (GGGGS) _n (SEQ ID NO: 301).

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

Exemplary non-cleavable linker sequences are provided in Table 105 below.

Table 105
Non-cleavable linker sequence Linker sequence design SEQ ID NO: GGGGSLALGPGGGGGSLALGPGGGGGSLALGPGGS NCL1 308 GGGGSGGGGSGGGGSGGGGSGGGGS NCL2 309 (GGGGS) _n , optionally n=1-10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 NCL3 174 (GGGS) _n , optionally n=1-10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 NCL4 175 (GGS) _n , optionally n=1-10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 NCL5 176 (GS) _n , optionally n=1-10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 NCL6 177 (GSGGS) _n , optionally n=1-10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 NCL7 178 ADAAP NCL8 310 ADAAPTVSIFP NCL9 311 ADAAPTVSIFPP NCL10 312 AKTTAP NCL11 313 AKTTAPSVYPLAP NCL12 314 AKTTPKLEEGEFSEARV NCL13 315 AKTTPKLGG NCL14 316 AKTTPP NCL15 317 AKTTPPSVTPLAP NCL16 318 ASTKGP NCL17 319 ASTKGPSVFPLAPASTKGPSVFPLAP NCL18 320 EGKSSGSGSESKST NCL19 321 GEGESGEGESGEGES NCL20 322 GEGESGEGESGEGESGEGES NCL21 323 GEGGSGEGGSGEGGS NCL22 324 GENKVEYAPALMALS NCL23 325 GGEGSGGEGSGGEGS NCL24 326 GGGESGGEGSGEGGS NCL25 327 GGGESGGGESGGGES NCL26 328 GGGGSGGGGS NCL27 179 GGGGSGGGGSGGGGS NCL28 180 GGGGSGGGGSGGGGSGGGGS NCL29 181 GGGKSGGGKSGGGKS NCL30 182 GGGKSGGKGSGKGGS NCL31 183 GGGS NCL32 184 GGGSG NCL33 185 GGKGSGGKGSGGKGS NCL34 186 GGSG NCL35 329 GGSGG NCL36 330 GGSGGGGSG NCL37 331 GGSGGGGSGGGGS NCL38 187 GHEAAAVMQVQYPAS NCL39 332 GKGGSGKGGSGKGGS NCL40 333 GKGKSGKGKSGKGKS NCL41 334 GKGKSGKGKSGKGKSGKGKS NCL42 335 GKPGSGKPGSGKPGS NCL43 336 GKPGSGKPGSGKPGSGKPSGS NCL44 337 GPAKELTPLKEAKVS NCL45 338 GSAGSAAGSGEF NCL46 339 GSGGG NCL47 190 GSGSG NCL48 191 GSS NCL49 GSSG NCL50 192 GSSGGSGGSG NCL51 193 GSSGGSGGSGG NCL52 194 GSSGGSGGSGGS NCL53 195 GSSGGSGGSGGSG NCL54 196 GSSGGSGGSGGSGGGS NCL55 197 GSSGGSGGSGS NCL56 198 GSSGT NCL57 199 GSSSG NCL58 200 GSTSGSGKPGSGEGSTKG NCL59 340 GTAAAGAGAAGGAAAGAAG NCL60 341 GTSGSSGSGSGGSGSGGGG NCL61 342 IRPRAIGGSKPRVA NCL62 343 KESGSVSSEQLAQFRSLD NCL63 344 KTTPKLEEGEFSEAR NCL64 345 PRGASKSGSASQTGSAPGS NCL65 346 QPKAAP NCL66 347 QPKAAPSVTLFPP NCL67 348 RADAAAA(G4S) ₄ NCL68 349 RADAAAAGGPGS NCL69 350 RADAAP NCL70 351 RADAAPTVS NCL71 352 SAKTTP NCL72 353 SAKTTPKLEEGEFSEARV NCL73 354 SAKTTPKLGG NCL74 355 STAGDTHLGGEDFD NCL75 356 TVAAP NCL76 357 TVAAPSVFIFPP NCL77 358 TVAAPSVFIFPPTVAAPSVFIFPP NCL78 359 AGSGNSSGSGGSGGSGNSSGSGGSPVPSTPPTPSPSTPPTPSPSAS NCL79 360 GGGGSAS NCL80 361 GGGGSGGGGSAS NCL81 362 GGGGSGGGGSGGGGSAS NCL82 363 GGGGSGGGGSGGGGSGGGGSGGGGSAS NCL83 364 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSAS NCL84 365 AGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSAS NCL85 366 AGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSAS NCL86 367 AGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSAS NCL87 368

In certain embodiments, the IFN proprotein of the present disclosure can comprise a polypeptide chain comprising, in N- to C-terminal direction, a targeting moiety (or targeting moiety chain), a hinge domain, a CH2 domain and a CH3 domain ( e.g. , as illustrated in FIG. 1a ), or a polypeptide chain comprising a targeting moiety (or targeting moiety chain), a hinge domain, followed by a protease cleavable linker ( e.g. , as illustrated in FIG. 1c ). Thus, the hinge domain can be referred to as a type of linker. Exemplary hinge domains are provided in Section 6.8.3.

6.6. Target Moiety

By incorporating a targeting moiety into the IFN precursor protein of the present invention, high concentrations of IFN can be delivered to the tumor microenvironment with reduced systemic exposure, thereby reducing side effects compared to those obtained with non-targeted IFN molecules.

Any type of target molecule that is present at a specific location or tissue or that can drive an IFN proprotein is expected to be targeted by the IFN proprotein of the present disclosure. In some embodiments, the IFN proprotein is for treating cancer by inducing a local immune response against the tumor tissue ( e.g. , inducing a local immune response against the tumor tissue). Thus, the target molecule can be any local tumor and associated target molecule. The target molecules recognized by the targeting moiety of the IFN proprotein of the present disclosure are typically found on the surface of activated T cells, the surface of tumor cells, the surface of dendritic or other antigen presenting cells, the surface of natural killer (NK) cells, the surface of virus-infected cells, the surface of other diseased cells, serum free, in the extracellular matrix (ECM), or on immune cells present at the target site ( e.g. , tumor-reactive lymphocytes).

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

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

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

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

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

In certain embodiments, the targeting molecule is on the surface of a dendritic cell or other antigen presenting cell (e.g., XCR1, Clec9a, CD1c, CD11c, CD14, PDL1, macrophage mannose receptor (CD206), and DEC-205).

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

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

Suitable targeting moiety formats are described in Section 6.7. The targeting moiety is preferably an antigen binding moiety, e.g., an antibody or an antigen binding portion of an antibody, e.g., a scFv as described in Section 6.7.2 or a Fab as described in Section 6.7.1.

In some embodiments, the targeting moiety targets an exemplary targeting molecule as set forth in Table 106 below, along with references to exemplary antibodies or antibody sequences that may be the basis for the targeting moiety.

Table 106
Exemplary target molecules Target Antibody name and/or binding sequence 1-92-LFA-3 Amevive™ (Alefacept) 5T4 GEN1044 Activin receptor type II Bimagrumab VH: SEQ ID NO:107, 109 of US Patent No. 8,388,968 B2
VL: SEQ ID NO:93, 95 of US Patent No. 8,388,968 B2 B7-H3 Obrindamab (MGD009) B7-H3 (CD276) Enoblituzumab (MGA271) B7-H3 (CD276) MGC018 B7-H3 (CD276) MGA012 B7-H3 (CD276) 8H9 B7-H3 (CD276) VH: VH sequence of the heavy chain of SEQ ID NO:21, 26 or 31 of US 2021/0171641 A1.
VL: VL sequence of the light chain of SEQ ID NO:20, 22 or 30 of US 2021/0171641 A1. B7-H3 (CD276) VH: VH sequence of the heavy chain of SEQ ID NO:21, 29 or 37 of US 2019/0002563 A1.
VL: VL sequence of the light chain of SEQ ID NO:17, 25 or 33 of US 2019/0002563 A1. B7-H3 (CD276) VH: The VH sequence of the heavy chain of SEQ ID NO:146, 147 or 148 of U.S. Patent No. 10,640,563.
VL: The VL sequence of the light chain of SEQ ID NO:143, 144 or 145 of U.S. Patent No. 10,640,563. BAFF/B Lymphocyte Stimulating Agent Benlysta™ (Belimumab) BAFF/B Lymphocyte Stimulating Agent VH: Amino acids 1-123 of SEQ ID NO:327 of U.S. Patent No. 7,138,501
VL: Amino acids 139-249 of SEQ ID NO:327 of U.S. Patent No. 7,138,501. BAFF/B Lymphocyte Stimulating Agent VH: Amino acids 1-126 of SEQ ID NO:1321 of U.S. Patent No. 7,605,236;
VL: Amino acids 143-251 of SEQ ID NO:1049 of U.S. Patent No. 7,605,236. BAFF/B Lymphocyte Stimulating Agent Belimumab BCMA VH: Heavy chain VL sequence of SEQ ID NO:126, US 2021/0206865 A1
VL: Light chain VL sequence of SEQ ID NO:129 or SEQ ID NO:132, US 2021/0206865 A1 CA125 Igobumab CA125 OvaRex™ (oregovumab) Cardherin The antibody is published under US Publication Number US 2006/0039915. N-cardherin An antibody that binds to the amino acid sequence of SEQ ID NO:10, 17 or 18 of US Publication No. US 2010/0278821 CD11a Raptiva™ (Efalizumab)
Sequences from Werther et al ., 1996, The Journal of Immunology 157(11): 4986-4995. CD19 Blincyto™ (blinatumomab) CD19 SGN-CD19A CD20 Bexxar™ (tositumomab)
VH: VH sequence of heavy chain of SEQ ID No:124, US 2017/0002060 A1
VL: VL sequence of light chain of SEQ ID No:125, US 2017/0002060 A1 CD20 Zevalin™ (ibritumomab tuxetan)
VH: SEQ ID NO:9, U.S. Patent No. 5,736,137
VL: SEQ ID NO:6, U.S. Patent No. 5,736,137 CD20 Rituxan™ (Rituximab)
VH: SEQ ID NO:9, U.S. Patent No. 5,736,137
VL: SEQ ID NO:6, U.S. Patent No. 5,736,137 CD20 Ocrevus™ (ocrelizumab) CD20 Okaratuzumab CD20 Arzerra™ (ofatumumab)
VH: SEQ ID NO:2, U.S. Patent No. 8,529,902
VL: SEQ ID NO:4, US Patent No. 8,529,902 CD20 Gazyva™ (obinutuzumab) CD20 VH: SEQ ID NO:4, US 2021/0206870 A1
VL of SEQ ID NO:6, US 2021/0206870 A1 CD20 Epcoritamab CD22 Belimumab CD22 Epratuzumab CD22 Besponsa™ (inotuzumab ozogamicin) CD22 Lumoxiti™ (Moxetumumab Pasudox) CD22 pinatuzumab vedotin CD25 Zenapax™ (daclizumab)
VH: SEQ ID NO:9, U.S. Patent No. 7,060,269
VL: SEQ ID NO:10, US Patent No. 7,060,269 CD30 Adcetris™ (brentuximab vedotin)
VH: SEQ ID NO:2, US Patent No. 7,090,843
VL: SEQ ID NO:10, US Patent No. 7,090,843 CD33 Myelotarg™ (Gemtuzumab)
Sequence of Man Sung et al. , 1993, Molecular immunology 30:1361-1367 CD33 Lintuzumab CD38 Darzalex™ (daratumumab) CD38 IB4, HB7 CS/2, Clone 90 and NIM-R5 are disclosed in PCT Publication No. WO2015/009726A2 and references cited therein. CD40 lucatumumab CD40 Dacetuzumab CD40L Hu5c8 (ruplizumab) CD44v6 vibatuzumab mertansine CD52 Campath™ (alemtuzumab)
VH: SEQ ID NO:1, US Patent Publication No. US 2017/0002060 A1
VL: SEQ ID NO:2, US Patent Publication No. US 2017/0002060 A1 CD70 Blenrep™ (borsetuzumab mafodotin) CD123 flotetuzumab CD206 Anti-CD206 antibody having VH a of SEQ ID NO:2 and VL of SEQ ID NO:4, WO2003/040169A2 CD221 Tepezza™ (teprotumumab) CEA Hybri-Ceaker® (altumomab pentetate) CEA Scintimun™ (Becilesomab) CEA CEA-CIDE™ (Labezutumab) CEA CEA-Scan™ (Arcitumomab) CEA hMN-15
CDR-H1, CDR-H2 and CDR-H3 sequences of SEQ ID NO:4-6, U.S. Patent No. 8,771,690 B2
CDR-L1, CDR-L2 and CDR-L3 sequences of SEQ ID NO:1-3, U.S. Patent No. 8,771,690 B2 CEA CEA binding portion of RO6958688/RG7802 in clinical trial NCT02324257 CEA sibisatamab CEA CEA binding portion of MEDI-565/MT110/AMG211 in clinical trials NCT01284231 and NCT02291614
VH: SEQ ID NO:49 or 51, PCT Publication No. WO 2013/012414 A1
VL: SEQ ID NO:48, PCT Publication No. WO 2013/012414 A1 CEA Labetuzumab CEA Atezolizumab CEA sibisatamab CEA MEDI-565(AMG211, MT111) CEA RO6958688 CEA VH: SEQ ID NO:9 described in WO2022/048883A1
VL: SEQ ID NO:10 described in WO2022/048883A1 CLDN18.2 AMG910 Clec9a Anti-Clec9a antibody having VH and VL amino acids of SEQ ID NO:43 and 48, PCT Publication No. WO2009/026660A1 Clec9a Anti-Clec9a antibody having VH a of SEQ ID NO:38 and VL of SEQ ID NO:37 or SEQ ID NO:43, PCT Publication No. WO2022/073062A1
Anti-Clec9a antibody having VH a of SEQ ID NO:8 and VL amino acid of SEQ ID NO:7, PCT Publication No. WO2022/073062A1 Collagen alpha-4 chain TRC093 (MT293) Collagen Collagen-binding antibody fragment described in Liang et al ., 2016, Sci. Rep. 5, 18205; doi: 10.1038/srep18205 (2016) Collagen Type I Cetuximab (Erbitux) Collagen Type X Amino acid sequence of SEQ ID NO: 1 or 2, PCT Publication No. WO 2019/020797 Collagen Type X Amino acid sequence of SEQ ID NO:1, PCT Publication No. WO 2014/180992 Collagen Type X Antibody X34 as described in the literature [I. Girkontaite et al., "Immunolocalization of type X collagen in normal fetal and adult osteoarthritic cartilage with monoclonal antibodies," Matrix Biol 15, 231-238 (1996)]. Collagen Type X X53 or 1H8 or ARC0659 or JF0961 Collagen X polyclonal antibody sold by ThermoFisher Scientific under catalog numbers PA5-115039 or PA5-116871 or PA5-97603 or PA5-49198 Collagen Type X An antibody sold by RDI under catalog number RDI-COLL10abr. Complement C5 Soliris™ (eculizumab)
VH: Amino acids of SEQ ID No:1-122 of U.S. Patent No. 6,355,245
VL: Amino acid of SEQ ID NO:3-110 of U.S. Patent No. 6,355,245 CTLA-4 Yervoy™ (ipilimumab)
SEQ ID NO:17 of VH: WO 2001/014424 A2
SEQ ID NO:7 of VL: WO 2001/014424 A2 CTLA-4 (Tremelimumab) CTLA-4 Orencia™ (Abatacept) DEC-205 Anti-DEC-205 antibody having VH/VL pairs of SEQ ID NO:4/10, 16/22, 28/34, 40/46, 52/58, 76/82, PCT Publication WO2009/061996A2 DLL3 AMG757 EGFR Erbitux™ (Cetuximab)
VH: SEQ ID NO:11, U.S. Patent No. 6,217,866
VL: SEQ ID NO: 13, U.S. Patent No. 6,217,866 EGFR Vectibix™ (panitumumab)
VH: SEQ ID NO:37, U.S. Patent No. 6,235,883
VL: SEQ ID NO:38, U.S. Patent No. 6,235,883 EGFR Zalutumumab
VH: SEQ ID NO:64, WO 2018/140831 A2
VL: SEQ ID NO:69, WO 2018/140831 A2 EGFR Mapatumumab EGFR Matuzumab EGFR Nimotuzumab
VH: SEQ ID NO:51 WO 2018/140831 A2
VL: SEQ ID NO:56, WO 2018/140831 A2 EGFR ICR62 EGFR mAb 528 EGFR CH806 EGFRv3 AMG596 EGFRv3 AMG404 EGFR/CD64 MDX-447 EpCAM Panorex™ (edrecolomab)
VH: SEQ ID NO:129, WO 2018/140831 A2
VL: SEQ ID NO:134, WO 2018/140831 A2 EpCAM Adecatumumab
VH: SEQ ID NO:142, WO 2018/140831 A2
VL: SEQ ID NO:147, WO 2018/140831 A2 EpCAM Tucotuzumab selmoleukin EpCAM Sitazutumab Bogatox EpCAM EP1629013 B1
VH: SEQ ID NO: 80, 84, 88, 92 or 96
VL: SEQ ID NO: 82, 86, 90, 94 or 98 EpCAM G8.8
HC: SEQ ID NO:4, US Patent Publication No. US 2020/0317806 A1
HL: SEQ ID NO:3, US Patent Publication No. US 2020/0317806 A1 EpCAM VH: SEQ ID NO:17-22, WO 2021/211510 A2
VL: SEQ ID NO:15-16, WO 2021/211510 A2 EpCAM Removab™ (catumaxomab) EpCAM Vicineum™ (Oportuzumab Monatox) EpCAM M701 F protein of RSV Synagic™ (Palivizumab) GD2 3F8 Glycoprotein receptor IIb/IIIa ReoPro™ (Abcixiimab) gpA33 MGD007 GPC3 ERY974 GUCY2C PF-07062119 Heparanase An antibody selected from the amino acid sequences of HP130, HP 239, HP 108.264, HP 115.140, HP 152.197, HP 110.662, HP 144.141, HP 108.371, HP 135.108, HP 151.316, HP 117.372, HP 37/33, HP3/17, HP 201 or HP 102 or SEQ ID NO:1-11, US Patent Publication No. US 2004/0170631. Her2 Herceptin™ (trastuzumab) Her2 Aldesleukin (Proleukin) Her2 Sargramus Team (Ryu Shin) Her2 M802 Her2 Lunimotamab (BTRC4017A, R07227780) Her2 ISB1302 Her2-neu Perjeta™ (Pertuzumab)
VH: SEQ ID NO:16, WO 2013/096812 A1
VL: SEQ ID NO:15, WO 2013/096812 A1 Her2-neu Rexomun™ (ertumaxomab) IgE Xolair™ (omalizumab) IGFIR (Fijitumumab) IL1β Ilaris™ (canakinumab)
VH: SEQ ID NO:1, US Patent No. 7,446,175
VL: SEQ ID NO:2 US Patent No. 7,446,175 IL12/IFN3 Stelara™ (ustekinumab) IL1Ra Antril™, Kineret™ IFNR Simulect™ (Basiliximab)
VH: SEQ ID NO:3, U.S. Patent No. 6,383,487
VL: SEQ ID NO:6, U.S. Patent No. 6,383,487 IL6 Clazakizumab IL6 receptor Actemra™ (tocilizumab)
VH: SEQ ID NO:31, U.S. Patent No. 7,479,543
VL: SEQ ID NO:29, U.S. Patent No. 7,479,543 IL12/IFN3 p40 subunit Stelara™ (ustekinumab)
VH: SEQ ID NO:7, U.S. Patent No. 6,902,734
VL: SEQ ID NO:8, US Patent No. 6,902,734 Integrin α4 Tysabri™ (natalizumab)
VH: SEQ ID NO:11-13, U.S. Patent No. 5,840,299
VL: SEQ ID NO:7-8, U.S. Patent No. 5,840,299 Integrin α4 β7 Entyvio™ (vedolizumab)
HC: SEQ ID NO:2, US Patent Publication No. US 2012/0282249
LC: SEQ ID NO:4, US Patent Publication No. US 2012/0282249 Integrin α5 β1 VH: SEQ ID NO:2, European Patent No. 1 755 659
VL: SEQ ID NO:4, European Patent No. 1 755 659 Integrin β1 VH: SEQ ID NO:2, 6, 8, 10, 12, 14, 29-43 or 91-100, US Patent Publication No. US 2022/0089744
VL: SEQ ID NO:4, 16, 18, 20, 22, 44-57 or 107-116, US Patent Publication No. US 2022/0089744 KIR Anti-KIR antibody having VH of SEQ ID NO:5 and VL of SEQ ID NO:3, PCT Publication WO2014/066532A1 KIR Anti-KIR antibody having VH of SEQ ID NO:1 and VL of SEQ ID No:2, PCT Publication WO2012/160448A2
Anti-KIR antibody having VH of SEQ ID NO:3 and VL of SEQ ID NO:4, PCT Publication WO2012/160448A2 LAG3 Relatrimab (BMS-98016) LAG3 Sym022 LAG3 HLX26 LAG3 TSR-033 LAG3 ABL501 LAG3 INCAGN02385 LAG3 Pianlimab (REGN3767) LAG3 RO7247669 LAG3 EMB-02 LAG3 FS118 LAG3 GSK2831781 LAG3 IBI323 LAG3 IBI110 LAG3 LAG525 LAG3 XmAb®22841 LAG3 LBL-007 LAG3 VH: SEQ ID NO:1, 8, 10 or 12, U.S. Patent No. 9,902,772
VL: SEQ ID NO:2, 3, 4, 5, 6, 7, 9, 11, 13 or 14, U.S. Patent No. 9,902,772 LAG3 VH: SEQ ID NO:182, United States Patent Publication No. US 2021/0095026.
VL: SEQ ID NO:88, United States Patent Publication No. US 2021/0095026. LAG3 An antibody having VH/VL amino acid sequences of SEQ ID NO:23/24, ¾ and 11/12, US2022/0056126A1 Laminin Lam-89 from Sigma Aldrich Mesothelin Amatuximab Mesothelin HPN536 MUC1 Clivatuzumab tetraxetan MUC1 Pankomab™ (gatipotuzumab) MUC1 Femtumumab MUC1 Cantuzumab Labtansine MUC16 (CA125) Anti-MUC16 antibodies having VH and VL sequences having any one of the following SEQ ID NO pairs: 18/26; 82/858; 98/170 in US 2018/0118848A1 MUC17 AMG199 nectin-4 Enfortumab (ASP7465, ASG-22CE, ASG-22ME)
VH: SEQ ID NO:3, US PCT Publication WO 2021/151984
VL: SEQ ID NO:4, US PCT Publication WO 2021/151984 nectin-4 SBT290 nectin-4 VH: SEQ ID NO:1, US Patent No. 11,274,160
VL: SEQ ID NO:2, US Patent No. 11,274,160 NGF (Tanezumab) NKH1A Monoclonal antibody described in U.S. Patent No. 4,772,552A and deposited with ATCC under accession number HB8564; NKP46 Anti-NKP46 antibody having CDR-H1, CDR-H2, CDR-H3 sequences of SEQ ID NO:4, 6, 8 and CDR-L1, CDR-L2, CDR-L3 sequences of SEQ ID NO:12, 14, 16, US PCT Publication WO2018/047154A1 Osteopontin HC: SEQ ID NO:22, US PCT Publication WO 2021/030209
LC: SEQ ID NO:24, US PCT Publication WO 2021/030209 PD1 MDX-1106/BMS-936558 (nivolumab) is a human IgG4 mAb having the structure described in WHO Drug Information, Vol. 27, No. 1, pp. 68-69 (2013), and the heavy and light chain sequences are disclosed in Figure 7 of U.S. Publication No. US20190270812A1.
HC: SEQ ID NO:23, U.S. Publication No. US20190270812A1
LC: SEQ ID NO:24, U.S. Publication No. US20190270812A1 PD1 MK-3475 (pembrolizumab) is a humanized IgG4 mAb having the structure described in WHO Drug Information, Vol. 27, No. 2, pp. 161-162 (2013), and the heavy and light chain sequences are disclosed in Figure 6 of U.S. Publication No. US20190270812A1.
HC: SEQ ID NO:21, US Publication Number US20190270812A1
LC: SEQ ID NO:22, US Publication Number US20190270812A1 PD1 REGN2810 (disclosed as H4H7798N in U.S. Publication No. 20150203579)
HC: SEQ ID NO:330, US Publication Number 20150203579
LC: SEQ ID NO:331, US Publication No. 20150203579 PD1 An anti-PD1 antibody having CDR H1-H3 and CDR L1-L3 sequences corresponding to the following SEQ ID NOs of U.S. Patent No. 11,034,765 B2:
a) SEQ ID NO: 18, 19, 20, 21, 22, and 23, respectively;
b) SEQ ID NO: 24, 25, 26, 27, 28, and 29, respectively;
c) SEQ ID NO: 30, 31, 32, 33, 34, and 35, respectively;
d) SEQ ID NO: 36, 37, 38, 39, 40, and 41, respectively;
e) SEQ ID NO: 42, 43, 44, 45, 46, and 47, respectively;
f) SEQ ID NO: 48, 49, 50, 51, 52, and 53, respectively;
g) SEQ ID NO: 54, 55, 56, 57, 58, and 59, respectively; and
h) SEQ ID NO: 60, 61, 62, 63, 64, and 65, respectively; PD1 Anti-PD1 antibodies are disclosed in Tables 1-3 of PCT Publication WO2015112800A1 and include, but are not limited to, anti-PD1 antibodies having a VH/VL pair of the following SEQ ID NO. SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 1 14/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/202, 218/202, 226/202, 234/202, 242/202, 250/202, 258/202, 266/202, 274/202, 282/202, 290/202, 298/186, 306/186, and 314/186, U.S. PCT Publication No. WO2015112800A1 PD1 Anti-PD1 antibodies disclosed in U.S. Patent No. 10,294,299 B2 having the following SEQ ID NO pairs for the heavy and light chain variable domains:
SEQ ID NO:164/178
SEQ ID NO:165/179
SEQ ID NO:166/180
SEQ ID NO:167/181
SEQ ID NO:168/182
SEQ ID NO:169/183
SEQ ID NO:170/184
SEQ ID NO:171/185
SEQ ID NO:172/186
SEQ ID NO:173/187
SEQ ID NO:174/188
SEQ ID NO:175/189
SEQ ID NO:176/190
SEQ ID NO:177/190 PD1 MEDI-0680 (AMP-514) PD1 PDR001 (spartalizumab), a humanized IgG4 mAb having heavy and light chain sequences disclosed as BAP049-Clone-E, US Patent No.: 9683048 B2
HC: SEQ ID NO: 91, U.S. Patent No: 9,683,048
LC: SEQ ID NO: 72, US Patent No: 9,683,048 PD1 BGB-108 PD1 h409A11 described in WO2008/156712
HC: SEQ ID NO:31, PCT Publication WO2008/156712
LC: SEQ ID NO:36, PCT Publication WO2008/156712 PD1 h409A16 described in WO2008/156712
HC: SEQ ID NO:31, PCT Publication WO2008/156712
LC: SEQ ID NO:37, PCT Publication WO2008/156712 PD1 h409A17 described in WO2008/156712
HC: SEQ ID NO:31, PCT Publication WO2008/156712
LC: SEQ ID NO:38, PCT Publication WO2008/156712 PD1 An anti-PD1 antibody described in U.S. Patent No. 7,488,802, having the following SEQ ID NO pairs for the heavy and light chain variable domains:
SEQ ID NO:2/4
SEQ ID NO:6/8
SEQ ID NO:10/12
SEQ ID NO:14/16
SEQ ID NO:47/49 PD1 An anti-PD1 antibody described in U.S. Patent No. 7,521,051 having the following SEQ ID NO pairs for the heavy and light chain variable domains:
SEQ ID NO:2/4
SEQ ID NO:6/8
SEQ ID NO:10/12
SEQ ID NO:14/16
SEQ ID NO:47/49 PD1 An anti-PD1 antibody described in U.S. Patent No. 8,000,8449 having the following SEQ ID NO pairs for the heavy and light chain variable domains:
SEQ ID NO:1/8
SEQ ID NO:2/9
SEQ ID NO:3/10
SEQ ID NO:4/11
SEQ ID NO:5/12
SEQ ID NO:6/13
SEQ ID NO:7/14 PD1 An anti-PD1 antibody described in U.S. Patent No. 8,354,509, having the following SEQ ID NO pairs for the heavy and light chain full-length domains:
SEQ ID NO:31/36
SEQ ID NO:31/37
SEQ ID NO:31/38 PD1 An anti-PD1 antibody described in U.S. Patent No. 8,168,757, having the following SEQ ID NO pairs for the heavy and light chain variable domains:
SEQ ID NO:4/5
SEQ ID NO:12/13
SEQ ID NO:18/19
SEQ ID NO:40/41
SEQ ID NO:47/48
SEQ ID NO:26/27
SEQ ID NO:34/35
SEQ ID NO:55/56
SEQ ID NO:67/68 PD1 Anti-PD1 antibody described in PCT Publication No. WO2004/004771 PD1 An anti-PD1 antibody described in PCT Publication No. WO2004/056875, having the following SEQ ID NO pairs for the heavy and light chain variable domains:
SEQ ID NO:2/4
SEQ ID NO:6/8
SEQ ID NO:10/12
SEQ ID NO:14/16
SEQ ID NO:47/49 PD1 Anti-PD1 antibody described in PCT Publication No. WO2004/072286 PD1 VH: SEQ ID NO:25, 26, 27, 28 or 29, U.S. Publication No. US2011/0271358
VL: SEQ ID NO:30, 31, 32 or 33 US Patent No. US2011/0271358 PD1 SHR-1210 (camrelizumab) is described in PCT Publication No. WO 2015/085847 and has the following heavy and light chain variable domains:
HC: SEQ ID NO: 9
LC: SEQ ID NO: 10 PDL1 Durvalumab (MEDI4736)
HC: SEQ ID NO:26, PC Application No. WO2020225552
LC: SEQ ID NO:27, PCT Application No. WO2020225552 PDL1 Atezolizumab (Tecentriq, MPDL3280A, RG7446)
HC: SEQ ID NO:20, US Patent No. 8,217,149
LC: SEQ ID NO:21, US Patent No. 8,217,149 PDL1 MDX 1105(BMS-936559) PDL1 An anti-PD-L1 antibody described in U.S. Patent No. 7,943,743, having the following SEQ ID NO pairs for the heavy and light chain variable domains:
SEQ ID NO: 1/11
SEQ ID NO: 2/12
SEQ ID NO: 3/13
SEQ ID NO: 4/14
SEQ ID NO: 5/15
SEQ ID NO: 6/16
SEQ ID NO: 7/17
SEQ ID NO: 8/18
SEQ ID NO: 9/19
SEQ ID NO: 10/20 PDL1 Avelumab, U.S. Patent No. 9,624,298, has heavy and light chain variable domains as follows:
HC: SEQ ID NO: 24
LC: SEQ ID NO: 25 PDL1 ZKAB001 (Socazolimab) PDL1 TQB2450(APL-502 or CBT-502) PDL1 HLX20
CDR-H1, CDR-H2 and CDR-H3 sequences: SEQ ID NO: 52, 56, 77, PCT Publication No. 2018/080812
CDR-L1, CDR-L2 and CDR-L3 sequences: SEQ ID NO: 65, 42, 71, PCT Publication No. 2018/080812 PDL1 KN035 (envapolimab) is a nanobody described as Hu56V2 in U.S. Patent No. 11,225,522, having VHH. SEQ ID NO: 34 PDL1 LY3434172 PDL1 LY3300054 (rhodapolymab), PCT Publication No. WO 2017/034916, has the following heavy and light chain variable domains:
HC: SEQ ID NO: 10
L: SEQ ID NO: 11 PDL1 LDP (Resabelimab, ADG104), Chinese patent number 114225023, has the following heavy and light chain variable domains.
HC: SEQ ID NO: 10
LC: SEQ ID NO: 9 PDL1 EMB-09 PDL1 ABL501 PDL1 INBRX-105 PDL1 STI-3031 (IMC-001), U.S. Patent No. 10,118,963, has the following heavy and light chain variable domains:
HC: SEQ ID NO: 1
LC: SEQ ID NO: 2 PDL1 BGB-A333 (garibulimab), U.S. Patent No. 11,512,132, has the following heavy and light chain variable domains:
HC: SEQ ID NO: 22
LC: SEQ ID NO: 23 PDL1 HLX301 PDL1 Y101D PDL1 ES101 PDL1 IBI322 PDL1 VH: SEQ ID NO:46, 48, 50 or 52, U.S. Patent No. 11,168,144
VL: SEQ ID NO:58, 137 or 12, US Patent No. 11, 168,144 PDL1 VH: SEQ ID NO:23, 124, 126, 127, 128, 130, 140 or 145, U.S. Patent No. 11,208,486
VL: SEQ ID NO:24 or 125, U.S. Patent No. 11,208,486 Phosphatidylserine (Bavituximab) PSCA GEM3PSCA PSMA huJ591 PSMA Anti-PSMA antibodies having VH and VL sequences having an amino acid sequence of one of the following SEQ ID NO pairs: 2/1642; 10/1642; 18/1642; 26/1642; 34/1642; 42/1642; 50/1642; 58/1642; 66/1642; 74/1642; 82/1642; 90/1642; 98/1642; 106/1642; 1 14/1642; 122/130; and 138/146 in WO 2017/023761A1 PSMA Antibodies such as: PSMA 3.7, PSMA 3.8, PSMA 3.9, PSMA 3.11, PSMA 5.4, PSMA 7.1, PSMA 7.3, PSMA 10.3, PSMA 1.8.3, PSMA A3.1.3, PSMA A3.3.1, Abgenix described in WO2003034903A2 4.248.2, Abgenix 4.360.3, Abgenix 4.7.1, Abgenix 4.4.1, Abgenix 4.177.3, Abgenix 4.16.1, Abgenix 4.22.3, Abgenix 4.28.3, Abgenix 4.40.2, Abgenix 4.48.3, Abgenix 4.49.1, Abgenix 4.209.3, Abgemx 4.219.3, Abgenix 4.288.1, Abgenix 4.333.1, Abgemx 4.54.1, Abgenix 4.153.1, Abgenix 4.232.3, Abgenix 4.292.3, Abgenix 4.304.1, Abgenix 4.78.1 and Abgenix 4.152.1
The following hybrid cell lines: PSMA 3.8, PSMA 3.9 (PTA-3258), PSMA 3.11 (PTA-3269), PSMA 5.4 (PTA-3268), PSMA 7.1 (PTA-3292), PSMA 7.3 (PTA-3293), PSMA 10.3 (PTA-3247), PSMA 1.8.3 (PTA-3906), PSMA A3.1.3 (PTA-3904), PSMA A3.3.1 (PTA-3905), Abgenix 4.248.2 (PTA-4427), Abgenix 4.360.3 (PTA-4428), Abgenix 4.7.1 (PTA-4429), Abgenix as described in WO 2003/034903A2 4.4.1 (PTA-4556), Abgenix 4.177.3 (PTA-4557), Abgenix 4.16.1 (PTA-4357), Abgenix 4.22.3 (PTA-4358), Abgenix 4.28.3 (PTA-4359), Abgenix 4.40.2 (PTA-4360), Abgenix 4.48.3 (PTA-4361), Abgenix 4.49.1 (PTA-4362), Abgenix 4.209.3 (PTA-4365), Abgenix 4.219.3 (PTA-4366), Abgenix 4.288.1 (PTA-4367), Abgenix 4.333.1 (PTA-4368), Abgenix 4.54.1 (PTA-4363), Abgenix 4.153.1 (PTA-4388), Abgenix 4.232.3 (PTA-4389), Abgenix 4.292.3 (PTA-4390), Abgenix 4.304.1 (PTA-4391), Abgenix 4.78.1 (PTA-4652), and Abgemx 4.152.1(PTA-4653)
VH SEQ ID NO:2-7, WO 2003/034903A2
VL SEQ ID NO: 8-13, WO 2003/034903A2 PMSA VH: SEQ ID NO: 225, 239, 253, 267, 281, 295, 309, 323, 337, 351, 365, 379, 393, 407, 421, 435, 449, 463, 477, 491, 505, 519, 533, 547, 561, 575, 589, 603 or 617, WO 2011/121110A1
VL SEQ ID NO: 230, 244, 258, 272, 286, 300, 314, 328, 342, 356, 370, 384, 398, 412, 426, 440, 454, 468, 482, 496, 510, 524, 538, 552, 566, 580, 594, 608 or 622, WO 2011/121110A1
VH and VL SEQ ID NO: 235, 249, 263, 277, 291, 305, 319, 333, 347, 361, 375, 389, 403, 417, 431, 445, 459, 473, 487, 501, 515, 529, 543, 557, 571, 585, 599, 613 or 627, WO 2011/121110A1 PMSA Anti-PMSA antibody having a VL amino acid sequence corresponding to one of US 2022/0119525 A1 SEQ ID NO:229-312 and a VH corresponding to US 2022/0119525 A1 SEQ ID NO: 217 PMSA ES414 PMSA BAY2010112 (Pasotuxizumab) PMSA CCW702 PMSA JNJ-63898081 PMSA CC-1 PMSA Acapatamab PSMA HPN424 RAAG12 RAV12 RANKL Prolia™ (denosumab)
VH: SEQ ID NO:51, US Patent Publication 2017/0002060
VL: SEQ ID NO:52, US Patent Publication 2017/0002060 SLAMF7 Empliciti™ (elotuzumab) SSTR2 XmAb®18087 STEAP1 VHCDR1 SEQ ID NO: 14, 33, 182, 184 or 185, US20210179731A1
VHCDR2 SEQ ID NO: 15, 21, 34, 182, 184 or 185, US20210179731A1
VHCDR3 SEQ ID NO: 16 and 35, US20210179731A1
VH SEQ ID NO: 182 or 184, US20210179731A1
VLCDR1 SEQ ID NO: 11 or 30, US20210179731A1
VLCDR2 SEQ ID NO: 12 or 31, US20210179731A1
VLCDR3 SEQ ID NO: 13 or 32, US20210179731A1
VL SEQ ID NO: 183 or 186, US20210179731A1 STEAP1 AMG509 STEAP2 An anti-STEAP 2 antibody having CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 sequences selected from the following SEQ ID NOs: (1) 4-6-8-12-14-16; (2) 20-22-24-28-30-32; (3) 36-38-40-44-46-48; (4) 52-54-56-60-62-64; (5) 68-70-72-60-62-64; (6) 76-78-80-60-62-64; (7) 84-86-88-60-62-64; (8) 92-94-96-60-62-64; (9) 100-102-104-60-62-64; (10) 108-110-112-116-118-120; (11) 124-126-128-132-134-136; (12) 140-142-144-148-150-152; (13) 156-158-160-164-166-168; (14) 172-174-176-180-182-184; (15) 188-190-192-196-198-200; (16) 204-206-208-212-214-216; (17) 220-222-224-228-230-232; (18) 236-238-240-244-246-248; (19) 252-254-256-260-262-264; (20) 268-270-272-276-278-280; (21) 284-286-288-292-294-296; (22) 300-302-304-308-310-312; (23) 316-318-320-324-326-328; (24) 332-334-336-340-342-344; (25) 348-350-352-356-358-360; (26) 364-366-368-372-374-376; and (27) 380-382-384-388-390-392, U.S. Patent No. 10,772,972 B2

An anti-STEAP 2 antibody comprising: (a) a VH having an amino acid sequence corresponding to one of the following SEQ ID NOs: 2, 18, 34, 50, 66, 74, 82, 90, 98, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, and 378, U.S. Patent No. 10,772,972 B2; and (b) a VL having an amino acid sequence corresponding to one of the following SEQ ID NOs: 10; 26; 42; 58; 114; 130; 146; 162; 178; 194; 210; 226, 242; 258; 274; 290; 306; 322; 338; 354; 370; and 386, U.S. Patent No. 10,772,972 B2

An anti-STEAP 2 antibody having a VH/VL pair comprising an amino acid sequence corresponding to one of the following SEQ ID NO pairs of U.S. Patent No. 10,772,972 B2: 2/10; 18/26; 34/42; 50/58; 66/58; 74/58; 82/58; 90/58; 98/58; 106/114; 122/130; 138/146; 154/162; 170/178; 186/194; 202/210; 218/226; 234/242; 250/258; 266/274; 282/290; 298/306; 314/322; 330/338; 346/354; 362/370; and 378/386 Syndekan-1 (CD 138) B-B4 antibody, Wijdenes et al. (1996) Br. J. Haematol., 94: 318-323 Syndekan-4 Amino acid sequence of amino acids 93 and 121 of SEQ ID NO:1 or amino acid sequence of amino acids 92 and 122 of SEQ ID NO:2, European Patent Publication No. EP 2 603 236 TGFβ GC1008 TNFR Enbrel™ (etanercept) TNFα Remicade™ (infliximab)
VH: SEQ ID NO:2, International patent application WO201/3087911 A1
VH: SEQ ID NO:3, International patent application WO2013/ A1087911 TNFα Humira™ (adalimumab)
VH: SEQ ID NO:4, US Patent No. 6,258,562
VL: SEQ ID NO:3, US Patent No. 6,258,562 TNFα Cimzia™ (Certolizumab pegol)
VH: SEQ ID NO:14, U.S. Patent No. 7,012,135
VL: SEQ ID NO:9, US Patent No. 7,012,135 TNFα Simponi™ (golimumab)
VH: SEQ ID NO:7, U.S. Patent No. 7,250,165
VL: SEQ ID NO:8, US Patent No. 7,250,165 VEGF Avastin™ (Bevacizumab)
VH: SEQ ID NO:9, U.S. Patent No. 7,060,269
VL: SEQ ID NO:10, US Patent No. 7,060,269 VEGF Lucentis™ (ranibizumab)
VH: SEQ ID NO:4, U.S. Patent No. 9,914,770
VL: SEQ ID NO:2, US Patent No. 9,914,770 XCR1 Anti-XCR1 antibodies disclosed in U.S. Patent No. 9,371,389 B2, comprising:
- Antibodies designated 2H6, 5G7, 11H2, HK1L2 and HK5L5
- An antibody having the CDR-H1, CDR-H2 and CDR-H3 sequences of SEQ ID NO:53-55 and an antibody having the CDR-L1, CDR-L2 and CDR-L3 sequences of SEQ ID NO:56-58
- An antibody having the CDR-H1, CDR-H2 and CDR-H3 sequences of SEQ ID NO:41-43 and an antibody having the CDR-L1, CDR-L2 and CDR-L3 sequences of SEQ ID NO:44-46

In some embodiments, the targeting moiety competes for binding to the target molecule with an antibody set forth in Table 106. In other embodiments, the targeting moiety comprises a CDR having a CDR sequence of an antibody set forth in Table 106. In some embodiments, the targeting moiety comprises all six CDR sequences of an antibody set forth in Table 106. In other embodiments, the targeting moiety further comprises at least a heavy chain CDR sequence (CDR-H1, CDR-H2, CDR-H3) of an antibody set forth in Table 106 and a light chain CDR sequence of a universal light chain. In a further embodiment, the targeting moiety comprises a VH comprising an amino acid sequence of a VH of an antibody set forth in Table 106. In some embodiments, the targeting moiety further comprises a VL comprising an amino acid sequence of a VL of an antibody set forth in Table 106. In other embodiments, the targeting moiety further comprises a universal light chain VL sequence.

In some embodiments, the target molecule is PDL1. Table 107 below provides exemplary anti-PDL1 antibodies and/or antibody sequences that may serve as the basis for targeting moieties that may be incorporated into the targeting moiety for use in, for example, the interferon precursor proteins of the present disclosure.

Table 107
Exemplary PDL1 antibodies and/or binding sequences Target Antibody name and/or binding sequence PDL1 Durvalumab (MEDI4736)
HC: SEQ ID NO: 26, PCT Application No. WO2020225552
LC: SEQ ID NO: 27, PCT Application No. WO2020225552 PDL1 Atezolizumab (Tecentriq, MPDL3280A, RG7446)
HC: SEQ ID NO: 20, U.S. Patent No. 8,217,149
LC: SEQ ID NO: 21, U.S. Patent No. 8,217,149 PDL1 MDX 1105(BMS-936559) PDL1 An anti-PD-L1 antibody described in U.S. Patent No. 7,943,743, having the following SEQ ID NO pairs for the heavy and light chain variable domains:
SEQ ID NO: 1/11
SEQ ID NO: 2/12
SEQ ID NO: 3/13
SEQ ID NO: 4/14
SEQ ID NO: 5/15
SEQ ID NO: 6/16
SEQ ID NO: 7/17
SEQ ID NO: 8/18
SEQ ID NO: 9/19
SEQ ID NO: 10/20 PDL1 Avelumab, U.S. Patent No. 9,624,298, has heavy and light chain variable domains as follows:
HC: SEQ ID NO: 24
LC: SEQ ID NO: 25 PDL1 ZKAB001 (Socazolimab) PDL1 TQB2450(APL-502 or CBT-502) PDL1 HLX20
CDR-H1, CDR-H2 and CDR-H3 sequences, SEQ ID NO: 52, 56, 77, PCT Publication No. 2018/080812
CDR-L1, CDR-L2 and CDR-L3 sequences, SEQ ID NO: 65, 42, 71, PCT Publication No. 2018/080812 PDL1 KN035 (envapolimab) is a nanobody described as Hu56V2 in U.S. Patent No. 11,225,522, having VHH. SEQ ID NO: 34 PDL1 LY3434172 PDL1 LY3300054 (rhodapolymab), PCT Publication No. WO 2017/034916, has the following heavy and light chain variable domains:
HC: SEQ ID NO: 10
L: SEQ ID NO: 11 PDL1 LDP (Resabelimab, ADG104), Chinese patent number 114225023, has the following heavy and light chain variable domains.
HC: SEQ ID NO: 10
LC: SEQ ID NO: 9 PDL1 EMB-09 PDL1 ABL501 PDL1 INBRX-105 PDL1 STI-3031 (IMC-001), U.S. Patent No. 10,118,963, has the following heavy and light chain variable domains:
HC: SEQ ID NO: 1
LC: SEQ ID NO: 2 PDL1 BGB-A333 (garibulimab), U.S. Patent No. 11,512,132, has the following heavy and light chain variable domains:
HC: SEQ ID NO: 22
LC: SEQ ID NO: 23 PDL1 HLX301 PDL1 Y101D PDL1 ES101 PDL1 IBI322 PDL1 VH: SEQ ID NO:46, 48, 50 or 52, U.S. Patent No. 11,168,144
VL: SEQ ID NO:58, 137 or 12, US Patent No. 11, 168,144 PDL1 VH: SEQ ID NO:23, 124, 126, 127, 128, 130, 140 or 145, U.S. Patent No. 11,208,486
VL: SEQ ID NO:24 or 125, U.S. Patent No. 11,208,486

In some embodiments, the targeting moiety competes for binding to PD1 with an anti-PD1 antibody set forth in Table 107. In other embodiments, the targeting moiety comprises a CDR having a CDR sequence of an anti-PD1 antibody set forth in Table 107. In some embodiments, the targeting moiety comprises all six CDR sequences of an anti-PD1 antibody set forth in Table 107. In other embodiments, the targeting moiety comprises at least a heavy chain CDR sequence (CDR-H1, CDR-H2, CDR-H3) and a light chain CDR sequence of a universal light chain of an anti-PD1 antibody set forth in Table 107. In other embodiments, the targeting moiety comprises a VH having an amino acid sequence of a VH of an anti-PD1 antibody set forth in Table 107. In some embodiments, the targeting moiety further comprises a VL comprising an amino acid sequence of a VL of an anti-PD1 antibody set forth in Table 107. In another embodiment, the targeting moiety further comprises a universal light chain VL sequence.

In some embodiments, the target molecule is PD1. Table 108 below provides exemplary anti-PD1 antibodies and/or antibody sequences that may serve as the basis for targeting moieties that may be incorporated into the targeting moiety for use in, for example, the interferon proproteins of the present disclosure.

Table 108
Exemplary PD1 antibodies and/or binding sequences Target Antibody name and/or binding sequence PD1 MDX-1106/BMS-936558 (nivolumab) is a human IgG4 mAb having the structure described in WHO Drug Information, Vol. 27, No. 1, pp. 68-69 (2013), and the heavy and light chain sequences are disclosed in Figure 7 of US Publication No. 20190270812A1.
HC: SEQ ID NO: U.S. Publication Number US20190270812A1
LC: SEQ ID NO: 24, U.S. Publication No. US20190270812A1 PD1 MK-3475 (pembrolizumab) is a humanized IgG4 mAb having the structure described in WHO Drug Information, Vol. 27, No. 2, pp. 161-162 (2013), and the heavy and light chain sequences are disclosed in Figure 6 of U.S. Publication No. US20190270812A1.
HC: SEQ ID NO: 21, U.S. Publication No. US20190270812A1
LC: SEQ ID NO: 22, U.S. Publication No. US20190270812A1 PD1 REGN2810 (disclosed as H4H7798N in U.S. Publication No. 20150203579)
HC: SEQ ID NO: 330, U.S. Publication No. 20150203579
LC: SEQ ID NO: 331, U.S. Publication No. 20150203579 PD1 An anti-PD1 antibody having CDR H1-H3 and CDR L1-L3 sequences corresponding to the following SEQ ID NOs of U.S. Patent No. 11,034,765 B2:
a) SEQ ID NO: 18, 19, 20, 21, 22, and 23, respectively;
b) SEQ ID NO: 24, 25, 26, 27, 28, and 29, respectively;
c) SEQ ID NO: 30, 31, 32, 33, 34, and 35, respectively;
d) SEQ ID NO: 36, 37, 38, 39, 40, and 41, respectively;
e) SEQ ID NO: 42, 43, 44, 45, 46, and 47, respectively;
f) SEQ ID NO: 48, 49, 50, 51, 52, and 53, respectively;
g) SEQ ID NO: 54, 55, 56, 57, 58, and 59, respectively; and
h) SEQ ID NO: 60, 61, 62, 63, 64, and 65, respectively; PD1 Anti-PD1 antibodies are disclosed in Tables 1-3 of PCT Publication WO2015112800A1 and include, but are not limited to, anti-PD1 antibodies having a VH/VL pair of the following SEQ ID NO. SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 1 14/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/202, 218/202, 226/202, 234/202, 242/202, 250/202, 258/202, 266/202, 274/202, 282/202, 290/202, 298/186, 306/186, and 314/186, U.S. Pat. WO2015112800A1 PD1 Anti-PD1 antibodies disclosed in U.S. Patent No. 10,294,299 B2 having the following SEQ ID NO pairs for the heavy and light chain variable domains:
SEQ ID NO:164/178
SEQ ID NO:165/179
SEQ ID NO:166/180
SEQ ID NO:167/181
SEQ ID NO:168/182
SEQ ID NO:169/183
SEQ ID NO:170/184
SEQ ID NO:171/185
SEQ ID NO:172/186
SEQ ID NO:173/187
SEQ ID NO:174/188
SEQ ID NO:175/189
SEQ ID NO:176/190
SEQ ID NO:177/190 PD1 MEDI-0680 (AMP-514) PD1 PDR001 (spartalizumab), a humanized IgG4 mAb having heavy and light chain sequences disclosed as BAP049-Clone-E, US Patent No. 9683048 B2
HC: SEQ ID NO: 91, U.S. Patent No. 9,683,048
LC: SEQ ID NO: 72, U.S. Patent No. 9,683,048 PD1 BGB-108 PD1 h409A11 described in WO2008/156712
HC: SEQ ID NO: 31, PCT Publication WO2008/156712
LC: SEQ ID NO: 36, PCT Publication WO2008/156712 PD1 h409A16 described in WO2008/156712
HC: SEQ ID NO: 31, PCT Publication WO2008/156712
LC: SEQ ID NO: 37, PCT Publication WO2008/156712 PD1 h409A17 described in WO2008/156712
HC: SEQ ID NO: 31, PCT Publication WO2008/156712
LC: SEQ ID NO: 38, PCT Publication WO2008/156712 PD1 An anti-PD1 antibody described in U.S. Patent No. 7,488,802, having the following SEQ ID NO pairs for the heavy and light chain variable domains:
SEQ ID NO:2/4
SEQ ID NO:6/8
SEQ ID NO:10/12
SEQ ID NO:14/16
SEQ ID NO:47/49 PD1 An anti-PD1 antibody described in U.S. Patent No. 7,521,051.
It has the following SEQ ID NO pairs for the heavy and light chain variable domains.
SEQ ID NO:2/4
SEQ ID NO:6/8
SEQ ID NO:10/12
SEQ ID NO:14/16
SEQ ID NO:47/49 PD1 Anti-PD1 antibody described in U.S. Patent No. 8008449
It has the following SEQ ID NO pairs for the heavy and light chain variable domains.
SEQ ID NO:1/8
SEQ ID NO:2/9
SEQ ID NO:3/10
SEQ ID NO:4/11
SEQ ID NO:5/12
SEQ ID NO:6/13
SEQ ID NO:7/14 PD1 An anti-PD1 antibody described in U.S. Patent No. 8,354,509, having the following SEQ ID NO pairs for the heavy and light chain full-length domains:
SEQ ID NO:31/36
SEQ ID NO:31/37
SEQ ID NO:31/38 PD1 An anti-PD1 antibody described in U.S. Patent No. 8,168,757, having the following SEQ ID NO pairs for the heavy and light chain variable domains:
SEQ ID NO:4/5
SEQ ID NO:12/13
SEQ ID NO:18/19
SEQ ID NO:40/41
SEQ ID NO:47/48
SEQ ID NO:26/27
SEQ ID NO:34/35
SEQ ID NO:55/56
SEQ ID NO:67/68 PD1 Anti-PD1 antibodies described in PCT Publication No. WO2004/004771 PD1 An anti-PD1 antibody described in PCT Publication No. WO2004/056875, having the following SEQ ID NO pairs for the heavy and light chain variable domains:
SEQ ID NO:2/4
SEQ ID NO:6/8
SEQ ID NO:10/12
SEQ ID NO:14/16
SEQ ID NO:47/49 PD1 Anti-PD1 antibodies described in PCT Publication No. WO2004/072286 PD1 VH: SEQ ID NO: 25, 26, 27, 28, or 29, U.S. Publication No. US2011/0271358
VL: SEQ ID NO: 30, 31, 32, or 33, U.S. Publication No. US2011/0271358 PD1 SHR-1210 (camrelizumab) is described in PCT Publication No. WO 2015/085847 and has the following heavy and light chain variable domains:
HC: SEQ ID NO: 9
LC: SEQ ID NO: 10

In some embodiments, the targeting moiety competes for binding to PD1 with an anti-PD1 antibody set forth in Table 108. In other embodiments, the targeting moiety comprises a CDR having a CDR sequence of an anti-PD1 antibody set forth in Table 108. In some embodiments, the targeting moiety comprises all six CDR sequences of an anti-PD1 antibody set forth in Table 108. In other embodiments, the targeting moiety comprises at least a heavy chain CDR sequence (CDR-H1, CDR-H2, CDR-H3) and a light chain CDR sequence of a universal light chain of an anti-PD1 antibody set forth in Table 108. In other embodiments, the targeting moiety comprises a VH having an amino acid sequence of a VH of an anti-PD1 antibody set forth in Table 108. In some embodiments, the targeting moiety further comprises a VL comprising an amino acid sequence of a VL of an anti-PD1 antibody set forth in Table 108. In another embodiment, the targeting moiety further comprises a universal light chain VL sequence.

Where the target molecule is a checkpoint inhibitor, in some embodiments, the checkpoint inhibitor targeting moiety does not block or does not block ligand-receptor binding very well. Examples of non-blocking or low blocking anti-PD1 antibodies include antibodies comprising SEQ ID NO: 2/10 of PCT Publication WO2015/112800A1, SEQ ID NO: 16/17 of U.S. Patent No. 11,034,765 B2, and SEQ ID NO: 164/178, 165/179, 166/180, 167/181, 168/182, 169/183, 170/184, 171/185, 172/186, 173/187, 174/188, 175/189, 176/190, 177/190 of U.S. Patent No. 10,294,299 B2. Examples of non-blocking or low-blocking anti-LAG3 antibodies include antibodies having the VH/VL amino acid sequences of SEQ ID NO: 23/24, 3/4, and 11/12 of U.S. Publication No. US2022/0056126A1.

Additional target molecules that can be targeted by IFN precursor proteins are disclosed in Table 1 below and in particular in Hafeez et al ., 2020, Molecules 25:4764, doi:10.3390/molecules25204764. Table 1 by Hafeez et al . is incorporated herein by reference in its entirety.

6.7. Target Moiety Format

In certain embodiments, the targeting moiety of the IFN precursor protein of the present disclosure can be any type of antibody or fragment thereof that retains specific binding to an antigenic determinant. In one embodiment, the targeting moiety is an immunoglobulin molecule or fragment thereof, particularly an IgG class immunoglobulin molecule, more particularly an IgG ₁ or IgG ₄ immunoglobulin molecule. Antibody fragments include, but are not limited to, VH (or V _H ) fragments, VL (or V _L ) fragments, Fab fragments, F(ab') ₂ fragments, scFv fragments, Fv fragments, minibodies, diabodies, triabodies, and tetrabodies.

6.7.1. Fab

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

A Fab domain typically comprises a CH1 domain attached to a VH domain, which pairs with a CL domain attached to a VL domain. In a 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. A disulfide bond between the two constant domains can further stabilize the Fab domain. When the targeting moiety is a Fab, for example, as shown in Figures 1B and 1C, the CH1 of the Fab can represent an unfavorable domain N-terminal to the IFN moiety.

For the IFN precursor proteins of the present disclosure, particularly when the light chain of the targeting moiety is not a common or universal light chain, it is advantageous to use a Fab dimerization strategy to allow correct binding of Fab domains belonging to the same targeting moiety and to minimize aberrant pairing of Fab domains belonging to different targeting moieties. For example, the Fab heterodimerization strategy shown in Table 109 below can be used:

Table 109
Fab heterodimerization strategy Strategy VH CH1 VL CL reference CrossMabCH1-CL WT CL domain WT CH1 domain Schaefer et al. , 2011, Cancer Cell 2011; 20:472-86; PMID:22014573. Orthogonal Fab VHVRD1CH1CRD2 - VLVRD1CλCRD2 39K, 62E H172A, F174G 1R, 38D, (36F) L135Y, S176W Lewis et al. , 2014, Nat Biotechnol 32:191-8 Orthogonal Fab VHVRD2CH1wt - VLVRD2Cλwt 39Y WT 38R WT Lewis et al. , 2014, Nat Biotechnol 32:191-8 TCR CαCβ 39K TCR Cα 38D TCR Cβ Wu et al ., 2015, Mabs 7:364-76 CR3 WT T192E WT N137K, S114A Golay et al ., 2016, J Immunol 196:3199-211 MUT4 WT L143Q, S188V WT V133T, S176V Golay et al ., 2016, J Immunol 196:3199-211 DuetMab WT F126C WT S121C Mazor et al ., 2015, Mabs 7:377-89; Mazor et al ., 2015, Mabs 7:461-669. Exchanged domain WT CH3 + Knob or Hole Mutation WT CH3 + hole or knob mutation Wozniak-Knopp et al ., 2018, PLoSONE13(4):e0195442

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

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

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

In one embodiment, the modifications introduced into the VH and CH1 and/or VL and CL domains are complementary to each other. Complementarity at the heavy and light chain interface can be achieved based on steric and hydrophobic contacts, electrostatic/charge interactions, or a combination of various interactions. Complementarity between protein surfaces is broadly described in the literature in terms of lock and key fit, height and hole, protrusion and cavity, donor and acceptor , etc. , which all refer to the properties of structural and chemical match between two interacting surfaces.

In one embodiment, one or more of the introduced modifications introduce a new hydrogen bond at the interface of the Fab component. In one embodiment, one or more of the introduced modifications introduce a new salt bridge at the interface of the Fab component. Exemplary substitutions are described in WO 2014/150973 and WO 2014/082179, the contents of which are incorporated herein by reference.

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

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

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

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

Fab domains can also be modified by replacing the CH1 and CL domains with alternative domains that promote correct assembly. For example, the literature [Wu et al ., 2015, Mabs 7:364-76] describes 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 introducing a 38D modification in the VL domain and a 39K modification in the VH domain, which domain replacements result in additional charge-charge interactions and pairing between the VL and VH domains.

Instead of, or in addition to, using a Fab heterodimerization strategy to promote correct VH-VL pairing, the VL of a common light chain (also referred to as a universal light chain) can be used for each unique ABD of the IFN precursor proteins of the present disclosure. In various embodiments, utilizing a common light chain as described herein reduces the number of inappropriate species of the IFN precursor protein compared to utilizing the original cognate VL. In various embodiments, the VL domain of the ABD is identified from a monospecific antibody comprising the common light chain. In various embodiments, the VH region of the ABD of the IFN precursor protein comprises human heavy chain variable gene segments rearranged in vivo in a mouse B cell previously engineered to express a restricted human light chain repertoire or a single human light chain, wherein in response to exposure to an antigen of interest, the antibody repertoire comprises a plurality of human VHs that are recognized by one or two possible human VLs, wherein the antibody repertoire specific for the antigen of interest is characterized by the generation of a plurality of human VHs that are recognized by the human light chain. The common light chain is derived from a rearranged human Vκ1-39Jκ5 sequence or a rearranged human Vκ3-20Jκ1 sequence, including somatically mutated ( e.g. , affinity matured) versions. See, e.g., U.S. Patent No. 10,412,940.

6.7.2. scFv

Single-chain Fv or "scFv" antibody fragments comprise the VH and VL domains of an antibody in a single polypeptide chain, can be expressed as a single chain polypeptide, and retain the specificity of the intact antibody from which it was derived. Typically, scFv polypeptides additionally comprise a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for target binding. Examples of suitable linkers for connecting the VH and VL chains of a scFv are the non-cleavable linkers identified in Section 6.5.

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

The scFv may comprise VH and VL sequences derived from any suitable species, e.g., murine, human or humanized VH and VL sequences.

To generate scFv-encoding nucleic acids, the VH and VL-encoding DNA fragments are operably linked to another fragment encoding a linker, for example one of the linkers described in Section 6.5 (typically a sequence comprising repeats of the amino acids glycine and serine, e.g., the amino acid sequence (Gly4-Ser)3 (SEQ ID NO: 182). This allows the VH and VL sequences to be expressed as a contiguous single-chain protein in which the VL and VH regions are connected by a flexible linker (see, e.g. , Bird et al. , 1988, Science 242:423-426; Huston et al ., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al. , 1990, Nature 348:552-554).

6.8. Fc area

The IFN precursor protein of the present disclosure typically comprises a pair of Fc domains that associate to form an Fc region. In a natural antibody, the Fc region comprises a hinge region at the N-terminus to form a constant domain. Throughout this disclosure, references to an Fc domain encompass an Fc domain having a hinge domain at the N-terminus, unless otherwise specified.

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

The Fc domain can be derived from any suitable antibody class, 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 lgG4. Exemplary sequences of Fc domains derived from IgG1, IgG2, IgG3, and IgG4 are provided in Table 110 below.

Table 110
Fc sequence Fc order SEQ ID NO hIgG1 Fc
(Amino acids 99-330 of UniprotKB P01857-1) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 392 hIgG2 Fc
(Amino acids 99-326 of UniprotKB P01859-1) ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE KTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 393 hIgG3 Fc (amino acids 99-377 of UniprotKB P01860-1) ELKTPLGDTTHTCPRCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK 394 hIgG4 Fc (amino acids 99-327 of UniprotKB P01861-1) ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 395

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

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

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

In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to SEQ ID NO:413. When the Fc domain has greater than 90% sequence identity and less than 100% sequence identity to SEQ ID NO:413 ( e.g. , 90% to 99% sequence identity to SEQ ID NO:413), the Fc domain may also comprise one or more amino acid substitutions described herein, e.g., one or more substitutions that decrease effector function ( e.g. , as described in Section 6.8.1) and/or one or more substitutions that promote Fc heterodimerization ( e.g. , as described in Section 6.8.2).

The two Fc domains within the Fc region may be identical or different. In a native antibody, the Fc domains are usually identical, but for the purpose of producing multispecific binding molecules, such as the IFN proproteins of the present disclosure and MBMs produced by their activation, the Fc domains may advantageously be different to allow heterodimerization, as described in section 6.8.2 below.

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 those of IgE and IgM are composed of three heavy chain constant domains (CH2, CH3, and CH4). These dimerize to form the Fc region.

In the IFN precursor protein of the present disclosure, the Fc region and/or the Fc domain therein may comprise heavy chain constant domains from one or more different classes of antibodies, for example, one, two or three different classes.

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

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

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

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

In one embodiment, the Fc region comprises a CH4 domain from IgM. The IgM CH4 domain is typically located C-terminal to the CH3 domain.

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

It will be appreciated that the heavy chain constant domain for use in generating an Fc region for an IFN precursor protein of the present disclosure may comprise a variant of a naturally occurring constant domain as described above. Such a variant may comprise one or more amino acid mutations as compared to a wild-type constant domain. In one embodiment, the Fc region of the present disclosure comprises at least one constant domain that differs in sequence from a wild-type constant domain. It will be appreciated that the variant constant domain may be longer or shorter than the wild-type constant domain. Preferably, the variant constant domain is at least 60% identical or similar to the wild-type constant domain. In another example, the variant constant domain is at least 70% identical or similar. In another example, the variant constant domain is at least 80% identical or similar. In another example, the variant constant domain is at least 90% identical or similar. In another example, the variant constant domain is at least 95% identical or similar.

IgM and IgA occur in humans as covalent multimers of common H2L2 antibody units. IgM occurs as a pentamer when incorporated into the J chain, or as a hexamer when the J chain is absent. IgA occurs in monomeric and dimeric forms. The heavy chains of IgM and IgA have an 18 amino acid extension with a C-terminal constant domain known as the tailpiece. The tailpiece contains cysteine residues that form disulfide bonds between the heavy chains of the polymer and are believed to play an important role in polymerization. The tailpiece also contains a glycosylation site. In certain embodiments, the IFN precursor protein of the present disclosure does not comprise a tailpiece.

The Fc domain incorporated into the IFN precursor protein of the present disclosure can comprise one or more modifications that alter a functional property of the protein, e.g., binding to an Fc receptor, e.g., FcRn or a leukocyte receptor, binding to complement, a modified disulfide bond structure, or an altered glycosylation pattern. Exemplary Fc modifications that alter effector function are described in Section 6.8.1.

The Fc domain may also be modified to include modifications that improve the manufacturability of asymmetric IFN precursor proteins, for example by allowing heterodimerization to preferentially form pairs of non-identical Fc domains over identical Fc domains. Heterodimerization allows for the production of IFN precursor proteins in which different polypeptide components are linked to each other by Fc regions comprising Fc domains of different sequences. Examples of heterodimerization strategies are exemplified in Section 6.8.2.

It will be appreciated that any of the above-mentioned modifications may be combined in any suitable manner to achieve the desired functional properties and/or may be combined with other modifications to alter the properties of the IFN precursor protein.

6.8.1. Fc domain with altered effector function

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

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

In one embodiment, the Fc domain ( e.g. , an Fc domain of an IFN proprotein half antibody) or the Fc region ( e.g. , one or both Fc domains of an IFN proprotein capable of combining to form an Fc region) comprises an amino acid substitution at a position selected from the group E233, L234, L235, N297, P331 and P329 (numbering according to the Kabat EU index). In a more specific embodiment, the Fc domain or Fc region comprises an amino acid substitution at a position selected from the group L234, L235 and P329 (numbering according to the Kabat EU index). In some embodiments, the Fc domain or Fc region comprises the amino acid substitutions L234A and L235A (numbering according to the Kabat EU index). In one such embodiment, the Fc domain or region is an Igd Fc domain or region, particularly a human Igd Fc domain or region. In one embodiment, the Fc domain or Fc region comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, in particular P329G (numbering according to the Kabat EU index). In one embodiment, the Fc domain or Fc region comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numbering according to the Kabat EU index). In a more specific 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 an amino acid substitution at positions P329, L234 and L235 (numbering according to the Kabat EU index). In a more specific embodiment, the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”, “PGLALA” or “LALAPG”).

Typically, the same one or more amino acid substitutions are present in each of the two Fc domains of the Fc region. Thus, in a particular embodiment, each Fc domain of the Fc region comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering). 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) (numbering according to the Kabat EU index).

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 mutant IgG1 having reduced effector function, including the D265A, N297A mutations (EU numbers).

In another embodiment, the Fc domain is an IgG4 Fc domain having reduced binding to an Fc receptor. An exemplary IgG4 Fc domain having reduced binding to an Fc receptor can comprise an amino acid sequence selected from Table 111 below. In some embodiments, the Fc domain comprises only the bold portion of the sequence shown below.

Table 111 Fc domain order SEQ ID NO: SEQ ID NO:1, WO2014/121087 Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 369 SEQ ID NO:2, WO2014/121087 Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 SEQ ID NO:30, WO2014/121087 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 371 SEQ ID NO:31, WO2014/121087 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 372 SEQ ID NO:37 of WO2014/121087 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 373 SEQ ID NO:38 of WO2014/121087 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 374

In certain embodiments, an IgG4 with reduced effector function comprises the bold portion of SEQ ID NO:31 of WO2014/121087 (sometimes referred to herein as IgG4s or hIgG4s) having the amino acid sequence ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 396).

For the heterodimeric Fc region, it is possible to incorporate a combination of the variant IgG4 Fc sequences described above, for example an Fc region comprising an Fc domain (or a bold portion thereof) comprising the amino acid sequence of SEQ ID NO:30 of WO2014/121087 and an Fc domain (or a bold portion thereof) comprising the amino acid sequence of SEQ ID NO:37 of WO2014/121087 or an Fc domain comprising an Fc domain (or a bold portion thereof) comprising the amino acid sequence of SEQ ID NO:31 of WO2014/121087 and an Fc domain (or a bold portion thereof) comprising the amino acid sequence of SEQ ID NO:38 of WO2014/121087.

6.8.2. Fc heterodimerization mutants

Certain IFN precursor proteins involve dimerization between two Fc domains that are operably linked at their non-identical N-terminal or C-terminal regions, unlike native immunoglobulins. Insufficient heterodimerization of the two Fc domains forming the Fc region can be an obstacle to increasing the yield of the desired heterodimeric molecule and presents a challenge for purification. A variety of approaches available to those skilled in the art can be used to enhance dimerization of the Fc domain that may be present in the IFN precursor protein of the present disclosure and are described, for example, in EP 1870459A1, U.S. Patent No. 5,582,996, U.S. Patent No. 5,731,168, U.S. Patent No. 5,910,573, U.S. Patent No. 5,932,448, U.S. Patent No. 6,833,441, U.S. Patent No. 7,183,076, U.S. Patent Application Publication No. 2006204493A1, and PCT Publication No. WO 2009/089004A1.

In some embodiments, the present disclosure provides an IFN precursor protein comprising an Fc heterodimer, i.e., an Fc region comprising heterologous, non-identical Fc domains. Typically, each Fc domain of the Fc heterodimer comprises a CH3 domain of an antibody. The CH3 domain is derived from the constant region of an antibody of any isotype, class or subtype, and preferably of the class IgG (IgG1, IgG2, IgG3 and IgG4) as described in the preceding sections.

Heterodimerization of two different heavy chains in the CH3 domain will produce the desired IFN proprotein, whereas homodimerization of the same heavy chain will reduce the yield of the desired IFN proprotein. Thus, in a preferred embodiment, a polypeptide that assembles to form an IFN proprotein of the present disclosure will comprise a CH3 domain with a modification that favors heterodimeric association relative to an unmodified Fc domain.

In a specific embodiment, the modification which promotes the formation of Fc heterodimers is a so-called "knob-into-hole" or "knob-in-hole" modification, which comprises a "knob" modification in one of the Fc domains and a "hole" modification in the other Fc domain. Knob-in-hole techniques are described 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 (the "knob") at the interface of a first polypeptide and a corresponding cavity (the "hole") at the interface of a second polypeptide, such that the protrusion can be positioned in the cavity so as to promote heterodimer formation and prevent homodimer formation. The protrusion is formed by replacing a small amino acid side chain at the interface of the first polypeptide with a larger side chain ( e.g. , tyrosine or tryptophan). By replacing the large amino acid side chain with a small amino acid side chain ( e.g. , alanine or threonine), a compensatory cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide.

Thus, 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 positioned 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 in which the protrusion in the CH3 domain of the first subunit can be positioned. Preferably, said 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, said 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 protrusions and cavities can be created by altering the nucleic acid encoding the polypeptide, such as by site-directed mutagenesis or peptide synthesis. An exemplary substitution is Y470T.

In such a specific embodiment, the threonine residue at position 366 in the first Fc domain is replaced with a tryptophan residue (T366W), the tyrosine residue at position 407 in the Fc domain 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) (numbering according to the Kabat EU index). In a further embodiment, additionally in the first Fc domain the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (in particular the serine residue at position 354 is replaced with a cysteine residue), and additionally in the second Fc domain the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C) (numbering according to the Kabat EU index). In a specific embodiment, the first Fc domain comprises the amino acid substitutions S354C and T366W, and the second Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to the Kabat EU index).

In some implementations, electrostatic steering ( e.g. , as described in Gunasekaran et al ., 2010, J Biol Chem 285(25)) can be used to promote binding of the first and second Fc domains of an Fc region.

Alternatively, or additionally, to the use of a modified Fc domain to promote heterodimerization, the Fc domain can be modified to enable a purification strategy that allows selection of Fc heterodimers. In one such embodiment, a polypeptide comprises a modified Fc domain that abolishes binding to protein A, thus enabling a purification method for producing heterodimeric proteins. See, e.g., U.S. Patent No. 8,586,713. Accordingly, an IFN proprotein comprises a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from each other by at least one amino acid, and wherein the at least one amino acid difference reduces binding of the IFN proprotein to protein A compared to a corresponding IFN proprotein without the amino acid difference. In one embodiment, the first CH3 domain binds protein A and the second CH3 domain comprises a mutation/modification that reduces or eliminates protein A binding (e.g., H95R modification (by IMGT exon numbering, H435R by EU numbering)). The second CH3 can further comprise a Y96F modification (by IMGT; Y436F by EU). Modifications of this class are referred to herein as “star” mutations.

In some embodiments, the Fc may comprise one or more mutations to promote heterodimerization ( e.g. , knob and hole mutations) as well as a star mutation to promote purification.

6.8.3. Hinge domain

The IFN precursor protein of the present disclosure can comprise an Fc domain comprising a hinge domain at its N-terminus. The hinge region can be a natural or modified hinge region. The hinge region is typically found at the N-terminus of the Fc region. Unless the context indicates otherwise, the term "hinge domain" refers to a naturally or non-naturally occurring hinge sequence which is a monomeric hinge domain in the context of a single or monomeric polypeptide chain and which can comprise two joined hinge sequences in separate polypeptide chains in the context of a dimeric polypeptide ( e.g. , a homodimeric or heterodimeric IFN precursor protein formed by the association of two Fc domains). Sometimes the two joined hinge sequences are referred to as a "hinge region." In certain embodiments of the IFN precursor protein, additional repeats of the hinge region can be incorporated into the polypeptide sequence.

A native hinge region is a hinge region that is normally found between the Fab and Fc domains in a naturally occurring antibody. A modified hinge region is any hinge that differs in length and/or composition from the native hinge region. Such hinges may include hinge regions derived from other species, such as human, mouse, rat, rabbit, shark, pig, hamster, camel, llama, or goat hinge regions. Other modified hinge regions may include a complete hinge region derived from a different class or subclass of antibody than the heavy chain Fc domain or Fc region. Alternatively, a modified hinge region may include a portion of a native hinge or repeat unit, wherein each unit of the repeat is derived from a native hinge region. In a further alternative, a native hinge region may be altered by changing one or more cysteines or other residues to neutral residues, such as serine or alanine, or by changing a suitably positioned residue to a cysteine residue. By such means, the number of cysteine residues in the hinge region can be increased or decreased. Other modified hinge regions can be entirely synthetic and designed to have desired properties, such as length, cysteine composition and availability.

A number of modified hinge regions have already been described, for example, in U.S. Patent No. 5,677,425, WO 99/15549, WO 2005/003170, WO 2005/003169, WO 2005/003170, WO 98/25971 and WO 2005/003171, which are incorporated herein by reference in their entirety.

In one embodiment, the IFN precursor protein of the present disclosure comprises an Fc region having one or two Fc domains having an intact hinge domain at the N-terminus.

In various implementations, positions 233-236 within the hinge region can be G, G, G and empty, G, G, empty and empty, G, empty and empty, empty and empty, or all empty and the positions can be numbered with EU numbers.

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

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

The hinge sequence incorporated into the IFN precursor protein of the present disclosure can be a full-length (“long”) or a truncated (“short”) sequence. An example of a full-length hinge sequence is ESKYGPPCPPCPAPPVA (SEQ ID NO: 377). An example of a truncated hinge sequence is ESKYGPPCPPC (SEQ ID NO: 378). ESKYGPPCPPC (SEQ ID NO: 378) is truncated by 6 amino acids compared to the full-length hinge sequence ESKYGPPCPAPPCA (SEQ ID NO: 379). In various embodiments, the truncated hinge can have a C-terminal deletion of 1, 2, 3, 4, 5, or 6 amino acids compared to the full-length hinge sequence, e.g. , a full-length hinge sequence disclosed herein. Without being bound by theory, it is believed that the truncated hinge sequence may impart enhanced steric constraints to the IFN moiety. In an IFN proprotein comprising two hinge domains in each half antibody ( e.g. , an IFN proprotein having the configuration depicted in Figures 1C, 2C, and 2F), both hinge domains can be full-length, truncated, or a combination thereof. Thus, the N-terminal hinge domain can be truncated, the C-terminal hinge domain can be truncated, or both the N-terminal and C-terminal hinge domains can be truncated.

6.8.3.1. Chimeric hinge sequence

The hinge domain may be a chimeric hinge domain.

For example, a chimeric hinge may comprise an “upper hinge” sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region, in combination with a “lower hinge” sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region.

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

6.8.3.2. Hinge sequence with reduced effector function

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

Position 236 is unoccupied in canonical human IgG2 but is occupied in other canonical human IgG isotypes. Positions 233-235 are occupied by residues other than G in all four human isotypes (as depicted in Figure 1 of WO2016161010A2).

The hinge mutation within positions 233 to 236 can be combined with position 228, which is occupied by P. Position 228 is naturally occupied by P in human IgG1 and IgG2, but is occupied by S in human IgG4 and by R in human IgG3. The S228P mutation in an IgG4 antibody is advantageous in stabilizing the IgG4 antibody and reducing the exchange of heavy chain light chain pairing between exogenous and endogenous antibodies. Preferably, positions 226-229 are occupied by C, P, P and C, respectively.

An exemplary hinge region has residues 226 to 236, sometimes referred to as the middle (or core) and lower hinge, which are occupied by variant hinge sequences designated GGG-(233-236), GG--(233-236), G---(233-236), and G-less(233-236). Optionally, the hinge domain amino acid sequence comprises CPPCPAPGGG-GPSVF (SEQ ID NO: 381) (previously published as SEQ ID NO:1 of WO2016161010A2), CPPCPAPGG--GPSVF (SEQ ID NO:382) (previously published as SEQ ID NO:2 of WO2016161010A2), CPPCPAPG---GPSVF (SEQ ID NO:383) (previously published as SEQ ID NO:3 of WO2016161010A2) or CPPCPAP----GPSVF (SEQ ID NO:384) (previously published as SEQ ID NO:4 of WO2016161010A2).

The modified hinge region described above can generally be incorporated into a heavy chain constant region comprising the CH2 and CH3 domains, and can have additional hinge segments flanking the designated region ( e.g. , an upper hinge). Such additional constant region segments are typically of the same isotype, preferably a human isotype, but can be hybrids of different isotypes. The isotype of such additional human constant region segments is preferably human IgG4, but can also be human IgG1, IgG2 or IgG3 or hybrids thereof having different isotypes in the domains. Exemplary sequences of human IgG1, IgG2 and IgG4 are shown in FIGS. 2 to 4 of WO2016161010A2.

In certain embodiments, the modified hinge sequence can be linked to an IgG4 CH2 region (e.g., by incorporating it into an IgG4 Fc domain, such as a human or murine Fc domain that can be further modified in the CH2 and/or CH3 domain to reduce effector function as described in Section 6.8.1).

6.9. Nucleic Acids and Host Cells

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

A single nucleic acid can encode an IFN precursor protein comprising a single polypeptide chain, an IFN precursor protein comprising two or more polypeptide chains, or a portion of an IFN precursor protein comprising two or more polypeptide chains (e.g., a single nucleic acid can encode two polypeptide chains of an IFN precursor protein comprising three, four or more polypeptide chains, or three polypeptide chains of an IFN precursor protein comprising four or more polypeptide chains). To control expression separately, the open reading frames encoding the two or more polypeptide chains can be under the control of separate transcriptional regulatory elements ( e.g. , promoters and/or enhancers). The open reading frames encoding the two or more polypeptides can be controlled by the same transcriptional regulatory element and separated by an internal ribosome entry site (IRES) sequence so that they are translated as separate polypeptides.

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

The nucleic acid of the present disclosure may be DNA or RNA ( e.g. , mRNA).

In another aspect, the present disclosure provides host cells and vectors comprising a nucleic acid of the present disclosure. The nucleic acid may be present in a single vector or in separate vectors, present in the same host cell or in separate host cells, as described in more detail herein below.

6.9.1. Vector

The present disclosure provides vectors comprising a nucleotide sequence encoding an IFN proprotein or a component of an IFN proprotein described herein, for example, one or two of the polypeptide chains of a half antibody of the IFN proprotein. The vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YAC).

A number of vector systems may be utilized. For example, one class of vectors utilizes DNA elements derived from animal viruses, such as, for example, poxvirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rouse sarcoma virus, MMTV or MOMLV), or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses, such as Semliki Forest virus, eastern equine encephalitis virus, and flaviviruses.

Additionally, cells that have stably integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. For example, the markers may provide protropism for a co-trophic host, resistance to fungicides ( e.g. , antibiotics), or resistance to heavy metals such as copper. The selectable marker gene may be directly linked to the DNA sequence to be expressed or introduced into the same cell via co-transformation. Additional elements may also be required for optimal synthesis of the mRNA. These elements may include transcription promoters, enhancers, and termination signals, as well as splice signals.

Once the expression vector or construct containing the DNA sequence is prepared for expression, the expression vector can be transfected or introduced into an appropriate host cell. A variety of techniques can be used to accomplish this, such as, for example, 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 varied or optimized based on the particular expression vector and mammalian host cell utilized, based on the present description.

6.9.2. Cells

The present disclosure also provides a host cell comprising a nucleic acid of the present disclosure.

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

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

The present disclosure also provides a host cell comprising a vector described herein.

The cell may be, but is not limited to, a eukaryotic cell, a bacterial cell, an insect cell, or a human cell. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells.

6.10. Pharmaceutical Compositions

The IFN precursor protein of the present disclosure may be in the form of a composition comprising the IFN precursor protein and one or more carriers, excipients, and/or diluents. The composition may be formulated for a particular use, such as veterinary use or human pharmaceutical use. The form of the composition ( e.g. , dry powder, liquid formulation , etc. ) and the excipients, diluents, and/or carriers used will vary depending on the intended use of the IFN precursor protein and the mode of administration for the therapeutic use.

For therapeutic use, the composition may be supplied as part of a sterile pharmaceutical composition comprising a pharmaceutically acceptable carrier. Such composition may be in any suitable form (depending on the desired method of administration to the patient). The pharmaceutical composition may be administered to the patient by a variety of routes, such as orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intratumorally, intrathecally, topically or locally. The most appropriate route of administration in a given case will depend on the particular IFN precursor protein, the subject, the nature and severity of the disease, and the physical condition of the subject. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

The pharmaceutical composition may conveniently be presented in unit dosage form containing a predetermined amount of the disclosed IFN precursor protein per dose. The amount of IFN precursor protein contained in a unit dosage will vary depending on the disease being treated as well as other factors well known to those skilled in the art. Such unit dosage may be in the form of a lyophilized powder containing an amount of IFN precursor protein suitable for a single administration, or in the form of a liquid. The dry powder unit dosage form may be packaged in a kit together with a syringe, a suitable amount of diluent, and/or other components useful for administration. The unit dosage in liquid form may conveniently be supplied in the form of a syringe prefilled with an amount of IFN precursor protein suitable for a single administration.

The pharmaceutical composition may also be supplied in bulk quantities containing IFN precursor protein in quantities suitable for multiple administrations.

Pharmaceutical compositions can be prepared for storage in lyophilized form or as aqueous solutions by mixing IFN precursor protein having the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (all referred to herein as "carriers"), such as buffers, stabilizers, preservatives, isotherms, nonionic detergents, antioxidants and other additives. See Remington's Pharmaceutical Sciences, 16th ed. (Osol, ed. 1980). Such additives should be nontoxic to recipients at the dosages and concentrations employed.

Buffers help maintain the pH in a range close to physiological conditions. They can be present in a wide range of concentrations, but will typically be present in a concentration range from about 2 mM to about 50 mM. Buffers suitable for use with the present disclosure include organic and inorganic acids and their salts, for example, citric acid buffers ( e.g. , monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, and the like ), succinate buffers ( e.g. , succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, and the like ), tartrate buffers ( e.g. , tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture , and the like ), fumarate buffers ( e.g. , fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture , and the like ), gluconate buffers ( e.g. , gluconic acid-sodium glycolate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium glycolate mixture, and the like ), Examples of buffers include oxalate buffers ( e.g. , oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc. ), lactate buffers ( e.g. , lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc. ) and acetate buffers ( e.g. , acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture , etc. ). Additionally, phosphate buffers, histidine buffers and trimethylamine salts such as Tris can be used.

Preservatives may be added to retard microbial growth and may be added in amounts ranging from about 0.2 % to 1 % (w/v). Preservatives suitable for use with the present disclosure include phenol, benzyl alcohol, metacresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides ( e.g. , the chloride, bromide, and iodide), hexamethonium chloride, and alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonic agents, sometimes known as "stabilizers," may be added to ensure isotonicity of the liquid compositions of the present disclosure and include polyhydric sugar alcohols, for example, trihydric or higher sugar alcohols such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a broad category of excipients whose functions can range from bulking agents to additives that help to dissolve the therapeutic agent or prevent denaturation or adhesion to the walls of the container. Typical stabilizers include polyhydric sugar alcohols (as listed above); amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, and the like ; organic sugars or sugar alcohols, such as cyclitols, such as inositol, lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinositol, galactitol, glycerol, and the like; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides ( e.g. , peptides of 10 residues or less); proteins, such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; monosaccharides, such as xylose, mannose, fructose, glucose; disaccharides, such as lactose, maltose, sucrose and trehalose; and trisaccharides, such as raffinose; and polysaccharides, such as dextran. The stabilizer can be present in an amount ranging from 0.5 to 10 wt % per weight of the IFN precursor protein.

A nonionic surfactant or detergent (also called a "wetting agent") may be added to aid in solubilizing the glycoprotein and protect the glycoprotein from aggregation due to agitation, allowing the formulation to be exposed to shear surface stresses without denaturing the protein. Suitable nonionic surfactants include polysorbates ( e.g. , 20, 80), polyoxamers ( e.g. , 184, 188), and pluronic polyols. The nonionic surfactant can be present in the range of about 0.05 mg/mL to about 1.0 mg/mL, for example, about 0.07 mg/mL to about 0.2 mg/mL.

Other excipients include thickeners ( e.g. , starch), chelating agents ( e.g. , EDTA), antioxidants ( e.g. , ascorbic acid, methionine, vitamin E), and surfactants.

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

For example, formulations of IFN precursor protein can be prepared by mixing IFN precursor protein with a physiologically acceptable carrier, excipient, or stabilizer in the form of a lyophilized powder, slurry, aqueous solution, lotion, or suspension ( see, e.g. , Hardman et al ., 2001, Goodman and Gilman, 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 Formulations: General Medications, Marcel Dekker, NY; Lieberman, et al. (eds.), 1990, Pharmaceutical Formulations: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.), 1990, Pharmaceutical Formulations: Dispersed Systems, Marcel Dekker, NY; Weiner and (See Kotkoskie, 2000, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

The effective dose for a particular subject may vary depending on factors such as the condition being treated, the subject's general health, the route and dose of administration, and the severity of adverse effects (see, e.g. , Maynard et al. (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).

The compositions of the present disclosure can be administered via one or more routes of administration using one or more of the various methods known in the art. As will be appreciated by those skilled in the art, the route and/or manner of administration will vary depending upon the desired result. Routes of administration of the IFN precursor protein include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other conventional routes of administration (e.g., injection or infusion). Conventional administration usually refers to modes of administration other than enteral and topical administration by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intrathecal, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Alternatively, the compositions of the present disclosure can be administered via non-conventional routes, such as topical, epidermal, or mucosal routes of administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical administration. In one embodiment, the IFN precursor protein is administered via injection. In another embodiment, the IFN precursor protein of the present disclosure is administered subcutaneously.

6.10.1. Pharmaceutical composition for delivering nucleic acid encoding IFN precursor protein

The IFN precursor protein (e.g., IFN receptor agonist) of the present disclosure can be delivered by any method useful for gene therapy, for example, as mRNA under the control of a suitable promoter or via a viral vector encoding the IFN precursor protein (e.g., IFN receptor agonist).

Representative viral vectors include recombinant adenovirus and adeno-associated virus vectors (rAAV). rAAV vectors are based on defective nonpathogenic parvovirus adeno-associated type 2 virus. Most of these vectors are derived from plasmids that contain only AAV inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery through integration into the genome of the transduced cell are key features of this vector system. IL27 transgene AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV8.2, AAV9 and AAV rh10 are AAV serotypes useful for delivering the transgene AAV2/8, AAV2/5 and AAV2/6.

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

Replication-deficient recombinant adenovirus vectors (Ad) can be produced at high titers and can readily infect a variety of cell types. Most adenovirus vectors are designed to have a transgene that replaces the Ad Ela, Elb, and/or E3 genes, and the replication-defective vector is then propagated in human 293 cells to supply the deleted gene function by transformation. Ad vectors can transduce many types of tissues in vivo, including non-dividing, differentiated cells found in the liver, kidney, and muscle. Conventional Ad vectors have a high carrying capacity.

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

Nucleic acid molecules (e.g., mRNA) or viruses may be formulated as the sole pharmacologically active ingredient in a pharmaceutical composition or may be combined with other active agents for the particular disorder being treated. Optionally, other drugs, pharmaceutical agents, carriers, adjuvants, diluents may be included in the compositions provided herein. For example, any one or more of wetting agents, emulsifying agents, and glidants, such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coating agents, sweeteners, flavoring and perfume agents, preservatives, antioxidants, chelating agents, and inert gases may also be present in the composition. Exemplary other agents and excipients that may be included in the composition include, for example, water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite; Oil soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

6.11. Treatment Indications and Method of Use

The present disclosure provides methods of using and applying the IFN precursor protein of the present disclosure.

The IFN precursor proteins (e.g., IFN receptor agonists) of the present disclosure can be used to stimulate immune responses in a variety of applications.

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

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

The present disclosure also provides a method for local delivery of an IFN proprotein, comprising administering to a subject an IFN proprotein or a pharmaceutical composition as described herein, wherein the IFN proprotein has one or more protease cleavable linkers, each linker comprising one or more substrates for one or more proteases expressed by a tissue to which the IFN proprotein is to be locally delivered. The term "local delivery" as used herein does not require local administration, but rather means that the active component of the IFN proprotein is activated at a site of interest by a protease activated at the intended site, and optionally targeted to the site of interest together with a targeting moiety that recognizes a target molecule expressed by the tissue.

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

Therefore, the above-described method enables IFN therapy with reduced off-target side effects by preferentially activating IFN precursor protein at the site where IFN therapy is intended.

In some embodiments of the methods described above, the IFN precursor protein also comprises one or more targeting moieties that recognize a target molecule expressed at the site for treatment ( e.g. , a tissue).

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

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

In some embodiments, administration is not local to the tissue. For example, if the target tissue is a cancer tissue, administration may be systemic or subcutaneous.

The IFN precursor proteins of the present disclosure can be used in the treatment of any proliferative disorder ( e.g. , cancer) that expresses the target molecule ( e.g. , tumor cells or tumor microenvironment, e.g., extracellular matrix or tumor lymphocytes). In certain embodiments, the cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, cholangiocarcinoma, bladder cancer, bone cancer, breast cancer, bronchial tumor, Burkitt lymphoma, primary carcinoma of unknown etiology, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasm, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, adhesive neuroblastoma, fibrous histiocytoma, Ewing's sarcoma, ocular cancer, germ cell tumor, gallbladder cancer, stomach cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hairy cell leukemia, hepatocellular carcinoma, Histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, renal cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cancer, liver cancer, lobular carcinoma in situ, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with NUT gene-associated occult primary midline carcinoma, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative neoplasms, nasal cavity and paranasal sinuses, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, Parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal tumor, stomach cancer, T-cell lymphoma, teratomas, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, or Wilms tumor.

Table 112 below presents exemplary indications for which IFN precursor proteins targeting specific target molecules may be used.

Table 112
Examples of target molecule indications Target Exemplary indications ADRB3 Ewing's sarcoma ALK NSCLC, ALCL, IMT, neuroblastoma B7H3 Melanoma, osteosarcoma, leukemia, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer BCMA Multiple myeloma, leukemia ( e.g. , acute lymphoblastic leukemia (“ALL”), acute myeloid leukemia (“AML”), chronic lymphocytic leukemia (“CLL”), chronic myeloid leukemia (“CML”), and hairy cell leukemia (“HCL”)); lymphoma ( e.g. , Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, including diffuse large B-cell lymphoma (“DLBCL”)) Cardherin 17 Gastric, pancreatic, and colorectal cancer CAIX Clear cell carcinoma, hypoxic solid tumor, head and neck squamous carcinoma CD123 Leukemia ( e.g. , ALL, CLL, AML, CML, HCL); Lymphoma ( e.g. , Hodgkin's lymphoma, non-Hodgkin's lymphoma, e.g. , DLBCL); Multiple myeloma. In a preferred embodiment, the indication is AML. CD171 Neuroblastoma, paraganglioma CD179a B cell malignancy CD19 Leukemia ( e.g. , ALL, CLL, AML, CML, HCL); Lymphoma ( e.g. , Hodgkin lymphoma, non-Hodgkin lymphoma, e.g. , DLBCL); Multiple myeloma. CD20 Leukemia ( e.g. , ALL, CLL, AML, CML, HCL); Lymphoma ( e.g. , Hodgkin lymphoma, non-Hodgkin lymphoma, e.g. , DLBCL); Multiple myeloma. CD22 Leukemia ( eg , ALL, CLL, AML, CML, HCL); Lymphoma ( eg , Hodgkin's lymphoma, non-Hodgkin's lymphoma, eg , DLBCL); Multiple myeloma; Lung cancer CD24 Ovarian cancer, breast cancer, prostate cancer, bladder cancer, kidney cancer, non-small cell carcinoma CD30 Anaplastic large cell lymphoma, embryonal carcinoma, Hodgkin's lymphoma CD32b B cell malignancies, gastric cancer, pancreatic cancer, esophageal cancer, glioblastoma, breast cancer, colorectal cancer CD33 Leukemia ( e.g. , ALL, CLL, AML, CML, HCL); Lymphoma ( e.g. , Hodgkin's lymphoma, non-Hodgkin's lymphoma, e.g. , DLBCL); Multiple myeloma. In a preferred embodiment, the indication is AML. CD38 Leukemia ( eg , ALL, CLL, AML, CML, HCL); Lymphoma ( eg , Hodgkin lymphoma, non-Hodgkin lymphoma, eg , DLBCL); Multiple myeloma CD44v6 Colon cancer, small cell carcinoma of the head and neck CD97 B cell malignancies, gastric cancer, pancreatic cancer, esophageal cancer, glioblastoma, breast cancer, colorectal cancer CEA (CEACAM5) Colorectal carcinoma, gastric carcinoma, pancreatic carcinoma, lung cancer, breast cancer, medullary thyroid carcinoma CLDN6 Ovarian cancer, breast cancer, lung cancer CLL-1 Leukemia ( e.g. , ALL, CLL, AML, CML, HCL); Lymphoma ( e.g. , Hodgkin's lymphoma, non-Hodgkin's lymphoma, e.g. , DLBCL); Multiple myeloma. In a preferred embodiment, the indication is AML. CS1 (SLAMF7) Multiple myeloma EGFR Squamous cell carcinoma of the lung, anal cancer, glioblastoma, epithelial tumor of the head and neck, colon cancer EGFRvIII Glioblastoma EPCAM Gastrointestinal cancer, colorectal cancer EphA2 Kaposi's sarcoma, glioblastoma, solid tumor, glioma Ephrin B2 Thyroid cancer, breast cancer, malignant melanoma ERBB2 (Her2/neu) Breast cancer, ovarian cancer, stomach cancer, lung adenocarcinoma, non-small cell lung cancer, uterine cancer, uterine serous endometrial carcinoma, salivary duct carcinoma FAP Pancreatic cancer, colorectal cancer, metastasis, epithelial cancer, soft tissue sarcoma FCRL5 Multiple myeloma FLT3 Leukemia ( e.g. , ALL, CLL, AML, CML, HCL), lymphoma ( e.g. , Hodgkin lymphoma, non-Hodgkin lymphoma, e.g. , DLBCL), multiple myeloma folate receptor alpha Ovarian cancer, breast cancer, kidney cancer, lung cancer, colorectal cancer, brain cancer folate receptor beta Ovarian cancer Fucosil GM1 AML, myeloma GD2 Malignant melanoma, neuroblastoma GD3 Melanoma GloboH Ovarian cancer, stomach cancer, prostate cancer, lung cancer, breast cancer, and pancreatic cancer gp100 Melanoma GPNMB Breast cancer, head and neck cancer GPR20 GIST GPR64 Ewing's sarcoma, prostate sarcoma, renal sarcoma and lung sarcoma GPRC5D Multiple myeloma HAVCR1 Kidney cancer HER2 HER-2 (+) adenocarcinoma of the gastroesophageal junction, HER-2 positive gastric adenocarcinoma, HER2 positive breast carcinoma HER3 Colon and stomach cancer HMWMAA Melanoma, glioblastoma, breast cancer IGF-I receptor Breast cancer, prostate cancer, lung cancer IL11Rα Papillary thyroid cancer, osteosarcoma, colorectal adenocarcinoma, lymphocytic leukemia IL13Rα2 Renal cell carcinoma, prostate cancer, glioma, head and neck cancer, astrocytoma KIT Myeloid leukemia, Kaposi sarcoma, erythroleukemia, gastrointestinal stromal tumor KLRG2 Breast cancer, lung cancer and ovarian cancer. LewisY Squamous cell lung carcinoma, lung adenocarcinoma, ovarian carcinoma, and colorectal adenocarcinoma LMP2 Prostate cancer, Hodgkin's lymphoma, nasopharyngeal carcinoma LRP6 Breast cancer LY6K Breast cancer, lung cancer, ovarian cancer, and cervical cancer LYPD8 Colorectal cancer and stomach cancer Mesothelin Mesothelioma, pancreatic cancer, ovarian cancer, stomach cancer, lung cancer, endometrial cancer MUC1 Breast cancer, ovarian cancer, lung cancer, stomach cancer, pancreatic cancer, and prostate cancer NCAM Melanoma, Wilms tumor, small cell lung cancer, neuroblastoma, myeloma, paraganglioma, pancreatic acinar cell carcinoma, myeloid leukemia NY-BR-1 Breast cancer o-Acetyl GD2 Neuroblastoma, melanoma OR51E2 Prostate cancer PANX3 Osteosarcoma PLAC1 Hepatocellular carcinoma Polysialic acid small cell lung cancer PDGFR-beta Myelomonocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, acute lymphoblastic leukemia PRSS21 Colon cancer, testicular cancer, ovarian cancer PSCA Prostate cancer, stomach cancer and bladder cancer PSMA Prostate cancer ROR1 Metastatic cancer, chronic lymphocytic leukemia, solid tumors of the lung, breast, ovary, colon, pancreas, and sarcoma SLC34A2 Bladder cancer SLC39A6 Breast cancer, esophageal cancer SLITRK6 Breast cancer, urothelial cancer, lung cancer SSEA-4 Breast cancer, cancer stem cells, epithelial ovarian cancer STEAP1 Prostate cancer STEAP2 Prostate cancer (including castration-resistant prostate cancer), bladder cancer, cervical cancer, lung cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, stomach cancer, uterine cancer, ovarian cancer, and preferably prostate cancer. TACSTD2 Carcinoma ( eg , non-small cell lung cancer) TAG72 Ovarian cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, stomach cancer TEM1/CD248 Colorectal cancer TEM7R Colorectal cancer Tn Colorectal cancer, breast cancer, cervical cancer, lung cancer, stomach cancer TSHR Thyroid cancer, multiple myeloma Tyrosinase Prostate cancer, melanoma UPK2 Bladder cancer VEGFR2 Ovarian and pancreatic cancer, renal cell carcinoma, colorectal cancer, medullary thyroid carcinoma

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

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

7. Numbered implementation examples

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

In the numbered embodiments that follow, the targeting moiety preferably binds to a mammalian targeting molecule, the IFN moiety is preferably derived from a mammalian IFN, the Fc domain is preferably derived from a mammalian antibody, and the subject is preferably a mammal. More preferably, the mammal is a human.

1. As a type I interferon (IFN) protein

(a) in the first polypeptide chain:

(i) a first immunoglobulin constant domain;

(ii) first linker;

(iii) a type I interferon (IFN) moiety;

(iv) a second linker; and

(v) first Fc domain;

(b) in the second polypeptide chain:

(i) a second immunoglobulin constant domain;

(ii) a third linker;

(iii) a type II interferon (IFN) moiety;

(iv) a fourth linker; and

(v) comprising a second Fc domain capable of combining with the first Fc domain to form an Fc region;

An IFN precursor protein, wherein at least two of the first linker, the second linker, the third linker and the fourth linker are protease cleavable linkers (PCLs), and optionally, the IFN moiety of the IFN precursor protein is sterically hindered from binding to an IFN receptor by the Fc domain.

2. In embodiment 1, an IFN precursor protein, wherein the first and second IFN moieties each comprise an amino acid sequence having at least about 90% sequence identity to (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ with at most a 15-amino acid truncation at the N-terminus and/or C-terminus.

3. In embodiment 1, the first and second IFN moieties each comprise an IFN precursor protein having an amino acid sequence having about 95% sequence identity to (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ having at most a 15-amino acid truncation at the N-terminus and/or C-terminus.

4. In embodiment 1, the first and second IFN moieties each comprise an IFN precursor protein having an amino acid sequence having about 98% sequence identity to (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ having at most a 15-amino acid truncation at the N-terminus and/or C-terminus.

5. An IFN precursor protein according to any one of embodiments 1 to 4, wherein the first and second IFN moieties each comprise an amino acid sequence having one or more attenuating mutations relative to mature human IFNα1 or IFNα2b.

6. An IFN precursor protein having one or more mutations selected from L26A, F27A, R33A, R33K, L30A, D35E, H57Y, E58N, Q61S, H57S, E58S, H57A, E58A, Q61A, Q90A, E96A, R120A, L135A, R144A, R144S, R144T, R144Y, R144I, R144L, A145D, A145H, A145K, A145M, A145V, A145Y, R149A, R149K, S152A, R162A and E165D, in any one of embodiments 1 to 5.

7. An IFN precursor protein comprising an amino acid substitution R33A according to any one of embodiments 1 to 6.

8. An IFN precursor protein comprising the amino acid substitution R33K according to any one of embodiments 1 to 6.

9. An IFN precursor protein comprising an amino acid substitution Q90A according to any one of embodiments 1 to 6.

10. An IFN precursor protein comprising the amino acid substitution E96A according to any one of embodiments 1 to 6.

11. An IFN precursor protein comprising an amino acid substitution R120A according to any one of embodiments 1 to 6.

12. An IFN precursor protein comprising the amino acid substitution A145M according to any one of embodiments 1 to 6.

13. An IFN precursor protein comprising an amino acid substitution R149A according to any one of embodiments 1 to 6.

14. An IFN precursor protein comprising an amino acid substitution R149K according to any one of embodiments 1 to 6.

15. An IFN precursor protein comprising the amino acid substitution S152A according to any one of embodiments 1 to 6.

16. An IFN precursor protein comprising amino acid substitutions R33A, H57Y, E58N and Q61S, according to any one of embodiments 1 to 6.

17. An IFN precursor protein comprising amino acid substitutions H57Y, E58N, Q61S and R144A, according to any one of embodiments 1 to 6.

18. An IFN precursor protein comprising amino acid substitutions Q90A and R120A according to any one of embodiments 1 to 6.

19. An IFN precursor protein comprising amino acid substitutions A145M and R149K, according to any one of embodiments 1 to 6.

20. An IFN precursor protein according to any one of embodiments 1 to 19, wherein the first linker, the second linker, the third linker and the fourth linker are protease-cleavable linkers (PCL).

21. An IFN precursor protein according to any one of embodiments 1 to 19, wherein the first linker and the third linker are protease cleavable linkers (PCLs), and optionally, the second linker and the fourth linker are non-cleavable linkers (NCLs).

22. An IFN precursor protein according to any one of embodiments 1 to 19, wherein the second linker and the fourth linker are protease cleavable linkers (PCL), and optionally, the first linker and the third linker are non-cleavable linkers (NCL).

23. An IFN precursor protein according to any one of embodiments 1 to 22, wherein the PCL comprises a substrate sequence cleavable by any of the proteases set forth in Table 101.

24. An IFN precursor protein according to any one of embodiments 1 to 23, wherein the PCL comprises one or more substrate sequences selected from the substrate sequences set forth in Table 102.

25. An IFN precursor protein according to any one of embodiments 1 to 24, wherein the PCL comprises at least one spacer sequence selected from the substrate sequences set forth in Table 103.

26. An IFN precursor protein according to any one of embodiments 1 to 25, wherein the PCL comprises any amino acid sequence of the PCL sequence set forth in Table 104, or a variant having up to 5 amino acid substitutions ( e.g. , a variant having 1 amino acid substitution, 2 amino acid substitutions, 3 amino acid substitutions, 4 amino acid substitutions or 5 amino acid substitutions).

27. An IFN precursor protein according to any one of embodiments 1 to 26, comprising two or four PCLs or consisting of the amino acid sequence ISSGLLSGRSDNH.

28. An IFN precursor protein according to any one of embodiments 1 to 26, comprising two or four PCLs comprising or consisting of the amino acid sequence GGGISSGLLSGRSDNHGGGISSGLLSGRSDNHGGS.

29. An IFN precursor protein according to any one of embodiments 1 to 26, comprising two or four PCLs comprising or consisting of the amino acid sequence GGSGGSIPVSLRSGGGISSGLLSGRSDNHGGSGGS.

30. An IFN precursor protein according to any one of embodiments 1 to 26, comprising two or four PCLs comprising or consisting of the amino acid sequence GGSGGSVPLSLYSGGGISSGLLSGRSDNHGGSGGS.

31. An IFN precursor protein comprising two or four PCLs comprising or consisting of the amino acid sequence GGSHPVGLLARGGGHPVGLLARGGGHPVGLLARGS, in any one of embodiments 1 to 26.

32. An IFN precursor protein according to any one of embodiments 1 to 26, comprising two or four PCLs comprising or consisting of the amino acid sequence GGSHPVGLLARGGGHPVGLLARGGSGRSAGGSGRSA.

33. An IFN precursor protein according to any one of embodiments 1 to 32, wherein the first and third linkers are the same and/or the third and fourth linkers are the same.

34. An IFN precursor protein according to embodiment 33, wherein the first, second, third and fourth linkers are identical.

35. An IFN precursor protein according to any one of embodiments 1 to 33, wherein (i) the first and third linkers are non-cleavable linkers or (ii) the second and fourth linkers are non-cleavable linkers.

36. An IFN precursor protein according to embodiment 35, wherein the non-cleavable linker comprises or consists of any NCL sequence set forth in Table 105.

37. An IFN precursor protein according to any one of embodiments 1 to 33, wherein the first Fc domain and/or the second Fc domain comprises a hinge domain.

38. An IFN precursor protein according to any one of embodiments 1 to 37, further comprising one or more targeting moieties that bind to one or more target molecules.

39. In embodiment 38, an IFN precursor protein comprising a first targeting moiety and a second targeting moiety

40. In embodiment 39, the IFN precursor protein, wherein the first targeting moiety and the second targeting moiety are antibodies or antigen-binding fragments thereof.

41. An IFN proprotein according to embodiment 40, wherein the first targeting moiety and the second targeting moiety are Fab.

42. An IFN proprotein according to embodiment 40 or embodiment 41, wherein the first targeting moiety and the second targeting moiety comprise a Fab domain.

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

44. In embodiment 43, the IFN precursor protein, wherein the first targeting moiety and the second targeting moiety each further comprise an Fc domain comprising a CH2 domain and a CH3 domain.

45. In embodiment 44, the Fc domain of the first targeting moiety and the Fc domain of the second targeting moiety are associated with each other, IFN precursor protein.

46. An IFN proprotein according to any one of embodiments 39 to 45, wherein the first and second targeting moieties are N-terminal to the first and third linkers, respectively.

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

48. In embodiment 40, the IFN precursor protein, wherein the first immunoglobulin constant domain is a CH3 domain.

49. In embodiment 40, the IFN precursor protein, wherein the first immunoglobulin constant domain is a CH1 domain.

50. An IFN precursor protein according to any one of embodiments 1 to 49, wherein the IFN precursor protein is configured as illustrated in FIG. 1a, FIG. 2a, or FIG. 2d.

51. In embodiment 50, an IFN precursor protein comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain and a fourth polypeptide chain:

(a) the first polypeptide chain:

(i) the first VH1 domain;

(ii) the first CH1 domain;

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

(iv) first linker;

(v) a type I interferon (IFN) moiety;

(vi) a second linker; and

(vii) first Fc domain;

(b) the second polypeptide chain:

(i) a second VH1 domain;

(ii) the second CH1 domain;

(iii) a fourth Fc domain comprising a hinge domain, a CH2 domain and a CH3 domain (the CH3 domain is a second immunoglobulin constant domain);

(iv) third linker;

(v) a type II interferon (IFN) moiety;

(vi) a fourth linker; and

(vii) comprising a third Fc domain;

(c) the third polypeptide chain:

(i) first VL domain;

(ii) comprising a first CL domain,

(d) the fourth polypeptide chain;

(i) a second VL domain; and

(ii) comprising a second CL domain,

An IFN precursor protein, wherein the first polypeptide chain is associated with a third polypeptide chain such that the first VH, CH1, VL and CL form a first targeting moiety, and the second polypeptide chain is associated with a fourth polypeptide chain such that the second VH, CH1, VL and CL form a second targeting moiety.

52. An IFN precursor protein according to embodiment 51, wherein the first, second, third and fourth linkers are protease-cleavable linkers (PCL).

53. An IFN precursor protein according to embodiment 51, wherein the first and third linkers are non-cleavable linkers (NCLs), and the second and fourth linkers are protease-cleavable linkers (PCLs).

54. An IFN precursor protein according to embodiment 51, wherein the first and third linkers are protease-cleavable linkers (PCLs) and the second and fourth linkers are non-cleavable linkers (NCLs).

55. An IFN precursor protein according to any one of embodiments 1 to 49, wherein the IFN precursor protein is configured as illustrated in FIG. 1b, FIG. 2b or FIG. 2e.

56. In implementation example 55,

(a) the first polypeptide chain:

(i) the first VH1 domain;

(ii) the first CH1 domain;

(iii) first linker;

(iv) a type I interferon (IFN) moiety;

(v) a second linker; and

(vi) first Fc domain;

(b) the second polypeptide chain:

(i) a second VH1 domain;

(ii) the second CH1 domain;

(iii) third linker;

(iv) a type II interferon (IFN) moiety;

(v) a fourth linker; and

(vi) a second Fc domain;

(c) the third polypeptide chain:

(i) a first VL domain; and

(ii) comprising a second CL domain, and

(d) the fourth polypeptide chain:

(i) the second VL domain;

(ii) comprising a second CL domain,

An IFN precursor protein, wherein the first polypeptide chain is associated with a third polypeptide chain such that the first VH, CH1, VL and CL form a first targeting moiety, and the second polypeptide chain is associated with a fourth polypeptide chain such that the second VH, CH1, VL and CL form a second targeting moiety.

57. In embodiment 56, the first, second, third and fourth linkers are protease-cleavable linkers (PCL), an IFN precursor protein.

58. An IFN precursor protein according to embodiment 56, wherein the first and third linkers are non-cleavable linkers (NCLs), and the second and fourth linkers are protease-cleavable linkers (PCLs).

59. An IFN precursor protein according to embodiment 56, wherein the first and third linkers are protease-cleavable linkers (PCLs) and the second and fourth linkers are non-cleavable linkers (NCLs).

60. An IFN precursor protein configured as illustrated in any one of embodiments 1 to 49, wherein the IFN precursor protein is configured as illustrated in FIG. 1c, FIG. 2c, or FIG. 2f.

61. In embodiment 60, an IFN precursor protein comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain and a fourth polypeptide chain:

(a) the first polypeptide chain:

(i) the first VH1 domain;

(ii) the first CH1 domain;

(iii) first hinge domain;

(iv) first linker;

(v) a type I interferon (IFN) moiety;

(vi) a second linker; and

(vii) first Fc domain;

(b) the second polypeptide chain:

(i) a second VH1 domain;

(ii) the second CH1 domain;

(iii) second hinge domain;

(iv) third linker;

(v) a type II interferon (IFN) moiety;

(vi) a fourth linker; and

(vii) a second Fc domain;

(c) the third polypeptide chain:

(i) first VL domain;

(ii) comprising a first CL domain, and

(d) the fourth polypeptide chain:

(i) a second VL domain; and

(ii) comprising a second CL domain,

An IFN precursor protein, wherein the first polypeptide chain is associated with a third polypeptide chain such that the first VH, CH1, VL and CL form a first targeting moiety, and the second polypeptide chain is associated with a fourth polypeptide chain such that the second VH, CH1, VL and CL form a second targeting moiety.

62. An IFN precursor protein according to embodiment 61, wherein the first, second, third and fourth linkers are protease-cleavable linkers (PCL).

63. An IFN precursor protein according to embodiment 61, wherein the first and third linkers are non-cleavable linkers (NCLs), and the second and fourth linkers are protease-cleavable linkers (PCLs).

64. An IFN precursor protein according to embodiment 61, wherein the first and third linkers are protease-cleavable linkers (PCLs) and the second and fourth linkers are non-cleavable linkers (NCLs).

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

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

67. An IFN proprotein according to any one of embodiments 38 to 66, wherein the first targeting moiety and/or the second targeting moiety comprises (a) (i) CDRs or (ii) VH and VL sequences of an antibody set forth in Table 106, or (b) competes for binding to the target molecule with an antibody set forth in Table 106.

68. An IFN precursor protein according to any one of embodiments 38 to 66, wherein the first targeting moiety and/or the second targeting moiety binds to an ECM antigen optionally selected from a syndecan, a heparanase, an integrin, an osteopontin, a link, a cadherin, a laminin, a laminin type EGF, a lectin, a fibronectin, a notch, a nectin ( e.g. , nectin-4), a tenascin, a collagen ( e.g. , collagen type X) and a matrixin.

69. In embodiment 68, the first targeting moiety and/or the second targeting moiety is an IFN precursor protein capable of binding to nectin ( e.g. , nectin 4).

70. In embodiment 68, the first targeting moiety and/or the second targeting moiety is an IFN precursor protein capable of binding to collagen ( e.g. , collagen X).

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

72. In embodiment 71, the antigen is a T cell co-stimulatory protein, IFN precursor protein.

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

74. In embodiment 73, the T cell co-stimulatory protein is B7-H3, an IFN precursor protein.

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

76. In embodiment 75, the checkpoint inhibitor is an IFN precursor protein which is CTLA-4, PD1, PDL1, PDL2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1 or CHK2.

77. In embodiment 76, the checkpoint inhibitor is PDL1, an IFN precursor protein.

78. In embodiment 76, the checkpoint inhibitor is PD1, an IFN precursor protein.

79. In embodiment 76, the checkpoint inhibitor is LAG3, an IFN precursor protein.

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

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

82. In embodiment 81, TAA is an IFN precursor protein, which is EGFR.

83. In embodiment 81, TAA is an IFN precursor protein that is HER2.

84. In embodiment 81, TAA is EPCAM, an IFN precursor protein.

85. In embodiment 81, TAA is an IFN precursor protein, which is CEACAM5.

86. In embodiment 81, TAA is CD20, an IFN precursor protein.

87. In any one of embodiments 38 to 68, the first targeting moiety and/or the second targeting moiety is an IFN precursor protein capable of binding to a dendritic cell (DC) antigen, wherein the IFN precursor protein is optionally selected from XCR1, Clec9a, CD1c, CD11c, CD14, PDL1, macrophage mannose receptor (CD206) and DEC-205.

88. In embodiment 87, the dendritic cell antigen is XCR1, an IFN precursor protein.

89. In embodiment 87, the dendritic cell antigen is Clec9a, an IFN precursor protein.

90. In embodiment 87, the dendritic cell antigen is DEC-205, an IFN precursor protein.

91. An IFN proprotein according to any one of embodiments 38 to 68, wherein the first targeting moiety and/or the second targeting moiety is capable of binding to a natural killer (NK) cell antigen.

92. In any one of embodiments 1 to 37, the IFN precursor protein further comprises one or more targeting moieties that bind to one or more target molecules.

93. An IFN precursor protein according to embodiment 92, comprising a first targeting moiety and a second targeting moiety, each comprising means for binding to a target molecule.

94. In embodiment 93, the IFN precursor protein, wherein the first targeting moiety and the second targeting moiety are antibodies or antigen-binding fragments thereof.

95. An IFN proprotein according to embodiment 94, wherein the first targeting moiety and the second targeting moiety are Fab.

96. In embodiment 94, the IFN proprotein wherein the first targeting moiety and the second targeting moiety comprise a Fab domain.

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

98. In embodiment 97, the IFN precursor protein, wherein the first targeting moiety and the second targeting moiety each further comprise an Fc domain comprising a CH2 domain and a CH3 domain.

99. In embodiment 98, the Fc domain of the first targeting moiety and the Fc domain of the second targeting moiety are associated with each other, IFN precursor protein.

100. An IFN proprotein according to any one of embodiments 92 to 99, wherein the first and second targeting moieties are N-terminal to the first and third linkers, respectively.

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

102. An IFN precursor protein according to embodiment 101, wherein the first immunoglobulin constant domain is a CH3 domain.

103. An IFN precursor protein according to embodiment 101, wherein the first immunoglobulin constant domain is a CH1 domain.

104. An IFN precursor protein according to any one of embodiments 92 to 103, wherein the IFN precursor protein is configured as illustrated in FIG. 1a, FIG. 2a or FIG. 2d.

105. In embodiment 104, an IFN precursor protein comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain and a fourth polypeptide chain:

(a) the first polypeptide chain:

(i) the first VH1 domain;

(ii) the first CH1 domain;

(iii) a third Fc domain comprising a hinge domain, a CH2 domain and a CH3 domain, the CH3 domain being a second immunoglobulin constant domain;

(iv) first linker;

(v) a type I interferon (IFN) moiety;

(vi) a second linker; and

(vii) first Fc domain;

(b) the second polypeptide chain:

(i) a second VH1 domain;

(ii) the second CH1 domain;

(iii) a fourth Fc domain comprising a hinge domain, a CH2 domain and a CH3 domain (the CH3 domain is a second immunoglobulin constant domain);

(iv) third linker;

(v) a type II interferon (IFN) moiety;

(vi) a fourth linker; and

(vii) comprising a third Fc domain;

(c) the third polypeptide chain:

(i) first VL domain;

(ii) comprising a first CL domain,

(d) the fourth polypeptide chain:

(i) a second VL domain; and

(ii) comprising a second CL domain,

An IFN precursor protein, wherein the first polypeptide chain is associated with a third polypeptide chain such that the first VH, CH1, VL and CL form a first targeting moiety, and the second polypeptide chain is associated with a fourth polypeptide chain such that the second VH, CH1, VL and CL form a second targeting moiety.

106. An IFN precursor protein according to embodiment 105, wherein the first, second, third and fourth linkers are protease-cleavable linkers (PCL).

107. An IFN precursor protein according to embodiment 105, wherein the first and third linkers are non-cleavable linkers (NCLs), and the second and fourth linkers are protease-cleavable linkers (PCLs).

108. An IFN precursor protein according to embodiment 105, wherein the first and third linkers are protease-cleavable linkers (PCLs) and the second and fourth linkers are non-cleavable linkers (NCLs).

109. An IFN precursor protein configured as illustrated in any one of embodiments 92 to 103, wherein the IFN precursor protein comprises a protein molecule as illustrated in FIG. 1b, FIG. 2b, or FIG. 2e.

110. In implementation example 109,

(a) the first polypeptide chain:

(i) the first VH1 domain;

(ii) the first CH1 domain;

(iii) first linker;

(iv) a type I interferon (IFN) moiety;

(v) a second linker; and

(vi) first Fc domain;

(b) the second polypeptide chain:

(i) a second VH1 domain;

(ii) the second CH1 domain;

(iii) third linker;

(iv) a type I interferon (IFN) moiety;

(v) a fourth linker; and

(vi) a second Fc domain;

(c) the third polypeptide chain:

(i) first VL domain;

(ii) comprising a second CL domain, and

(d) the fourth polypeptide chain:

(i) the second VL domain;

(ii) comprising a second CL domain,

An IFN precursor protein, wherein the first polypeptide chain is associated with a third polypeptide chain such that the first VH, CH1, VL and CL form a first targeting moiety, and the second polypeptide chain is associated with a fourth polypeptide chain such that the second VH, CH1, VL and CL form a second targeting moiety.

111. An IFN precursor protein according to embodiment 110, wherein the first, second, third and fourth linkers are protease-cleavable linkers (PCL).

112. An IFN precursor protein according to embodiment 110, wherein the first and third linkers are non-cleavable linkers (NCLs), and the second and fourth linkers are protease-cleavable linkers (PCLs).

113. An IFN precursor protein according to embodiment 110, wherein the first and third linkers are protease-cleavable linkers (PCL) and the second and fourth linkers are non-cleavable linkers (NCL).

114. An IFN precursor protein according to any one of embodiments 92 to 103, wherein the IFN precursor protein is configured as illustrated in FIG. 1c, FIG. 2c or FIG. 2f.

115. In embodiment 114, an IFN precursor protein comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain and a fourth polypeptide chain:

(a) the first polypeptide chain:

(i) the first VH1 domain;

(ii) the first CH1 domain;

(iii) first hinge domain;

(iv) first linker;

(v) a type I interferon (IFN) moiety;

(vi) a second linker; and

(vii) first Fc domain;

(b) the second polypeptide chain:

(i) a second VH1 domain;

(ii) the second CH1 domain;

(iii) second hinge domain;

(iv) third linker;

(v) a type II interferon (IFN) moiety;

(vi) a fourth linker; and

(vii) a second Fc domain;

(c) the third polypeptide chain:

(i) first VL domain;

(ii) comprising a first CL domain, and

(d) the fourth polypeptide chain:

(i) a second VL domain; and

(ii) comprising a second CL domain,

An IFN precursor protein, wherein the first polypeptide chain is associated with a third polypeptide chain such that the first VH, CH1, VL and CL form a first targeting moiety, and the second polypeptide chain is associated with a fourth polypeptide chain such that the second VH, CH1, VL and CL form a second targeting moiety.

116. An IFN precursor protein according to embodiment 115, wherein the first, second, third and fourth linkers are protease-cleavable linkers (PCL).

117. An IFN precursor protein according to embodiment 115, wherein the first and third linkers are non-cleavable linkers (NCLs), and the second and fourth linkers are protease-cleavable linkers (PCLs).

118. An IFN precursor protein according to embodiment 115, wherein the first and third linkers are protease-cleavable linkers (PCLs) and the second and fourth linkers are non-cleavable linkers (NCLs).

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

120. An IFN proprotein according to any one of embodiments 92 to 119, wherein the first targeting moiety and/or the second targeting moiety comprises means for binding to a target molecule identified in section 6.6.

121. In any one of embodiments 92 to 119, the first targeting moiety and/or the second targeting moiety of the IFN precursor protein can bind to an ECM antigen optionally selected from syndecan, heparanase, integrin, osteopontin, link, cadherin, laminin, laminin type EGF, lectin, fibronectin, notch, nectin ( e.g. , nectin-4), tenascin, collagen ( e.g. , collagen type X) and matrixin.

122. An IFN precursor protein according to embodiment 121, wherein the first targeting moiety and/or the second targeting moiety comprises means for binding to a nectin ( e.g. , nectin 4).

123. An IFN precursor protein according to embodiment 121, wherein the first targeting moiety and/or the second targeting moiety comprises means for binding to collagen ( e.g. , collagen X).

124. In any one of embodiments 92 to 119, the first targeting portion and/or the second targeting portion of the IFN precursor protein can bind to a cell surface molecule of a tumor or viral lymphocyte.

125. In embodiment 124, the cell surface molecule is a T cell co-stimulatory protein, IFN precursor protein.

126. In embodiment 125, the IFN precursor protein is CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C or B7-H3.

127. In embodiment 126, the T cell co-stimulatory protein is B7-H3, an IFN precursor protein.

128. In any one of embodiments 92 to 119, the first targeting portion and/or the second targeting portion of the IFN precursor protein can bind to an immune checkpoint inhibitor.

129. In embodiment 128, the immune checkpoint inhibitor of the IFN precursor protein is CTLA-4, PD1, PDL1, PDL2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, or CHK2.

130. In embodiment 129, the checkpoint inhibitor is PDL1, an IFN precursor protein.

131. In embodiment 129, the checkpoint inhibitor is PD1, an IFN precursor protein.

132. In embodiment 129, the checkpoint inhibitor is LAG3, an IFN precursor protein.

133. In any one of embodiments 92 to 119, the first targeting portion and/or the second targeting portion of the IFN precursor protein can bind to a tumor-associated antigen (TAA).

134. In embodiment 133, the TAA is selected from the group consisting of AFP, ALK, BAGE protein, BIRC5 (survivin), BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CEACAM5 (also known as carcinoembryonic antigen or CEA), CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE protein ( e.g. , GAGE-1 or -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT, LMP2, MAGE proteins ( e.g. MAGE-1, -2, -3, -4, -6 and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-βTMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase or uroplakin-3, IFN precursor protein.

135. In embodiment 134, TAA is an IFN precursor protein, which is EGFR.

136. In embodiment 134, TAA is an IFN precursor protein that is HER2.

137. In embodiment 134, TAA is EPCAM, an IFN precursor protein.

138. In embodiment 134, TAA is an IFN precursor protein, which is CEACAM5.

139. In embodiment 134, TAA is CD20, an IFN precursor protein.

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

141. In embodiment 140, the dendritic cell antigen is XCR1, an IFN precursor protein.

142. In embodiment 140, the dendritic cell antigen is Clec9a, an IFN precursor protein.

143. In embodiment 140, the dendritic cell antigen is DEC-205, an IFN precursor protein.

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

145. In any one of embodiments 1 to 144, the Fc region of the IFN precursor protein is a homodimer.

146. In any one of embodiments 1 to 144, the Fc region of the IFN precursor protein is a heterodimer.

147. A nucleic acid or a plurality of nucleic acids encoding the IL2 precursor protein of any one of embodiments 1 to 146.

148. A host cell engineered to express the IFN precursor protein of any one of embodiments 1 to 146 or the nucleic acid(s) of embodiment 147.

149. A method for producing an IFN precursor protein of any one of embodiments 1 to 146, comprising the step of culturing a host cell of embodiment 148 and recovering the IFN precursor protein expressed thereby.

150. A pharmaceutical composition comprising an IFN precursor protein and an excipient according to any one of embodiments 1 to 146.

151. A method of treating cancer, comprising administering to a subject in need thereof an IFN precursor protein of any one of embodiments 1 to 146 or a pharmaceutical composition of embodiment 150.

152. A method according to embodiment 151, wherein the IFN precursor protein comprises at least one targeting moiety capable of binding to a target molecule.

153. A method according to embodiment 151, wherein the IFN precursor protein comprises at least one means for binding to a target molecule.

154. A method according to any one of embodiments 150 to 153, wherein the cancer is associated with expression of a target molecule, e.g. , a TAA as set forth in Table 112, and an associated cancer.

155. A method according to any one of embodiments 150 to 154, wherein one or more protease-cleavable linkers in an IFN precursor protein are cleaved by one or more proteases within the cancer tissue to produce an activated IFN protein comprising an IFN moiety.

156. A method according to embodiment 155, wherein the IFN protein is selectively activated in cancer tissue.

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

158. A method according to embodiment 157, wherein the IFN precursor protein comprises one or more targeting moieties that recognize a target molecule expressed by the tissue.

159. A method according to embodiment 158, wherein the IFN precursor protein comprises two targeting moieties, each of which recognizes a target molecule expressed by the tissue.

160. A method according to embodiment 157, wherein the IFN precursor protein comprises one or more means for binding to a target molecule expressed by the tissue.

161. A method according to embodiment 160, wherein the IFN precursor protein comprises two means for binding to a target molecule expressed by the tissue.

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

163. In embodiment 162, the target molecule expressed by the tissue is an extracellular matrix (ECM) antigen, a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or viral lymphocyte, a T cell antigen (TCA), a checkpoint inhibitor, a tumor-associated antigen (TAA), a dendritic cell (DC) or other antigen-presenting cell (APC) antigen, or a natural killer (NK) cell antigen.

164. A method according to any one of embodiments 157 to 163, wherein one or more protease-cleavable linkers in an IFN precursor protein are cleaved by one or more proteases within a target tissue to generate an activated IFN protein comprising an IFN moiety.

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

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

167. A method according to embodiment 166, wherein the IFN precursor protein comprises two targeting moieties each recognizing a target molecule expressed by the cancer tissue.

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

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

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

171. A method according to any one of embodiments 165 to 170, wherein one or more protease-cleavable linkers in an IFN precursor protein are cleaved by one or more proteases within the cancer tissue to produce an activated IFN protein comprising an IFN moiety.

172. A method of administering to a subject an IFN therapy having reduced systemic exposure and/or reduced systemic toxicity, comprising administering to the subject an IFN therapy in the form of an IFN proprotein according to any one of embodiments 1 to 146 having one or more protease-cleavable linkers (or a pharmaceutical composition comprising the IL2 proprotein and an excipient), each comprising one or more substrates for one or more proteases expressed by a tissue in which the IFN therapy is desired and/or intended.

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

174. A method according to embodiment 173, wherein the IFN precursor protein comprises two targeting moieties, each of which recognizes a target molecule expressed by the tissue.

175. A method according to embodiment 172, wherein the IFN precursor protein comprises one or more means for binding to a target molecule expressed by the tissue.

176. A method according to embodiment 175, wherein the IFN precursor protein comprises two means for binding to a target molecule expressed by the tissue.

177. A method according to any one of embodiments 172 to 176, wherein the tissue is cancer tissue or associated immune cells.

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

179. A method according to any one of embodiments 172 to 178, wherein one or more protease-cleavable linkers in an IFN precursor protein are cleaved by one or more proteases within a target tissue to generate an activated IFN protein comprising an IFN moiety.

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

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

182. A method according to embodiment 181, wherein the IFN precursor protein comprises two targeting moieties each recognizing a target molecule expressed by the cancer tissue.

183. A method according to embodiment 180, wherein the IFN precursor protein comprises one or more means for binding to a target molecule expressed by the cancer tissue or associated immune cells.

184. A method according to embodiment 183, wherein the IFN precursor protein comprises two means for binding to a target molecule expressed by the cancer tissue or associated immune cells.

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

186. A method according to any one of embodiments 179 to 184, wherein one or more protease-cleavable linkers in an IFN precursor protein are cleaved by one or more proteases within the cancer tissue to produce an activated IFN protein comprising an IFN moiety.

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

(a) (i) one or more targeting moieties that recognize a target molecule expressed by a cancer tissue or associated immune cells, or (ii) a means for binding to a target molecule expressed by a cancer tissue or associated immune cells; and

(b) a method having one or more protease-cleavable linkers each comprising one or more substrates for one or more proteases expressed by a tissue in which IFN therapy is desired and/or intended.

188. A method according to embodiment 187, wherein the IFN precursor protein comprises (i) two targeting moieties each recognizing a target molecule expressed by the cancer tissue or associated immune cells, or (ii) two means for binding to a target molecule expressed by the cancer tissue or associated immune cells.

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

190. A method according to any one of embodiments 187 to 189, wherein one or more protease-cleavable linkers in an IFN precursor protein are cleaved by one or more proteases within the cancer tissue to produce an activated IFN protein comprising an IFN moiety.

191. A method of locally inducing an immune response in a target tissue, comprising administering to a subject an IFN precursor protein according to any one of embodiments 1 to 146 (or a pharmaceutical composition comprising the IFN precursor protein and an excipient) having at least one targeting moiety capable of binding a target molecule expressed in the target tissue and at least one protease-cleavable linker, each of which comprises at least one substrate for at least one protease expressed in the target tissue.

192. A method according to embodiment 191, wherein the IFN precursor protein comprises (i) two targeting moieties, each of which recognizes a target molecule expressed by a target tissue or an associated immune cell, or (ii) two means for binding to a target molecule expressed by a target tissue or an associated immune cell.

193. A method according to embodiment 191 or embodiment 192, wherein the target tissue is cancer tissue.

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

195. A method according to any one of embodiments 190 to 194, wherein one or more protease-cleavable linkers in an IFN precursor protein are cleaved by one or more proteases within a target tissue to produce an activated IFN protein comprising an IFN moiety.

196. A method according to embodiment 195, wherein the IFN precursor protein induces an immune response against at least one cell type in a target tissue.

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

198. In embodiment 197, the method wherein the administration of the immunogenic agent and the IFN precursor protein (e.g., the IFN receptor agonist) is performed simultaneously or separately, but may be performed simultaneously or sequentially.

199. A method according to embodiment 197 or embodiment 198 or embodiment 1, wherein the immunogenic agent is a vaccine, and optionally, the vaccine is a cancer vaccine or a vaccine against an infectious agent.

200. A method according to any one of embodiments 151 to 199, wherein the administration is non-local.

201. A method according to embodiment 200, wherein the administration is systemic.

8. Yes

8.1. Protein composition sequence

Table 3 below provides the sequences of the IFN precursor proteins and control constructs used in the studies described herein.

Table 3 Structure order Anti-PD1-PCL-IFN-PCL-Fc

Chain 1: anti-hPD1-VH-CH1-CH2-CH3-PCL-IFN-PCL-CH2-CH3

Chain 2: anti-hPD1-VH-CH1-CH2-CH3-PCL-IFN-PCL-CH2-CH3


Chain 3: Anti-hPD1 light chain

Chain 4: Anti-hPD1 light chain Chain 1: QVQLVQSGAEVKRPGSSVKVSCKVSGVTFRNFAIIWVRQAPGQGLEWMGGIIPFFSAANYAQSFQGRVTITPDESTSTAFMELASLRSEDTAVYYCAREGERGHTYGF DYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVD KRVESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGKISSGLLSGRSDNHCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLL DKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSSLSTNLQESLRSKEISSGLLSGRSDNHESKYGPPCPPC PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 385)

Chain 2: Qvqlvqsgaevkrpgssvkvsckvsgvtfrnfaiiwvrqapgqglewmggiipffsaanyaqsfqgrvtitpdeststafmelaslrsedtavyycaregerghtygfdywgqgtlvtvss
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKISSGLLSGRSDNHCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDR HDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEISSGLLSGRSDNHESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 385)


Chain 3: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 386)

Chain 4: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 386)
Anti-PD1-VH-CH1-PCL-IFN-PCL-Hinge-Fc

Chain 1: anti-PD1-VH-CH1-PCL-IFN-PCL-Hinge-CH2-CH3

Chain 1: anti-PD1-VH-CH1-PCL-IFN-PCL-Hinge-CH2-CH3


Chain 3: Anti-hPD1 light chain

Chain 4: Anti-hPD1 light chain


Chain 1: QVQLVQSGAEVKRPGSSVKVSCKVSGVTFRNFAIIWVRQAPGQGLEWMGGIIPFFSAANYAQSFQGRVTITPDESTSTAFMELASLRSEDTAVYYCAREGERGHTYGFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYISSGLLSGRSDNHCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDK FYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEISSGLLSGRSDNHGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 387)

Chain 2: QVQLVQSGAEVKRPGSSVKVSCKVSGVTFRNFAIIWVRQAPGQGLEWMGGIIPFFSAANYAQSFQGRVTITPDESTSTAFMELASLRSEDTAVYYCAREGERGHTYGFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYISSGLLSGRSDNHCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDK FYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEISSGLLSGRSDNHGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 387)

Chain 3: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 386)

Chain 4: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 386)






Chain 3: Anti-hPD1 light chain

Chain 4: Anti-hPD1 light chain


Chain 1: QVQLVQSGAEVKRPGSSVKVSCKVSGVTFRNFAIIWVRQAPGQGLEWMGGIIPFFSAANYAQSFQGRVTITPDESTSTAFMELASLRSEDTAVYYCAREGERGHTYGFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYISSGLLSGRSDNHTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLD KFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQISSGLLSGRSDNHGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 388)

Chain 2: QVQLVQSGAEVKRPGSSVKVSCKVSGVTFRNFAIIWVRQAPGQGLEWMGGIIPFFSAANYAQSFQGRVTITPDESTSTAFMELASLRSEDTAVYYCAREGERGHTYGFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYISSGLLSGRSDNHTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLD KFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQISSGLLSGRSDNHGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 388)

Chain 3: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 386)
Chain 4: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 386)






Chain 3: Anti-hPD1 light chain

Chain 4: Anti-hPD1 light chain
Chain 1: QVQLVQSGAEVKRPGSSVKVSCKVSGVTFRNFAIIWVRQAPGQGLEWMGGIIPFFSAANYAQSFQGRVTITPDESTSTAFMELASLRSEDTAVYYCAREGERGHTYGFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYISSGLLSGRSDNHCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDET LLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQISSGLLSGRSDNHGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 389)

Chain 2: QVQLVQSGAEVKRPGSSVKVSCKVSGVTFRNFAIIWVRQAPGQGLEWMGGIIPFFSAANYAQSFQGRVTITPDESTSTAFMELASLRSEDTAVYYCAREGERGHTYGFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYISSGLLSGRSDNHCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDET LLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQISSGLLSGRSDNHGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 389)

Chain 3: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 386)

Chain 4: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 386)
Anti-PD1-VH-CH1-LongHinge-PCL-IFN-PCL-LongHinge-Fc

Chain 1: anti-PD1-VH-CH1-LongHinge-PCL-IFN-PCL-LongHinge-CH2-CH3

Chain 2: anti-PD1-VH-CH1-LongHinge-PCL-IFN-PCL-LongHinge-CH2-CH3

Chain 3: Anti-hPD1 light chain

Chain 4: Anti-hPD1 light chain

Chain 1: QVQLVQSGAEVKRPGSSVKVSCKVSGVTFRNFAIIWVRQAPGQGLEWMGGIIPFFSAANYAQSFQGRVTITPDESTSTAFMELASLRSEDTAVYYCAREGERGHTYGFDYWGQGTLVTVSS
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPPVAISSGLLSGRSDNHCDLPQ THSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCA WEVVRAEIMRSFSLSTNLQESLRSKEISSGLLSGRSDNHESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 390)

Chain 2: QVQLVQSGAEVKRPGSSVKVSCKVSGVTFRNFAIIWVRQAPGQGLEWMGGIIPFFSAANYAQSFQGRVTITPDESTSTAFMELASLRSEDTAVYYCAREGERGHTYGFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPPVAISSGLLSGRSDNHCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDE TLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEISSGLLSGRSDNHESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 390)

Chain 3: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 386)

Chain 4: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 386)
Anti-PD1-VH-CH1-ShortHinge-PCL-IFN-PCL-ShortHinge-Fc

Chain 1: anti-PD1-VH-CH1-ShortHinge-PCL-IFN-PCL-ShortHinge-CH2-CH3

Chain 2: anti-PD1-VH-CH1-ShortHinge-PCL-IFN-PCL-ShortHinge-CH2-CH3

Chain 3: Anti-hPD1 light chain

Chain 4: Anti-hPD1 light chain
Chain 1: QVQLVQSGAEVKRPGSSVKVSCKVSGVTFRNFAIIWVRQAPGQGLEWMGGIIPFFSAANYAQSFQGRVTITPDESTSTAFMELASLRSEDTAVYYCAREGERGHTYGFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCISSGLLSGRSDNHCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDE TLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEISSGLLSGRSDNHCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 391)

Chain 2: QVQLVQSGAEVKRPGSSVKVSCKVSGVTFRNFAIIWVRQAPGQGLEWMGGIIPFFSAANYAQSFQGRVTITPDESTSTAFMELASLRSEDTAVYYCAREGERGHTYGFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCISSGLLSGRSDNHCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDE TLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEISSGLLSGRSDNHCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 391)

Chain 3: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 386)

Chain 4: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 386)

8.2. Materials and Methods

8.2.1. Preparation of type I IFN constructs

Constructs encoding antibodies and sterically attenuated IFN fusion proteins were generated in standard mammalian protein expression DNA vectors (pcDNA3.4 or similar) suitable for high-yield protein production and containing standard elements such as promoter sequence, polyA sequence, regulatory elements, and resistance genes. Where applicable, sequences were codon optimized. The 29 amino acid signal sequence of the rat inactive tyrosine-protein kinase transmembrane receptor ROR1 (mROR1) was added to the N-terminus of the constructs to function as a secretion signal. All IFN fusion proteins were expressed as preproteins containing the signal sequence that is cleaved by intracellular processing to generate the mature protein. The constructs were expressed in Expi293FTM cells via transient infection (Thermo Fisher Scientific). Proteins from Expi293F supernatants were purified using the ProteinMaker system (Protein BioSolutions, Gaithersburg, MD) with HiTrapTM Protein G HP or MabSelect SuRe pcc columns (Cytiva). After single-step elution, the protein was neutralized and dialyzed against a final buffer of phosphate-buffered saline (PBS) containing 5% glycerol and stored as-is at -80 °C. Samples were further analyzed by SE-UPLC to determine the presence of high- or low-molecular-weight species relative to the species of interest.

8.2.2. IFN structure In a test tube Cut

Protease-cleavable linkers were enzymatically cleaved by incubating the protein constructs with uPA or MMP enzymes. For enzymatic cleavage using uPA, 8 μg of protein construct was incubated with 100 ng of uPA enzyme in 200 μL of uPA buffer (50 mM Tris pH 8.5, 0.01% (v/v) Tween20) at 37 °C for 20 h. For enzymatic cleavage using MMP, 8 μg of protein construct was incubated with 200 μL of MMP buffer (50 mM Tris pH 7.5, 150 mM NaCl, ₁₀ mM CaCl2, 0.05% Brij35) at 37 °C for 20 h with 200 MMP2 and MMP9, respectively.

8.2.3. Manipulation of reporter KG-1a cells

The promyelocytic macrophage cell line KG-1a was transfected with an ISRE-based luciferase reporter construct and maintained in Iscove's modified Dulbecco's medium supplemented with 2 mM L-glutamine/penicillin/streptomycin + 20% FBS + 1 μg/mL puromycin. A single-cell clone with high responsiveness to IFNα2b was identified, renamed KG-1a/ISRE-Luc cl.2F5, and used for analyses as described previously.

8.2.4. Luciferase assay setup

Cell suspensions and protein dilutions were prepared using RPMI1640 medium supplemented with 2 mM L-glutamine/penicillin/streptomycin + 10% fetal bovine serum (FBS) as the assay medium.

On the day of assay, cells were centrifuged and suspended in assay medium at a density of 5 x 10 ⁵ /mL. Recombinant IFNα2b and/or IFN fusion proteins were diluted 1:5 over an 11-point dilution range (100 nM to 10.2 fM for recombinant interferons and 500 nM to 51.2 fM for fusion proteins), with a 12th point containing no recombinant protein. 2.5 x 10 ⁴ reporter cells were added to 96-well white flat-bottom plates and incubated with serially diluted recombinant IFN or IFN fusion proteins. After incubation for 5 h at 37 °C, 5% CO ₂ , cells were lysed by adding 100 μL ONE-Glo™ (Promega) reagent, and luciferase activity was detected. The emitted light was measured in relative light units (RLU) with a multilabel reader Envision (PerkinElmer).

8.3. Example 1: SE-UPLC profile of Fc-conjugated interferon molecules

SE-UPLC was performed to evaluate IFN molecules linked to the Fc domain at the C-terminus or N-terminus. Three exemplary constructs analyzed by SE-UPLC, Fc-IFNα1 (Figure 4a), Fc-IFNα2b (Figure 4b), and IFNα2b-Fc (Figure 4c), each exhibit individual major peaks with varying levels of high molecular weight species. The major peak percent area of Fc-IFNα1 was calculated to be 37.43, whereas those of Fc-IFNα2b and IFNα2b-Fc showed larger percent values, calculated to be 57.66 and 56.4, respectively.

8.4. Example 2: Activity of interferon molecules

An interferon-stimulated response element (ISRE)-based luciferase reporter was incorporated into the promyelocytic macrophage cell line KG-1a as described in Section 8.2.3 and used to assess the ISRE-inducing ability of IFN receptor agonist constructs as described in Section 8.2.4.

The results shown in Figure 5 showed that the recombinant proteins of IFN variants and Fc fusions showed varying degrees of attenuation in the in vitro luciferase assay of the interferon sensitive response element (ISRE). The first evaluation was to test whether there was a difference in the activity of IFN molecules linked to the Fc molecule at the N-terminus or the C-terminus (Figure 5a). Compared to IFNα2b, both Fc-IFNα2b and IFNα2b-Fc showed weaker interferon signals. However, the levels of attenuation were similar for both molecules (Figure 5b). Next, the in vitro activities of the two Fc-IFN constructs, Fc-IFNα2b and Fc-IFNα1, were compared with those of the three IFN variants, IFNα2b, IFNα1, and IFNβ (Figure 5c). Among the IFN variants, the highest levels of activity were observed for IFNβ and IFNα2b, whereas the activity of IFNα1 was relatively lower. Recombinant proteins with Fc fusions showed attenuated levels of activity compared to IFN mutants. In conclusion, Fc fusions lead to attenuation of interferon signaling compared to free interferon.

8.5. Example 3: SE-UPLC profile of mutant IFN constructs

SE-UPLC was performed to evaluate mutant IFN molecules linked to the C-terminal Fc domain. Four exemplary constructs analyzed by SE-UPLC, Fc-IFNα2bR33A (Fig. 6a), Fc-IFNα2bR149A (Fig. 6b), Fc-IFNα2bR120A (Fig. 6c), and Fc-IFNα2bS152A (Fig. 6d), each exhibit individual major peaks with varying levels of high molecular weight species.

8.6. Example 4: Activity of mutant IFN constructs

The ISRE-based luciferase reporter assay was incorporated into the promyelocytic macrophage cell line KG-1a as described in Section 8.2.3 and used as described in Section 8.2.4 to assess the ISRE-inducing ability of mutant IFN receptor agonist constructs.

The activity of IFN mutants is correlated with their affinity for IFNAR. Therefore, mutations that affect IFN-IFNAR binding could affect the activity of Fc-IFN constructs. A series of mutations were introduced into IFNα2b at the IFNAR1 or IFNAR2 interface (Fig. 7A and 7B). Compared with wild-type Fc-IFNα2b, most mutations that disrupted IFNAR1 or IFNAR2 binding of Fc-IFNα2b attenuated ISRE-luciferase activity. Furthermore, the degree of this attenuation varied, with some mutations causing only a slight attenuation of activity, while others caused a very high degree of attenuation. Nevertheless, there was minimal difference in the degree of attenuation between mutations that disrupted IFNAR1 or IFNAR2 binding.

8.7. Example 5: SE-UPLC profile of an exemplary interferon protein construct

After generating constructs encoding sterically attenuated IFN fusion proteins, SE-UPLC was performed to assess the presence of high or low molecular weight species in the samples as described in Section 8.2.1. Figure 8 shows the profiles of three exemplary IFN precursor protein constructs: a single hinge IFN construct (Figure 8a), a double hinge IFN construct (Figure 8b), and an IFN construct with two Fc domains (Figure 8c). While the single and double hinge constructs displayed separate main peaks, the SEC profile of the IFN construct with two Fc domains was less distinct. Therefore, subsequent analyses were performed with the single hinge and double hinge IFN constructs, excluding the IFN construct with two Fc domains.

8.8. Example 6: Enzymatic cleavage of the IFN precursor protein construct

An exemplary IFN precursor protein construct was cleaved with uPA and MMP enzymes as described in Section 8.2.2 (Fig. 9). Cleavage of the protein construct was assessed by the presence of bands corresponding to the Fc and Fab components, which are expected to appear between 38 and 49 kDa. The identities of the other fragments were further confirmed by Western blot detecting the Fc portion.

Cleavage of the single-hinge and double-hinge IFN proprotein structures by uPA generated additional bands that appeared at higher molecular weights than the expected bands in the SDS-PAGE images (Fig. 9a), indicating incomplete cleavage of these structures by uPA in vitro. In contrast, MMP2/9 completely cleaved the single-hinge IFN proprotein. However, an additional high-molecular-weight band appeared in the lane loaded with MMP2/9-cleaved double-hinge IFN proprotein (Fig. 9b), indicating incomplete MMP cleavage of the double-hinge structure.

8.9. Example 7: Activation of IFN precursor protein construct

The ISRE-luciferase activities of three IFN precursor proteins were evaluated in comparison with IFNα2b and Fc-IFNα2b. Single-hinge full-length IFN and double-hinge IFN constructs showed minimal attenuation compared with Fc-IFNα2b. However, the best attenuation was associated with single-hinge truncated IFN (Fig. 10a).

To assess whether in vitro cleavage of the IFN precursor protein constructs would restore the activity of IFN, we first incubated the same three IFN precursor protein constructs in MMP buffer control or with MMP2 and MMP9 enzymes in MMP buffer as described in Section 8.2.2. Constructs incubated only with MMP buffer attenuated the activity of the IFN precursor protein constructs (Fig. 10B, dotted lines) similar to the attenuation of activity of the uncleaved constructs as seen in Fig. 10A. The presence of MMP buffer was generally associated with a decrease in bioassay signal. However, when the constructs were incubated with MMP enzyme in the same buffer, the release of IFN was associated with an increase in activity compared to the uncleaved construct (Fig. 10B, dotted lines). More specifically, the activities of single-hinge full-length IFN and double-hinge IFN constructs were similar to those of IFNα2b, and the activity of single-hinge truncated IFN was intermediate between those of IFNα2b and Fc-IFNα2b.

9. References Citation

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

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Claims (50)

It is a type I interferon (IFN) precursor protein composed of:
(a) in the first polypeptide chain:
(i) a first immunoglobulin constant domain;
(ii) first linker;
(iii) a type I interferon (IFN) moiety;
(iv) a second linker; and
(v) comprising a first Fc domain;
(b) in the second polypeptide chain:
(i) a second immunoglobulin constant domain;
(ii) a third linker;
(iii) a type II interferon (IFN) moiety;
(iv) a fourth linker; and
(v) comprising a second Fc domain capable of combining with the first Fc domain to form an Fc region;
An IFN precursor protein, wherein at least two of the first linker, the second linker, the third linker and the fourth linker are protease cleavable linkers (PCLs), and optionally, the IFN moiety of the IFN protein is sterically hindered from binding to the IFN receptor by the Fc domain. In claim 1, the first and second IFN moieties each comprise an IFN precursor protein having an amino acid sequence having at least about 90%, at least about 95%, or at least about 98% sequence identity to (a) full-length mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ, or (b) mature human IFNα1, IFNα2b, IFNβ, IFNω, IFNε or IFNκ having at most a 15-amino acid truncation at the N-terminus and/or C-terminus. An IFN precursor protein, wherein the first and second IFN moieties each comprise an amino acid sequence having one or more attenuating mutations compared to mature human IFNα1 or IFNα2b. An IFN precursor protein of any one of claims 1 to 3, wherein the first and second IFN moieties each comprise one or more mutations selected from L26A, F27A, R33A, R33K, L30A, D35E, H57Y, E58N, Q61S, H57S, E58S, H57A, E58A, Q61A, Q90A, E96A, R120A, L135A, R144A, R144S, R144T, R144Y, R144I, R144L, A145D, A145H, A145K, A145M, A145V, A145Y, R149A, R149K, S152A, R162A and E165D. An IFN precursor protein according to any one of claims 1 to 4, wherein the first linker, the second linker, the third linker and the fourth linker are protease-cleavable linkers (PCL). An IFN precursor protein according to any one of claims 1 to 4, wherein the first linker and the third linker are protease cleavable linkers (PCL), and optionally, the second linker and the fourth linker are non-cleavable linkers (NCL). An IFN precursor protein according to any one of claims 1 to 4, wherein the second linker and the fourth linker are protease-cleavable linkers (PCL), and optionally, the first linker and the third linker are non-cleavable linkers (NCL). An IFN precursor protein according to any one of claims 1 to 7, wherein the PCL comprises a substrate sequence cleavable by any protease specified in Table 101. An IFN precursor protein according to any one of claims 1 to 8, wherein the PCL comprises one or more substrate sequences selected from the substrate sequences set forth in Table 102. An IFN precursor protein according to any one of claims 1 to 9, wherein the PCL comprises at least one spacer sequence selected from the sequences set forth in Table 103. An IFN precursor protein according to any one of claims 1 to 10, wherein the PCL comprises an amino acid sequence of any PCL sequence set forth in Table 104 or a variant having at most 5 amino acid substitutions. An IFN precursor protein according to any one of claims 1 to 11, wherein the first and third linkers are the same and/or the third and fourth linkers are the same. An IFN precursor protein according to any one of claims 1 to 12, wherein the first Fc domain and/or the second Fc domain comprises a hinge domain. An IFN precursor protein according to any one of claims 1 to 13, further comprising a first targeting moiety and a second targeting moiety. An IFN precursor protein in claim 14, wherein the first targeting moiety and the second targeting moiety are antibodies or antigen-binding fragments thereof. An IFN precursor protein in claim 15, wherein the first targeting moiety and the second targeting moiety comprise a Fab domain. An IFN proprotein according to any one of claims 14 to 16, wherein the first and second targeting moieties are N-terminal to the first and third linkers, respectively. An IFN proprotein according to any one of claims 14 to 17, wherein the first immunoglobulin constant domain is part of the first targeting moiety and the second immunoglobulin constant domain is part of the second targeting moiety. An IFN precursor protein in claim 18, wherein the first immunoglobulin constant domain is a CH3 domain or a CH1 domain. An IFN precursor protein, comprising: a composition as illustrated in FIG. 1a, FIG. 2a or FIG. 2d, according to any one of claims 1 to 19. An IFN precursor protein, comprising: a composition as illustrated in FIG. 1b, FIG. 2b or FIG. 2e, according to any one of claims 1 to 19. An IFN precursor protein, comprising: a composition as illustrated in FIG. 1c, FIG. 2c or FIG. 2f according to any one of claims 1 to 19. An IFN proprotein according to any one of claims 14 to 22, wherein the first targeting moiety and/or the second targeting moiety is capable of binding to an extracellular matrix (ECM) antigen, a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or viral lymphocyte, a T cell antigen (TCA), a checkpoint inhibitor, a tumor-associated antigen (TAA), a dendritic cell (DC) or other antigen-presenting cell (APC) antigen, or a natural killer (NK) cell antigen. An IFN proprotein according to any one of claims 14 to 23, wherein the first targeting moiety and/or the second targeting moiety comprises (a) (i) CDRs or (ii) VH and VL sequences of an antibody specified in Table 106 or (b) competes with an antibody specified in Table 106 for binding to the target molecule. An IFN precursor protein according to any one of claims 14 to 23, wherein the first targeting moiety and/or the second targeting moiety binds to an ECM antigen optionally selected from syndecan, heparanase, integrin, osteopontin, link, cadherin, laminin, laminin type EGF, lectin, fibronectin, notch, nectin ( e.g. , nectin-4), tenascin, collagen ( e.g. , collagen type X) and matrixin. An IFN proprotein according to any one of claims 14 to 23, wherein the first targeting moiety and/or the second targeting moiety is capable of binding to a cell surface molecule of a tumor or viral lymphocyte. In claim 26, the antigen is a T cell co-stimulatory protein, IFN precursor protein, selectively selected from CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C and B7-H3. An IFN proprotein according to any one of claims 14 to 23, wherein the first targeting moiety and/or the second targeting moiety is capable of binding to a checkpoint inhibitor. In claim 28, the checkpoint inhibitor is PDL1, an IFN precursor protein. In claim 28, the checkpoint inhibitor is PD1, an IFN precursor protein. In any one of claims 14 to 23, the first targeting moiety and/or the second targeting moiety is selected from the group consisting of AFP, ALK, BAGE protein, BIRC5 (survivin), BIRC7, β-catenin, bcr-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, casspace-8, CALR, CEACAM5 (also called carcinoembryonic antigen or CEA), CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE protein ( e.g. , GAGE-1 or -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT, LMP2, MAGE proteins ( e.g. , MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, An IFN precursor protein capable of binding to a tumor-associated antigen (TAA) selectively selected from among TAG-72, TGF-β, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase and uroplakin-3. An IFN precursor protein according to any one of claims 14 to 23, wherein the first targeting moiety and/or the second targeting moiety is capable of binding to a dendritic cell (DC) antigen selectively selected from XCR1, Clec9a, CD1c, CD11c, CD14, PDL1, macrophage mannose receptor (CD206), and DEC-205. An IFN proprotein according to any one of claims 14 to 23, wherein the first targeting moiety and/or the second targeting moiety is capable of binding to a natural killer (NK) cell antigen. An IFN precursor protein according to any one of claims 1 to 33, wherein the Fc region is a homodimer. A nucleic acid or a plurality of nucleic acids encoding an IFN precursor protein of any one of claims 1 to 34. A host cell engineered to express the IFN precursor protein of any one of claims 1 to 34 or the nucleic acid of claim 35. A method for producing an IFN precursor protein of any one of claims 1 to 34, comprising the step of culturing a host cell of claim 36 and recovering the IFN precursor protein expressed thereby. A pharmaceutical composition comprising an IFN precursor protein and an excipient according to any one of claims 1 to 34. A method for treating cancer, comprising administering to a subject in need thereof an IFN precursor protein of any one of claims 1 to 34 or a pharmaceutical composition of claim 38. A method in claim 39, wherein the IFN precursor protein comprises at least one targeting moiety capable of binding to a target molecule. In claim 39, the cancer is associated with the expression of the target molecule, for example , TAA and related cancers specified in Table 111. A method according to any one of claims 39 to 41, wherein the activated IFN proprotein comprising the IFN moiety is generated by cleaving one or more protease-cleavable linkers in the IFN proprotein expressed by the cancer tissue. A method for local delivery of an IFN precursor protein, comprising administering to a subject an IFN precursor protein (or a pharmaceutical composition comprising the IFN precursor protein and an excipient) of any one of claims 1 to 34, wherein each linker comprises at least one substrate for at least one protease expressed by a tissue to which the IFN precursor protein is to be locally delivered. A method of treating cancer with an IFN proprotein that is selectively activated in cancer tissue, comprising administering to a subject in need an IFN proprotein of any one of claims 1 to 34 (or a pharmaceutical composition comprising said IFN proprotein and an excipient), said IFN proprotein comprising one or more protease-cleavable linkers, each of said linkers comprising one or more substrates for one or more proteases expressed by cancer tissue targeted by said IFN proprotein. A method of administering to a subject an IFN therapy with reduced systemic exposure and/or reduced systemic toxicity, comprising administering to the subject an IFN therapy in the form of an IFN proprotein of any one of claims 1 to 34 (or a pharmaceutical composition comprising the IFN proprotein and an excipient), wherein each of the linkers comprises one or more substrates for one or more proteases expressed by a tissue for which IFN therapy is desired and/or intended. A method of treating cancer with an IFN proprotein that is selectively activated in cancer tissue, comprising administering to a subject in need an IFN proprotein of any one of claims 1 to 34 (or a pharmaceutical composition comprising said IFN proprotein and an excipient), said IFN proprotein comprising one or more protease-cleavable linkers, each of said linkers comprising one or more substrates for one or more proteases expressed by said cancer tissue. A method for targeted delivery of an activated IFN precursor protein to cancer tissue, comprising administering to a subject an IFN precursor protein of any one of claims 1 to 34 (or a pharmaceutical composition comprising the IFN precursor protein and an excipient), wherein the IFN precursor protein comprises:
(a) comprising one or more targeting moieties that recognize a target molecule expressed by said cancer tissue or associated immune cells, and
(b) a method having one or more protease-cleavable linkers, each having one or more substrates for one or more proteases expressed in a tissue in which IFN therapy is desired and/or intended. A method of inducing an immune response locally in a target tissue, comprising administering to a subject an IFN proprotein of any one of claims 1 to 34 (or a pharmaceutical composition comprising the IFN proprotein and an excipient), the IFN proprotein having one or more targeting moieties capable of binding a target molecule expressed in the target tissue and one or more protease-cleavable linkers, each of the linkers comprising one or more substrates for one or more proteases expressed in the target tissue. A method of enhancing an immune response to an antigen, comprising administering to a subject an immunogenic agent that induces an immune response to said antigen together with an IFN proprotein of any one of claims 1 to 34 (or a pharmaceutical composition comprising said IFN proprotein and an excipient) or a nucleic acid encoding such an IFN proprotein ( e.g. , as described in Section 6.10.1). A method according to any one of claims 39 to 49, wherein the administration is non-local.