Targeted modified interferon and its use

Abstract

To provide chimeric proteins and the like useful for treating diseases, etc. [Solution] The present invention relates to a chimeric protein comprising a consensus interferon as a signaling molecule. In some embodiments, the consensus interferon is modified to include one or more mutations. In one embodiment, the modified consensus interferon includes one or more mutations that reduce affinity for IFNAR1 and one or more mutations that reduce affinity for IFNAR2. The present invention provides pharmaceutical compositions comprising the chimeric protein and their use in treating various diseases. The present invention relates to a method for treating cancer, comprising administering an effective amount of the chimeric protein.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 454,992, filed on February 6, 2017, the entire content of which is incorporated herein by reference.

[0002] Technical Field The present invention relates in part to chimeric proteins comprising consensus interferon and their use as therapeutic agents.

[0003] Sequence Listing This application includes a sequence listing submitted in ASCII format via EFS - Web, the entire content of which is incorporated herein by reference. The ASCII copy created on January 29, 2018, is named ORN - 024PC_ST25.txt and is 327,680 bytes in size.

Background Art

[0004] Type I interferons (IFNs) constitute a family of multifunctional cytokines that play a major role in the immune response. Human type I IFNs include 13 distinct non - allelic genetic α subtypes, 1 β subtype, and 1 ω subtype. All type I interferons appear to bind to a common receptor, the type I interferon α / β receptor (IFNAR), which is composed of the IFNAR1 subunit and the IFNAR2 subunit. When type I IFN binds, IFNAR activates the JAK - STAT signaling pathway to induce various biological effects. Differential activities have been reported for IFN subtypes and, for example, IFN - α2 is used clinically for the treatment of various diseases and disorders, including viral hepatitis.

[0005] Consensus interferon (CIFN, also known as interferon alpha con-1 or Infergen®) is a synthetic recombinant type I interferon modified to contain the most commonly seen amino acids among several allelic IFN-α subtypes. In vitro studies have demonstrated that consensus interferon has antiviral activity 10-fold higher than wild-type IFN-α2a. Consensus interferon has been approved by the US Food and Drug Administration (FDA) for the treatment of chronic hepatitis C infection.

[0006] Due to its pharmacokinetic properties, the clinical use of consensus interferon is disadvantaged by severe side effects and low patient compliance. In the United States, consensus interferon is administered three times a week at a concentration of 15 μg. After subcutaneous injection, consensus interferon reaches high levels, but the serum concentration drops to nearly undetectable levels by the next administration. This kinetic profile provides the virus with opportunities to recover and proliferate. Protocols involving higher doses and / or more frequent administrations have been tested, but they are associated with severe side effects including influenza-like symptoms, myalgia, leukopenia, thrombocytopenia, neutropenia, depression, and weight loss. A high patient dropout rate has also been reported.

[0007] Therefore, there is a need for a safe and effective consensus interferon-based therapy with improved pharmacokinetic and therapeutic properties and a minimal toxicity profile. SUMMARY OF THE INVENTION

[0008] In one aspect, the present invention relates to a chimeric protein comprising consensus interferon as a signaling substance. In certain embodiments, the consensus interferon comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof. In some embodiments, the consensus interferon is modified and comprises one or more mutations. In some embodiments, the biological activity of the consensus interferon is reduced by one or more mutations. For example, the affinity of the consensus interferon for a therapeutic receptor may be reduced by one or more mutations. In one embodiment, the therapeutic receptor is the interferon α / β receptor (IFNAR), which is composed of the IFNAR1 subunit and the IFNAR2 subunit. In one embodiment, the modified consensus interferon comprises one or more mutations that reduce the affinity for IFNAR1. In another embodiment, the modified consensus interferon comprises one or more mutations that reduce the affinity for IFNAR2. In one embodiment, the modified consensus interferon comprises one or more mutations that reduce the affinity for IFNAR1 and one or more mutations that reduce the affinity for IFNAR2.

[0009] In some embodiments, the chimeric protein may comprise one or more additional signaling substances that may be modified, such as, but not limited to, interferon, interleukin, and tumor necrosis factor. In various embodiments, the chimeric protein of the present disclosure provides improved safety, and / or therapeutic activity, and / or pharmacokinetic profile (e.g., increased serum half-life) compared to non-targeted consensus interferon or unmodified wild-type IFN-α such as IFN-α2a or IFN-α2b.

[0010] In various embodiments, the chimeric protein comprises one or more targeting moieties having a recognition domain that specifically binds to a target of interest (e.g., an antigen, a receptor), such as an antigen recognition domain including various antibody formats, such as, but not limited to, single domain antibodies. In various embodiments, the targeting moiety comprises a recognition domain that specifically binds to a target of interest (e.g., an antigen, a receptor) and is found on one or more types of immune cells, including, but not limited to, T cells, cytotoxic T lymphocytes, helper T cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g., M1 macrophages), B cells, and dendritic cells. In some embodiments, the recognition domain specifically binds to a target of interest (e.g., an antigen, a receptor) and effectively recruits one or more types of immune cells. In some embodiments, the target of interest (e.g., an antigen, a receptor) may be present on one or more types of tumor cells. In some embodiments, the chimeric protein of the present disclosure can recruit immune cells, such as immune cells that can kill and / or suppress tumor cells, to the site of action (e.g., the tumor microenvironment, as a non-limiting example). In some embodiments, the recognition domain specifically binds to a target of interest (e.g., an antigen, a receptor) that is part of a non-cellular structure.

[0011] In various embodiments, the chimeric proteins of the present disclosure are used for the treatment of various diseases or disorders, such as cancer, infectious diseases, immune disorders, autoimmune diseases, cardiovascular diseases, wound healing, ischemia-related diseases, neurodegenerative diseases, metabolic diseases, and many other diseases and disorders, and the present invention encompasses various treatment methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0012]

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DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention is based in part on the discovery that a targeted chimeric protein comprising consensus interferon exhibits beneficial therapeutic properties and reduced side effects. For example, the chimeric proteins of the present invention are highly active and / or long acting, yet induce minimal side effects. Pharmaceutical compositions comprising the chimeric proteins and their use in the treatment of various diseases are provided by the present invention.

[0014] Modified (e.g., consensus) interferon or variants thereof In one aspect, the present invention provides a chimeric protein comprising a modified interferon. In one aspect, the present invention provides a chimeric protein comprising consensus interferon. Consensus interferon is created by examining in detail the sequences of several human allelic IFN-α subtypes and selecting the amino acids most frequently observed at the corresponding positions. Consensus interferon differs from IFN-α2b in 20 out of 166 amino acids (88% homology) and shows identity in more than 30% of the amino acid positions when compared to IFN-β. In various embodiments, consensus interferon comprises the following amino acid sequence.

Chemical formula

[0015] In some embodiments, consensus interferon comprises the amino acid of SEQ ID NO: 2 that differs from the amino acid sequence of SEQ ID NO: 1 by one amino acid, i.e., SEQ ID NO: 2 does not include the starting methionine residue of SEQ ID NO: 1.

Chemical formula

[0016] In various embodiments, the chimeric protein of the present invention comprises a modified consensus interferon, i.e., a consensus interferon variant, as a signaling substance. In various embodiments, the consensus interferon variant includes functional derivatives, analogs, precursors, isoforms, splice variants, or fragments of the consensus interferon.

[0017] In certain embodiments, the chimeric protein comprises any one of the consensus interferon variants disclosed in U.S. Patent Nos. 4,695,623, 4,897,471, 5,541,293, and 8,496,921, the entire contents of which are incorporated herein by reference. For example, the consensus interferon variant may comprise the amino acid sequence of IFN-CON2 or IFN-CON3 as disclosed in U.S. Patent Nos. 4,695,623, 4,897,471, and 5,541,293. In certain embodiments, the consensus interferon variant comprises the amino acid sequence of IFN-CON2 (SEQ ID NO: 3).

[0018] In certain embodiments, the consensus interferon variant comprises the amino acid sequence of IFN-CON3 (SEQ ID NO: 4).

[0019] In certain embodiments, the consensus interferon variant comprises the amino acid sequence of any one of the variants disclosed in U.S. Patent No. 8,496,921. For example, the consensus variant may comprise the amino acid sequence of SEQ ID NO: 5.

[0020] In another embodiment, the consensus interferon variant may comprise the amino acid sequence of SEQ ID NO: 6.

[0021] In some embodiments, the consensus interferon variant may be PEGylated, i.e., it contains a PEG moiety. In certain embodiments, the consensus interferon variant may contain a PEG moiety attached at the position of S156C of SEQ ID NO: 6.

[0022] In some embodiments, the modified interferon has an insertion of Asp at the 41st position within the sequence Glu-Glu-Phe-Gly-Asn-Gln (SEQ ID NO: 277) to obtain the sequence Glu-Glu-Phe-Asp-Gly-Asn-Gln (SEQ ID NO: 278) (thus resulting in a re-numbering of the sequence relative to the IFN-α2a sequence), and is a variant of human IFN-α2a having the following mutations: Arg23Lys, Leu26Pro, Glu53Gln, Thr54Ala, Pro56Ser, Asp86Glu, Ile104Thr, Gly106Glu, Thr110Glu, Lys117Asn, Arg125Lys, and Lys136Thr. All embodiments described herein for consensus interferon apply equally to this modified interferon.

[0023] Other consensus interferon variant sequences are known in the art. In various embodiments, the modified consensus interferon comprises an amino acid sequence having at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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%, or at least about 99% sequence identity (e.g., about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity) with any known amino acid sequence of a consensus interferon variant.

[0024] In some embodiments, the consensus interferon variant comprises an amino acid sequence having at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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%, or at least about 99% sequence identity (e.g., about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity) to the consensus interferon or consensus interferon variant sequences disclosed herein, such as any of SEQ ID NOs: 1-6.

[0025] In various embodiments, the consensus interferon variant comprises an amino acid sequence having one or more amino acid mutations. In some embodiments, the one or more amino acid mutations can be independently selected from substitutions, insertions, deletions, and truncations.

[0026] In some embodiments, the amino acid mutation is an amino acid substitution and can include conservative substitutions and / or non-conservative substitutions.

[0027] "Conservative substitution" may be performed, for example, based on similarities in the polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the amino acid residues involved. The 20 natural amino acids can be grouped into six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0028] As used herein, "conservative substitution" is defined as an exchange of an amino acid with another amino acid described in the same group among the above six standard amino acid groups. For example, one negative charge is maintained in the polypeptide so modified by the exchange of Asp with Glu. Further, glycine and proline can be substituted for each other based on their ability to disrupt their α-helices.

[0029] As used herein, "non-conservative substitution" is defined as an exchange of an amino acid with another amino acid described in a different group among the above six standard amino acid groups (1)-(6).

[0030] In various embodiments, substitutions can include non-canonical amino acids (e.g., generally, selenocysteine, pyrrolysine, N-formylmethionine, β-alanine, GABA, and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids such as β-methylamino acids, Cα-methylamino acids, Nα-methylamino acids, and amino acid analogs).

[0031] In various embodiments, consensus interferon is modified to have one or more mutations. In some embodiments, due to those mutations, the consensus interferon variant has a decrease in one or more activities, such as a decrease in binding affinity, a decrease in intrinsic activity, and a decrease in a specific biological activity, compared to the unmutated form, e.g., wild-type consensus interferon (e.g., consensus interferon having the amino acid sequence of SEQ ID NO: 1 or 2). For example, the decrease in one or more of the activities such as a decrease in binding affinity, a decrease in intrinsic activity, and a decrease in a specific biological activity with respect to the unmutated form, e.g., wild-type consensus interferon, can occur in a therapeutic receptor such as IFNAR. As a result, in various embodiments, due to those mutations, the consensus interferon variant has reduced systemic toxicity, reduced side effects, and reduced off-target effects compared to the unmutated form, e.g., wild-type consensus interferon.

[0032] In various embodiments, the consensus interferon is modified to have mutations that reduce its binding affinity or activity at therapeutic receptors such as IFNAR. In some embodiments, the activity conferred by the consensus interferon is receptor activation activity at the therapeutic receptor (e.g., activation of cellular effects at the treatment site). For example, the consensus interferon may activate the therapeutic receptor. In such embodiments, the mutation causes the consensus interferon variant to have reduced activation activity at the therapeutic receptor.

[0033] In some embodiments, the reduction in affinity or activity at the therapeutic receptor can be restored by binding to a targeting moiety. In other embodiments, the reduction in affinity or activity at the therapeutic receptor is not substantially restored by binding to a targeting moiety. In various embodiments, the therapeutic chimeric protein of the present invention reduces off-target effects because the consensus interferon variant has mutations that weaken its binding affinity or activity at the therapeutic receptor. In various embodiments, this reduces, for example, the side effects seen with wild-type consensus interferon. In various embodiments, the consensus interferon variant is substantially inactive en route to the therapeutic active site, acts substantially on specifically targeted cell types, and greatly reduces unwanted side effects.

[0034] In various embodiments, the consensus interferon variant has one or more mutations that attenuate or reduce the affinity of the consensus interferon variant for one or more therapeutic receptors, e.g., binding (e.g., K D ) and / or activation (e.g., K A and / or EC 50 as measurable). In various embodiments, the reduction in affinity at the therapeutic receptor attenuates the activity and / or signal transduction from the therapeutic receptor.

[0035] In various embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity for the IFNAR1 subunit of IFNAR. In certain embodiments, the consensus interferon variant has reduced affinity and / or activity in IFNAR1. In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity for the IFNAR2 subunit of IFNAR. In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity for both the IFNAR1 subunit and the IFNAR2 subunit of IFNAR.

[0036] In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity for IFNAR1 and one or more mutations that substantially reduce or eliminate its binding or affinity for IFNAR2. In some embodiments, a chimeric protein having such a consensus interferon variant can provide target-selective IFNAR1 activity (e.g., IFNAR1 activity can be restored by targeting via a targeting moiety).

[0037] In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity for IFNAR2 and one or more mutations that substantially reduce or eliminate its binding or affinity for FNAR1. In some embodiments, a chimeric protein comprising such a consensus interferon variant can provide target-selective IFNAR2 activity (e.g., IFNAR2 activity can be restored by targeting via a targeting moiety).

[0038] In some embodiments, the consensus interferon variant has one or more mutations that reduce binding to or affinity for IFNAR1 and one or more mutations that reduce binding to or affinity for IFNAR2. In some embodiments, a chimeric protein having such a consensus interferon variant can provide target-selective IFNAR1 and / or IFNAR2 activity (e.g., IFNAR1 and / or IFNAR2 activity can be restored by targeting via a targeting moiety).

[0039] In various embodiments, the consensus interferon variant has an affinity for a therapeutic receptor (e.g., any one of IFNAR or its subunits IFNAR1 and / or IFNAR2) that is about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10% - 20%, about 20% - 40%, about 50%, about 40% - 60%, about 60% - 80%, about 80% - 100% lower than that of wild-type consensus interferon. In some embodiments, the binding affinity is at least about 2-fold lower, about 3-fold lower, about 4-fold lower, about 5-fold lower, about 6-fold lower, about 7-fold lower, about 8-fold lower, about 9-fold lower, at least about 10-fold lower, at least about 15-fold lower, at least about 20-fold lower, at least about 25-fold lower, at least about 30-fold lower, at least about 35-fold lower, at least about 40-fold lower, at least about 45-fold lower, at least about 50-fold lower, at least about 100-fold lower, at least about 150-fold lower, or about 10 - 50-fold lower, about 50 - 100-fold lower, about 100 - 150-fold lower, about 150 - 200-fold lower, or more than 200-fold lower than that of wild-type consensus interferon.

[0040] In some embodiments, the consensus interferon variant comprises one or more mutations that reduce the affinity of the consensus interferon variant for its receptor. In some embodiments, the binding affinity of the consensus interferon variant for its receptor is lower than the binding affinity of the targeting moiety for its receptor. In some embodiments, this difference in binding affinity is between the binding affinity of the consensus interferon variant / receptor and the binding affinity of the targeting moiety / receptor in the same cell. In some embodiments, this difference in binding affinity allows the consensus interferon variant to localize its on-target effects and minimize off-target effects underlying the side effects seen with wild-type consensus interferon. In some embodiments, this binding affinity is at least about 2-fold lower, at least about 5-fold lower, at least about 10-fold lower, at least about 15-fold lower, at least about 25-fold lower, at least about 50-fold lower, at least about 100-fold lower, or at least about 150-fold lower.

[0041] Receptor binding activity can be measured using methods known in the art. For example, affinity and / or binding activity can be evaluated by Scatchard plot analysis of binding data and computer fitting (e.g., Scatchard, 1949), or by reflectometric interference spectroscopy under flow-through conditions as described by Brecht et al. (1993), the entire contents of those references being incorporated herein by reference.

[0042] In various embodiments, attenuation of activity in a therapeutic receptor, i.e., diminished affinity in a therapeutic receptor, can be restored by binding to a targeting moiety (e.g., an antibody or antibody format described herein) having a high affinity for an antigen at a therapeutic active site. Targeting is achieved by linking consensus interferon or a variant thereof to the targeting moiety. In certain embodiments, consensus interferon or a variant thereof is linked to the targeting moiety via the amino terminus. In another embodiment, consensus interferon or a variant thereof is linked to the targeting moiety via the carboxy terminus. Thus, the chimeric proteins of the present disclosure, in some embodiments, achieve a localized, on-target, controlled therapeutic effect in a therapeutic receptor.

[0043] In some embodiments, consensus interferon is modified to have a mutation at one or more of amino acids 33 and / or 145 - 155 in SEQ ID NO:2, e.g., at amino acid positions 145, 146, 149, 150, and / or 154. In some embodiments, consensus interferon is modified to have a mutation at one or more of amino acids 33 and / or 145 - 155 in SEQ ID NO:2, e.g., at amino acid positions 145, 146, 149, 150, and / or 154, and those substitutions may be hydrophobic and are selected from alanine, valine, leucine, and isoleucine. In some embodiments, the consensus interferon variant comprises one or more mutations selected from R33A, R145X1, A146X2, M149A, R150A, and L154A in SEQ ID NO:2, wherein X1 is selected from A, S, T, Y, L, and I, and X2 is selected from G, H, Y, K, and D.

[0044] In certain embodiments, the consensus interferon is modified to have a mutation at amino acid 121 in SEQ ID NO:2 (i.e., K121). In certain embodiments, the consensus interferon comprises the K121E mutation in SEQ ID NO:2.

[0045] A therapeutic agent comprising a modified (e.g., consensus) interferon or a variant thereof Cell recruitment of the targeting moiety In various embodiments, the chimeric proteins of the invention further comprise one or more targeting moieties having recognition domains that specifically bind to a target of interest (e.g., an antigen, a receptor). In some embodiments, the chimeric protein may comprise two, three, four, five, six, seven, eight, nine, ten, or more targeting moieties. In an exemplary embodiment, the chimeric proteins of the invention comprise two or more targeting moieties. In such embodiments, those chimeric proteins can target two different cells (e.g., to create synapses) or the same cell (e.g., to obtain a more concentrated signaling effect).

[0046] In various embodiments, a target of interest (e.g., an antigen, a receptor) can be found on one or more types of immune cells, and such immune cells include, but are not limited to, T cells, cytotoxic T lymphocytes, helper T cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g., M1 macrophages), B cells, dendritic cells, or subsets thereof. In some embodiments, the recognition domain specifically binds to a target of interest (e.g., an antigen, a receptor) and effectively mobilizes one or more types of immune cells directly or indirectly. In some embodiments, a target of interest (e.g., an antigen, a receptor) can be found on one or more types of tumor cells. In some embodiments, the chimeric protein of the present disclosure can mobilize immune cells directly or indirectly, for example, in some embodiments, it can be mobilized to a treatment site (e.g., a location containing one or more types of diseased cells or cells whose therapeutic effect is to be regulated). In some embodiments, the chimeric protein of the present disclosure can directly or indirectly mobilize immune cells, such as immune cells that can kill and / or suppress tumor cells, to an action site (as a non-limiting example, a tumor microenvironment, etc.).

[0047] In various embodiments, the chimeric protein has a targeting moiety having a recognition domain that specifically binds to a target that is a non-cellular structure moiety (e.g., an antigen, a receptor). In some embodiments, the antigen or receptor is not an essential component of a complete cell or cell structure. In some embodiments, the antigen or receptor is an extracellular antigen or receptor. In some embodiments, the target is a non-proteinaceous non-cellular marker, which includes, but is not limited to, nucleic acids containing DNA or RNA, such as DNA released from necrotic tumor cells, or extracellular deposits such as cholesterol.

[0048] In some embodiments, the target of interest (e.g., antigen, receptor) is part of the non-cellular components of the stroma or extracellular matrix (ECM) or their associated markers. As used herein, stroma refers to the connective supportive framework of a tissue or organ. Stroma may include aggregates of cells such as fibroblasts / myofibroblasts, glial cells, epithelial cells, adipocytes, immune cells, vascular cells, smooth muscle cells, and immune cells with the extracellular matrix (ECM) and extracellular molecules. In various embodiments, the target of interest (e.g., antigen, receptor) is part of the non-cellular components of the stroma, such as the extracellular matrix and extracellular molecules. As used herein, ECM refers to the non-cellular components present in all tissues and organs. The ECM is composed of many biochemically distinct components including, but not limited to, proteins, glycoproteins, proteoglycans, and polysaccharides. These components of the ECM are usually produced by adjacent cells and secreted into the ECM by exocytosis. Once secreted, the ECM components often aggregate to form a complex network structure of macromolecules. In various embodiments, the chimeric protein of the invention comprises a targeting moiety that recognizes a target (e.g., antigen or receptor or non-proteinaceous molecule) that localizes on any component of the ECM. Examples of components of the ECM include, but are not limited to, proteoglycans, non-proteoglycan polysaccharides, fibers, and other ECM proteins, or ECM non-proteins such as polysaccharides and / or lipids, or ECM-binding molecules (e.g., proteins or non-proteins such as polysaccharides, nucleic acids, and / or lipids).

[0049] In some embodiments, the targeting moiety recognizes a target (e.g., antigen, receptor) on an ECM proteoglycan. A proteoglycan is a glycosylated protein. A basic proteoglycan unit contains one or more covalently linked glycosaminoglycan (GAG) chains along with a core protein. Proteoglycans have a net negative charge that attracts positively charged sodium ions (Na+), thereby attracting water molecules via osmosis and keeping the ECM and resident cells in a hydrated state. Proteoglycans may also serve to capture and store growth factors within the ECM. Examples of proteoglycans that the chimeric proteins of the present invention may target include, but are not limited to, heparan sulfate, chondroitin sulfate, and keratan sulfate. In certain embodiments, the targeting moiety recognizes a target (e.g., antigen, receptor) on a non-proteoglycan polysaccharide such as hyaluronic acid.

[0050] In some embodiments, the targeting moiety recognizes a target (e.g., antigen, receptor) on an ECM fiber. Examples of ECM fibers include collagen fibers and elastin fibers. In some embodiments, the targeting moiety recognizes one or more epitopes on collagen or collagen fibers. Collagen is the most abundant protein in the ECM. Collagen exists in the ECM as a fibrous protein, providing structural support to resident cells. In one or more embodiments, the targeting moiety recognizes and binds to various types of collagen present within the ECM, including but not limited to fibrillar collagens (type I, II, III, V, XI), FACIT collagens (type IX, XII, XIV), short-chain collagens (type VIII, X), basement membrane collagen (type IV), and / or type VI, VII, or XIII collagen. Elastin fibers give tissues elasticity, thereby allowing the tissue to return to its original state after stretching when needed. In some embodiments, the targeting moiety recognizes one or more epitopes on elastin or elastin fibers.

[0051] In some embodiments, the targeting moiety recognizes one or more ECM proteins including, but not limited to, tenascin, fibronectin, fibrin, laminin, or nidogen / entactin.

[0052] In one embodiment, the targeting moiety recognizes and binds tenascin. The tenascin (TN) family of glycoproteins includes at least four members: tenascin-C, tenascin-R, tenascin-X, and tenascin-W. The primary structure of tenascin proteins contains several common motifs arranged in the same sequential order: an amino-terminal 7-amino acid repeat, an epidermal growth factor (EGF)-like repeat, a fibronectin type III domain repeat, and a carboxyl-terminal fibrinogen globular domain. Each protein member is associated with typical diversity in the number and nature of the EGF-like repeats and fibronectin type III repeats. There are also isoform variants, particularly with respect to tenascin-C. Over 27 splice variants and / or isoforms of tenascin-C are known. In certain embodiments, the targeting moiety recognizes and binds tenascin-CA1. Similarly, tenascin-R also has various splice variants and isoforms. Tenascin-R typically exists as a dimer or trimer. Tenascin-X is the largest member of the tenascin family and is known to exist as a trimer. Tenascin-W exists as a trimer. In some embodiments, the targeting moiety recognizes one or more epitopes on the tenascin protein. In some embodiments, the targeting moiety recognizes monomeric, and / or dimeric, and / or trimeric, and / or hexameric tenascin proteins.

[0053] In some embodiments, the targeting moiety recognizes and binds to fibronectin. Fibronectin is a glycoprotein that links collagen fibers and cells in the ECM and enables cells to move through the ECM. When bound to integrin, fibronectin unfolds to form a functional dimer. In some embodiments, the targeting moiety recognizes monomeric and / or dimeric fibronectin. In some embodiments, the targeting moiety recognizes one or more epitopes on fibronectin. In an exemplary embodiment, the targeting moiety recognizes fibronectin extracellular domain A (EDA) or fibronectin extracellular domain B (EDB). Elevated EDA levels are associated with various diseases and disorders, including psoriasis, rheumatoid arthritis, diabetes, and cancer. In some embodiments, the targeting moiety recognizes fibronectin containing the EDA isoform and can be used to target chimeric proteins to diseased cells, including cancer cells. In some embodiments, the targeting moiety recognizes fibronectin containing the EDB isoform. In various embodiments, such targeting moieties can be used to target chimeric proteins to tumor cells, including tumor neovessels.

[0054] In some embodiments, the targeting moiety recognizes and binds to fibrin. Fibrin is another proteinaceous substance often found in the matrix network structure of the ECM. Fibrin is formed by the action of the thrombin protease on fibrinogen, which causes polymerization of fibrin. In some embodiments, the targeting moiety recognizes one or more epitopes on fibrin. In some embodiments, the targeting moiety recognizes monomeric and multimeric fibrin.

[0055] In certain embodiments, the targeting moiety recognizes and binds laminin. Laminin is a major component of the basement membrane, which is the protein network foundation for cells and organs. Laminin is a heterotrimeric protein containing an α-chain, a β-chain, and a γ-chain. In some embodiments, the targeting moiety recognizes one or more epitopes on laminin. In some embodiments, the targeting moiety recognizes monomeric, dimeric, and trimeric laminin.

[0056] In certain embodiments, the targeting moiety recognizes and binds nidogen or entactin. Nidogen / entactin is a family of highly conserved sulfated glycoproteins. These glycoproteins constitute major structural components of the basement membrane and function to link laminin and collagen IV network structures in the basement membrane. Members of this family include nidogen-1 and nidogen-2. In various embodiments, the targeting moiety recognizes epitopes on nidogen-1 and / or nidogen-2.

[0057] In various embodiments, the targeting moiety includes an antigen recognition domain that recognizes an epitope present on any of the targets (e.g., ECM proteins) described herein. In certain embodiments, the antigen recognition domain recognizes one or more linear epitopes present on the protein. As used herein, a linear epitope refers to any continuous sequence of amino acids present on the protein. In another embodiment, the antigen recognition domain recognizes one or more conformational epitopes present on the protein. As used herein, a conformational epitope refers to one or more sections of amino acids that form a three-dimensional surface (which may be discontinuous) having features and / or shape and / or tertiary structure that are recognizable by the antigen recognition domain.

[0058] In various embodiments, the targeting moiety may bind to the full length, and / or mature form, and / or isoform, and / or splice variant, and / or fragment, and / or any other native or synthetic analog, variant, or mutant of any of the targets described herein (e.g., ECM proteins). In various embodiments, the targeting moiety may bind to any form of protein described herein, including monomers, dimers, trimers, tetramers, heterodimers, multimers, and aggregates. In various embodiments, the targeting moiety may bind to a post-translationally modified form of protein, such as a glycosylated form and / or phosphorylated form, described herein.

[0059] In various embodiments, the targeting moiety includes an antigen recognition domain that recognizes extracellular molecules such as DNA. In some embodiments, the targeting moiety includes an antigen recognition domain that recognizes DNA. In one embodiment, the DNA is released into the extracellular space from necrotic tumor cells or apoptotic tumor cells or other diseased cells.

[0060] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes one or more acellular structures associated with atherosclerotic plaques. Two types of atherosclerotic plaques are known. Fibro-lipid (fibro-fatty) plaques are characterized by the accumulation of lipid-containing cells in the subintimal layer of arteries. There is a fibrous capsule covering the atheromatous core of the plaque beneath the endothelium. The core contains lipid-containing cells (macrophages and smooth muscle cells) with high tissue cholesterol and cholesterol ester content, fibrin, proteoglycans, collagen, elastin, and cell debris. In advanced plaques, the central core of the plaque usually contains extracellular cholesterol deposits (released from dead cells), thereby forming a region containing cholesterol crystals with empty needle-like fissures. Relatively young foam cells and capillaries are present at the edges of the plaque. Fibrous plaques localize beneath the intima within the arterial wall, causing the wall to thicken and enlarge, and sometimes the localized plaque-like lesions narrow the lumen and somewhat atrophy the muscular layer. Fibrous plaques contain collagen fibers (eosinophilic), calcium deposits (hematoxylinophilic), and lipid-containing cells. In some embodiments, the targeting moiety recognizes and binds to one or more of these non-cellular components of the plaques, such as fibrin, proteoglycans, collagen, elastin, cell debris, and calcium or other mineral deposits or precipitates. In some embodiments, the cell debris is nucleic acid released from dead cells, such as DNA or RNA.

[0061] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes one or more non-cellular structures found in brain plaques associated with neurodegenerative diseases. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures localized within amyloid plaques found in the brains of patients with Alzheimer's disease. For example, the targeting moiety may recognize and bind to amyloid-β peptide, which is a major component of amyloid plaques. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures localized within brain plaques found in patients with Huntington's disease. In various embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures found in plaques associated with other neurodegenerative or musculoskeletal diseases such as Lewy body dementia and inclusion body myositis.

[0062] In some embodiments, the chimeric protein of the present invention may have two or more targeting moieties that bind to non-cellular structures. In some embodiments, there are two targeting moieties, one targeting cells and the other targeting non-cellular structures. In various embodiments, the targeting moiety can directly or indirectly recruit cells, such as diseased cells and / or effector cells. In some embodiments, the chimeric protein of the present disclosure can alter the balance of immune cells that is advantageous for an immune attack on a tumor or is used in methods involving such alteration. For example, the chimeric protein of the present disclosure can be advantageous for cells that can kill and / or suppress tumors (e.g., T cells, cytotoxic T lymphocytes, helper T cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g., M1 macrophages), B cells, dendritic cells, or subsets thereof), and can change the ratio of immune cells at clinically important sites to be resistant to cells that protect tumors (e.g., myeloid-derived suppressor cells (MDSC), regulatory T cells (Treg), tumor-associated neutrophils (TAN), M2 macrophages, tumor-associated macrophages (TAM), or subsets thereof). In some embodiments, the chimeric protein of the present disclosure can increase the ratio of effector T cells to regulatory T cells.

[0063] For example, in some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, a receptor) associated with T cells. In some embodiments, the recognition domain directly or indirectly recruits T cells. In one embodiment, the recognition domain specifically binds to effector T cells. In some embodiments, the recognition domain directly or indirectly recruits effector T cells, for example, in some embodiments, to a treatment site (e.g., a location containing one or more types of diseased cells or cells whose therapeutic effect is to be modulated). Examples of effector T cells include cytotoxic T cells (e.g., αβTCR, CD3 + , CD8 + , CD45RO + ); CD4 +Effector T cells (e.g., αβ TCR, CD3 + , CD4 + , CCR7 + , CD62Lhi, IL-7R / CD127 + ); CD8 + Effector T cells (e.g., αβ TCR, CD3 + , CD8 + , CCR7 + , CD62Lhi, IL-7R / CD127 + ); Effector memory T cells (e.g., CD62Llow, CD44 + , TCR, CD3 + , IL-7R / CD127 + , IL-15R + , CCR7low); Central memory T cells (e.g., CCR7 + , CD62L + , CD27 + , or CCR7hi, CD44 + , CD62Lhi, TCR, CD3 + , IL-7R / CD127 + , IL-15R + ); CD62L + Effector T cells; Early effector memory T cells (CD27 + CD62L - ) and late effector memory T cells (CD27 - CD62L - )(TemE and TemL, respectively) CD8 + Effector memory T cells (TEM); CD127( + ) CD25(low / -) effector T cells; CD127( - ) CD25( - ) effector T cells; CD8 + Stem cell memory effector cells (TSCM) (e.g., CD44(low) CD62L(high) CD122(high) sca( + )); TH1 effector T cells (e.g., CXCR3 + , CXCR6 + and CCR5 + , or αβ TCR, CD3 + , CD4 +, IL-12R + , IFNγR + , CXCR3 + ); TH2 effector T cells (e.g., CCR3 + , CCR4 + and CCR8 + , or αβ TCR, CD3 + , CD4 + , IL-4R + , IL-33R + , CCR4 + , IL-17RB + , CRTH2 + ); TH9 effector T cells (e.g., αβ TCR, CD3 + , CD4 + ); TH17 effector T cells (e.g., αβ TCR, CD3 + , CD4 + , IL-23R + , CCR6 + , IL-1R + ); CD4 + CD45RO + CCR7 + effector T cells, ICOS + effector T cells; CD4 + CD45RO + CCR7( - ) effector T cells; and effector T cells that secrete IL-2, IL-4 and / or IFN-γ are included.

[0064] Examples of target T cell antigens (and, where applicable, also including extracellular domains) include, for example, CD8, CD3, SLAMF4, IL-2Rα, 4-1BB / TNFRSF9, IL-2Rβ, ALCAM, B7-1, IL-4R, B7-H3, BLAME / SLAMFS, CEACAM1, IL-6R, CCR3, IL-7Rα, CCR4, CXCR1 / IL-SRA, CCR5, CCR6, IL-10Rα, CCR7, IL-10Rβ, CCRS, IL-12Rβ1, CCR9, IL-12Rβ2, CD2, IL-13Rα1, IL-13, CD3, CD4, ILT2 / CDS5j, ILT3 / CDS5k, ILT4 / CDS5d, ILT5 / CDS5a, integrin α4 / CD49d, CD8, integrin αE / CD103, CD6, integrin αM / CD11b, CD8, integrin αX / CD11c, integrin β2 / CD18, KIR / CD158, CD27 / TNFRSF7, KIR2DL1, CD28, KIR2DL3, CD30 / TNFRSF8, KIR2DL4 / CD158d, CD31 / PECAM-1, KIR2DS4, CD40 ligand / TNFSF5, LAG-3, CD43, LAIR1, CD45, LAIR2, CD83, leukotriene B4-R1, CD84 / SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229 / SLAMF3, NKG2D, CD2F-10 / SLAMF9, NT-4, CD69, NTB-A / SLAMF6, common γ chain / IL-2Rγ, osteopontin, CRACC / SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4, RANK / TNFRSF11A, CX3CR1, CX3CL1, L-selectin, CXCR3, SIRPβ1, CXCR4, SLAM, CXCR6, TCCR / WSX-1, DNAM-1, thrombopoietin, EMMPRIN / CD147, TIM-1, EphB6, TIM-2, Fas / TNFRSF6, TIM-3, Fas ligand / TNFSF6, TIM-4, FcγRIII / CD16, TIM-6, TNFR1 / TNFRSF1A, granulysin, TNFRIII / TNFRSF1B, TRAIL R1 / TNFRSF10A, ICAM-1 / CD54, TRAILR2 / TNFRSF10B, ICAM-2 / CD102, TRAILR3 / TNFRSF10C, IFN-γR1, TRAILR4 / TNFRSF10D, IFN-γR2, TSLP, IL-1R1, and TSLP R. In various embodiments, the targeting portion of the chimeric protein binds to one or more of these examples of T cell antigens.

[0065] By way of non-limiting example, in various embodiments, the chimeric proteins of the disclosure have a targeting portion that targets one or more of the checkpoint markers expressed on T cells, such as PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, TIM3, and A2aR.

[0066] For example, in some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, a receptor) associated with B cells. In some embodiments, the recognition domain recruits B cells directly or indirectly, e.g., in some embodiments, to a site of treatment (e.g., a location containing one or more types of diseased cells or cells whose therapeutic effect is to be modulated). Examples of B cell antigens of interest include, for example, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD70, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a / b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138, CDw150, and B cell maturation antigen (BCMA). In various embodiments, the targeting portion of the chimeric protein binds to one or more of these examples of B cell antigens.

[0067] As another example, in some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, a receptor) associated with natural killer cells. In some embodiments, the recognition domain directly or indirectly recruits natural killer cells, for example, in some embodiments, to a treatment site (e.g., a location containing one or more types of diseased cells or cells whose therapeutic effect is modulated). Examples of natural killer cell antigens of interest include, for example, TIGIT, 2B4 / SLAMF4, KIR2DS4, CD155 / PVR, KIR3DL1, CD94, LMIR1 / CD300A, CD69, LMIR2 / CD300c, CRACC / SLAMF7, LMIR3 / CD300LF, DNAM-1, LMIR5 / CD300LB, Fc-εRII, LMIR6 / CD300LE, Fc-γR1 / CD64, MICA, Fc-γRIIB / CD32b, MICB, Fc-γRIIC / CD32c, MULT-1, Fc-γRIIA / CD32a, nectin-2 / CD112, Fc-γRIII / CD16, NKG2A, FcRH1 / IRTA5, NKG2C, FcRH2 / IRTA4, NKG2D, FcRH4 / IRTA1, NKp30, FcRH5 / IRTA2, NKp44, Fc receptor-like molecule 3 / CD16-2, NKp46 / NCR1, NKp80 / KLRF1, NTB-A / SLAMF6, Rae-1, Rae-1α, Rae-1β, Rae-1δ, H60, Rae-1ε, ILT2 / CD85j, Rae-1γ, ILT3 / CD85k, TREM-1, ILT4 / CD85d, TREM-2, ILT5 / CD85a, TREM-3, KIR / CD158, TREML1 / TLT-1, KIR2DL1, ULBP-1, KIR2DL3, ULBP-2, KIR2DL4 / CD158d, and ULBP-3. In various embodiments, the targeting portion of the chimeric protein binds to one or more of these examples of NK cell antigens.

[0068] In addition, in some embodiments, the recognition domain specifically binds to targets (e.g., antigens, receptors) associated with macrophages / monocytes. In some embodiments, the recognition domain directly or indirectly recruits macrophages / monocytes, for example, in some embodiments, to a treatment site (e.g., a location containing one or more types of diseased cells or cells whose therapeutic effect is to be modulated). Examples of target macrophage / monocyte antigens include, for example, SIRP1a, B7-1 / CD80, ILT4 / CD85d, B7-H1, ILT5 / CD85a, common beta chain, integrin alpha4 / CD49d, BLAME / SLAMF8, integrin alphaX / CD11c, CCL6 / C10, integrin beta2 / CD18, CD155 / PVR, integrin beta3 / CD61, CD31 / PECAM-1, Latexin, CD36 / SR-B3, Leukotriene B4R1, CD40 / TNFRSF5, LIMPIIISR-B2, CD43, LMIR1 / CD300A, CD45, LMIR2 / CD300c, CD68, LMIR3 / CD300LF, CD84 / SLAMF5, LMIR5 / CD300LB, CD97, LMIR6 / CD300LE, CD163, LRP-1, CD2F-10 / SLAMF9, MARCO, CRACC / SLAMF7, MD-1, ECF-L, MD-2, EMMPRIN / CD147, MGL2, Endoglin / CD105, Osteoactivin / GPNMB, Fc-gammaRI / CD64, Osteopontin, Fc-gammaRIIB / CD32b, PD-L2, Fc-gammaRIIC / CD32c, Siglec-3 / CD33, Fc-gammaRIIA / CD32a, SIGNR1 / CD209, Fc-gammaRIII / CD16, SLAM, GM-CSFRα, TCCR / WSX-1, ICAM-2 / CD102, TLR3, IFN-γR1, TLR4, IFN-γR2, TREM-1, IL-1RII, TREM-2, ILT2 / CD85j, TREM-3, ILT3 / CD85k, TREML1 / TLT-1, 2B4 / SLAMF4, IL-10Rα, ALCAM, IL-10Rβ, aminopeptidase N / ANPEP, ILT2 / CD85j, common β chain, ILT3 / CD85k, C1qR1 / CD93, ILT4 / CD85d, CCR1, ILT5 / CD85a, CCR2, integrin α4 / CD49d, CCR5, integrin αM / CD11b, CCR8, integrin αX / CD11c, CD155 / PVR, integrin β2 / CD18, CD14, integrin β3 / CD61, CD36 / SR-B3, LAIR1, CD43, LAIR2, CD45, leukotriene B4-R1, CD68, LIMPIIISR-B2, CD84 / SLAMF5, LMIR1 / CD300A, CD97, LMIR2 / CD300c, LMIR3 / CD300LF, coagulation factor III / tissue factor, LMIR5 / CD300LB, CX3CR1, CX3CL1, LMIR6 / CD300LE, CXCR4, LRP-1, CXCR6, M-CSF R, DEP-1 / CD148, MD-1, DNAM-1, MD-2, EMMPRIN / CD147, MMR, endoglin / CD105, NCAM-L1, Fc-γRI / CD64, PSGL-1, Fc-γRIIIICD16, RP105, G-CSF R, L-selectin, GM-CSF Rα, siglec-3 / CD33, HVEM / TNFRSF14, SLAM, ICAM-1 / CD54, TCCR / WSX-1, ICAM-2 / CD102, TREM-1, IL-6R, TREM-2, CXCR1 / IL-8RA, TREM-3, and TREML1 / TLT-1. In various embodiments, the targeting portion of the chimeric protein binds to one or more of these examples of macrophage / monocyte antigens.

[0069] Also, in some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, a receptor) associated with dendritic cells. In some embodiments, the recognition domain directly or indirectly recruits dendritic cells, for example, in some embodiments, to a treatment site (e.g., a location containing one or more types of diseased cells or cells whose therapeutic effect is modulated). Examples of dendritic cell antigens of interest include, for example, CLEC9A, XCR1, RANK, CD36 / SRB3, LOX-1 / SR-E1, CD68, MARCO, CD163, SR-A1 / MSR, CD5L, SREC-1, CL-P1 / COLEC12, SREC-II, LIMPIIISRB2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBB ligand / TNFSF9, IL-12 / IL-23 p40, 4-amino-1,8-Naphthalimide, ILT2 / CD85j, CCL21 / 6Ckine, ILT3 / CD85k, 8-oxo-dG, ILT4 / CD85d, 8D6A, ILT5 / CD85a, A2B5, Integrin α4 / CD49d, Aag, Integrin β2 / CD18, AMICA, Langerin, B7-2 / CD86, Leukotriene B4R1, B7-H3, LMIR1 / CD300A, BLAME / SLAMF8, LMIR2 / CD300c, C1qR1 / CD93, LMIR3 / CD300LF, CCR6, LMIR5 / CD300LB CCR7, LMIR6 / CD300LE, CD40 / TNFRSF5, MAG / Siglec-4-a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1 / CLEC5A, CD84 / SLAMF5, MMR, CD97, NCAML1, CD2F-10 / SLAMF9, Osteoactivin GPNMB, Chern23, PD-L2, CLEC-1, RP105, CLEC-2, CLEC-8, Siglec-2 / CD22, CRACC / SLAMF7, Siglec-3 / CD33, DC-SIGN, Siglec-5, DC-SIGNR / CD299, Siglec-6, DCAR, Siglec-7, DCIR / CLEC4A, Siglec-9, DEC-205, Siglec-10, Dectin-1 / CLEC7A, Siglec-F, Dectin-2 / CLEC6A, SIGNR1 / CD209, DEP-1 / CD148, SIGNR4, DLEC, SLAM, EMMPRIN / CD147, TCCR / WSX-1, Fc-γR1 / CD64, TLR3, Fc-γRIIB / CD32b, TREM-1, Fc-γRIIC / CD32c, TREM-2, Fc-γRIIA / CD32a, TREM-3, Fc-γRIII / CD16, TREML1 / TLT-1, ICAM-2 / CD102, and Vanilloid R1. In various embodiments, the targeting portion of the chimeric protein binds to one or more of these examples of DC antigens.,

[0070] In some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, a receptor) on an immune cell selected from, but not limited to, megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, or a subset thereof. In some embodiments, the recognition domain directly or indirectly mobilizes megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, or a subset thereof, for example, in some embodiments, to a treatment site (e.g., a location containing one or more types of diseased cells or cells whose therapeutic effect is being modulated).

[0071] In some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, a receptor) associated with megakaryocytes and / or platelets. Examples of megakaryocyte antigens and / or platelet antigens of interest include, for example, GPIIb / IIIa, GPIb, vWF, PF4, and TSP. In various embodiments, the targeting portion of the chimeric protein binds to one or more of these examples of megakaryocyte antigens and / or platelet antigens.

[0072] In some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, a receptor) associated with erythrocytes. Examples of erythrocyte antigens of interest include, for example, CD34, CD36, CD38, CD41a (platelet glycoprotein IIb / IIIa), CD41b (GPIIb), CD71 (transferrin receptor), CD105, glycoprotein A, glycoprotein C, c-kit, HLA-DR, H2 (MHC-II), and Rh antigens. In various embodiments, the targeting portion of the chimeric protein binds to one or more of these examples of erythrocyte antigens.

[0073] In some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, a receptor) associated with mast cells. Examples of mast cell antigens of interest include, for example, SCFR / CD117, Fc εExamples include RI, CD2, CD25, CD35, CD88, CD203c, C5R1, CMA1, FCER1A, FCER2, and TPSAB1. In various embodiments, the targeting portion of the chimeric protein binds to one or more of these examples of mast cell antigens.

[0074] In some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, a receptor) associated with basophils. Examples of basophil antigens of interest include, for example, Fc ε Examples include RI, CD203c, CD123, CD13, CD107a, CD107b, and CD164. In various embodiments, the targeting portion of the chimeric protein binds to one or more of these examples of basophil antigens.

[0075] In some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, a receptor) associated with neutrophils. Examples of neutrophil antigens of interest include, for example, 7D5, CD10 / CALLA, CD13, CD16 (FcRIII), CD18 protein (LFA-1, CR3, and p150,95), CD45, CD67, and CD177. In various embodiments, the targeting portion of the chimeric protein binds to one or more of these examples of neutrophil antigens.

[0076] In some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, a receptor) associated with eosinophils. Examples of eosinophil antigens of interest include, for example, CD35, CD44, and CD69. In various embodiments, the targeting portion of the chimeric protein binds to one or more of these eosinophil antigens.

[0077] In various embodiments, the recognition domain may bind to any suitable target, antigen, receptor, or cell surface marker known to those of skill in the art. In some embodiments, the antigen or cell surface marker is a tissue-specific marker. Examples of tissue-specific markers include endothelial cell surface markers such as ACE, CD14, CD34, CDH5, ENG, ICAM2, MCAM, NOS3, PECAM1, PROCR, SELE, SELP, TEK, THBD, VCAM1, VWF; smooth muscle cell surface markers such as ACTA2, MYH10, MYH11, MYH9, MYOCD; fibroblast (stromal) cell surface markers such as ALCAM, CD34, COL1A1, COL1A2, COL3A1, FAP, PH-4; epithelial cell surface markers such as CD1D, K6IRS2, KRT10, KRT13, KRT17, KRT18, KRT19, KRT4, KRT5, KRT8, MUC1, TACSTD1; angiogenesis markers such as CD13, TFNA, alpha-v-beta-3 (α V β3), E-selectin; and adipocyte surface markers such as ADIPOQ, FABP4, and RETN, but are not limited thereto. In various embodiments, the targeting portion of the chimeric protein binds to one or more of these antigens. In various embodiments, the targeting portion of the chimeric protein binds to one or more of the cells having these antigens.

[0078] In some embodiments, the recognition domain specifically binds to a target (e.g., an antigen, a receptor) associated with tumor cells. In some embodiments, the recognition domain directly or indirectly mobilizes tumor cells. For example, in some embodiments, the direct or indirect mobilization of tumor cells is towards one or more effector cells (e.g., immune cells as described herein) that can kill and / or suppress the tumor cells.

[0079] Tumor cells or cancer cells are associated with uncontrolled proliferation of cells or tissues, and / or abnormal increase in cell survival, and / or suppression of apoptosis, which interfere with the normal functions of the body's organs and body systems. For example, tumor cells include benign and malignant cancers, polyps, hyperplasia, and dormant tumors or micrometastases. Examples of tumor cells include basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain and central nervous system cancers, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive tract cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma, hepatocellular carcinoma, liver cancer, intraepithelial neoplasia, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell lung cancer), melanoma, myeloma, neuroblastoma, oral cancer (lips, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory tract cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary tract cancer, vulvar cancer, lymphomas including Hodgkin lymphoma and non-Hodgkin lymphoma and B-cell lymphoma (including low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, large tumor lesion NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenström macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, and other cancers and sarcomas, and post-transplant lymphoproliferative disorder (PTLD), and abnormal vascular proliferation associated with nevus syndrome, edema (e.g., edema associated with brain tumors), and cells of the MEGS syndrome, but are not limited thereto.

[0080] Examples of tumor cells or cancer cells include, but are not limited to, cancer (e.g., various subtypes including adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, and transitional cell carcinoma), sarcoma (e.g., including osteosarcoma and soft tissue sarcoma), leukemia (e.g., including acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, and hairy cell leukemia), lymphoma and myeloma (e.g., including Hodgkin and non-Hodgkin lymphoma, light chain myeloma, non-secretory myeloma, MGUS, and plasmacytoma), and central nervous system cancer (e.g., brain cancer (e.g., glioma (e.g., astrocytoma, oligodendroglioma, and ependymoma), meningioma, pituitary adenoma, and neuroma), and spinal cord tumors (e.g., meningioma and neurofibroma)).

[0081] Examples of tumor antigens include MART-1 / Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal cancer-related antigen (CRC)-0017-1A / GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T cell receptor / CD3ζ chain, MAGE family tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE family tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn, gp100Viral products such as Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig idiotype, p15, gp75, GM2 ganglioside and GD2 ganglioside, human papillomavirus proteins, Smad family tumor antigens, lmp-1, NA, Epstein-Barr virus-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1CT-7, c-erbB-2, CD19, CD20, CD22, CD30, CD33, CD37, CD56, CD70, CD74, CD138, AGS16, MUC1, GPNMB, Ep-CAM, PD-L1, PD-L2, PMSA, and BCMA (TNFRSF17), but are not limited to these. In various embodiments, the targeting portion of the chimeric protein binds to one or more of these tumor antigens. In one embodiment, the chimeric protein binds to HER2. In another embodiment, the chimeric protein binds to PD-L2.

[0082] In some embodiments, the chimeric proteins of the present disclosure have one or more of the targeting moieties that target immune cells selected from (i) T cells, B cells, dendritic cells, macrophages, NK cells, or subsets thereof, and (ii) one or more of the targeting moieties that target tumor cells together with any of the signaling agents described herein (e.g., consensus interferon or variants thereof). In certain embodiments, the chimeric proteins of the present disclosure have (i) a targeting moiety that targets T cells (including, but not limited to, effector T cells), and (ii) a targeting moiety that targets tumor cells together with any of the signaling agents described herein. In certain embodiments, the chimeric proteins of the present disclosure have (i) a targeting moiety that targets B cells, and (ii) a targeting moiety that targets tumor cells together with any of the signaling agents described herein. In certain embodiments, the chimeric proteins of the present disclosure have (i) a targeting moiety that targets dendritic cells, and (ii) a targeting moiety that targets tumor cells together with any of the signaling agents described herein. In certain embodiments, the chimeric proteins of the present disclosure have (i) a targeting moiety that targets macrophages, and (ii) a targeting moiety that targets tumor cells together with any of the signaling agents described herein. In certain embodiments, the chimeric proteins of the present disclosure have (i) a targeting moiety that targets NK cells, and (ii) a targeting moiety that targets tumor cells together with any of the signaling agents described herein.

[0083] As non-limiting examples, in various embodiments, the chimeric proteins of the present disclosure include (i) for example, CD8, SLAMF4, IL-2Rα, 4-1BB / TNFRSF9, IL-2Rβ, ALCAM, B7-1, IL-4R, B7-H3, BLAME / SLAMFS, CEACAM1, IL-6R, CCR3, IL-7Rα, CCR4, CXCR1 / IL-SRA, CCR5, CCR6, IL-10Rα, CCR7, IL-10Rβ, CCRS, IL-12Rβ1, CCR9, IL-12Rβ2, CD2, IL-13Rα1, IL-13, CD3, CD4, ILT2 / CDS5j, ILT3 / CDS5k, ILT4 / CDS5d, ILT5 / CDS5a, integrin α4 / CD49d, CDS, integrin αE / CD103, CD6, integrin αM / CD11b, CDS, integrin αX / CD11c, integrin β2 / CDlS, KIR / CD15S, CD27 / TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30 / TNFRSFS, KIR2DL4 / CD15Sd, CD31 / PECAM-1, KIR2DS4, CD40 ligand / TNFSF5, LAG-3, CD43, LAIR1, CD45, LAIR2, CDS3, leukotriene B4-R1, CDS4 / SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229 / SLAMF3, NKG2D, CD2F-10 / SLAMF9, NT-4, CD69, NTB-A / SLAMF6, common γ chain / IL-2Rγ, osteopontin, CRACC / SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4, RANK / TNFRSF11A, CX3CR1, CX3CL1, L-selectin, CXCR3, SIRPβ1, CXCR4, SLAM, CXCR6, TCCR / WSX-1, DNAM-1, thrombopoietin, EMMPRIN / CD147, TIM-1, EphB6, TIM-2, Fas / TNFRSF6, TIM-3, Fas ligand / TNFSF6, TIM-4, FcγRIII / CD16, TIM-6, TNFR1 / TNFRSF1A, granulysin, TNFRIII / TNFRSF1B, TRAIL R1 / TNFRSF10A, ICAM-1 / CD54, TRAILIt has a targeting moiety directed to T cells that is mediated to target R2 / TNFRSF10B, ICAM-2 / CD102, TRAILR3 / TNFRSF10C, IFN-γR1, TRAILR4 / TNFRSF10D, IFN-γR2, TSLP, IL-1R1, or TSLP R, and (ii) has a targeting moiety directed to tumor cells together with any of the signaling substances described herein (e.g., consensus interferon or variants thereof).

[0084] As non-limiting examples, in various embodiments, the chimeric proteins of the present disclosure have (i) a targeting moiety directed to one or more checkpoint markers expressed on T cells, such as PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, TIM3, and A2aR, and (ii) a targeting moiety directed to tumor cells together with any of the signaling substances described herein.

[0085] In various embodiments, the chimeric proteins of the present disclosure have one or more targeting moieties directed to PD-1. In some embodiments, the chimeric protein has one or more targeting moieties that selectively bind to the PD-1 polypeptide. In some embodiments, the chimeric protein comprises one or more antibodies, antibody derivatives or antibody formats, peptides or polypeptides, or fusion proteins that selectively bind to the PD-1 polypeptide.

[0086] In one embodiment, the targeting moiety comprises pembrolizumab (MK-3475, also known as Keytruda), an anti-PD-1 antibody, or a fragment thereof. Pembrolizumab and other humanized anti-PD-1 antibodies are disclosed in Hamid, et al. (2013) New England Journal of Medicine, 369(2): pp. 134-44, U.S. Patent No. 8,354,509, and International Publication No. 2009 / 114335, the disclosures of which are hereby incorporated by reference in their entirety. In an exemplary embodiment, pembrolizumab, or an antigen-binding fragment for use in the methods described herein, comprises a heavy chain comprising the amino acid sequence of (SEQ ID NO: 7), and / or a light chain comprising the amino acid sequence of (SEQ ID NO: 8).

[0087] In one embodiment, the targeting moiety comprises nivolumab (BMS-936558, MDX-1106, ONO-4538, also known as OPDIVO), an anti-PD-1 antibody, or a fragment thereof. Nivolumab (clone 5C4), and other human monoclonal antibodies that specifically bind to PD-1 are disclosed in U.S. Patent No. 8,008,449 and International Publication No. 2006 / 121168, the disclosures of which are hereby incorporated by reference in their entirety. In an exemplary embodiment, nivolumab or an antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of (SEQ ID NO: 9), and / or a light chain comprising the amino acid sequence of (SEQ ID NO: 10).

[0088] In certain embodiments, the targeting moiety comprises pidilizumab (also known as CT-011, hBAT or hBAT-1), an anti-PD-1 antibody, or a fragment thereof. Pidilizumab and other humanized anti-PD-I monoclonal antibodies are disclosed in U.S. Patent Application Publication No. 2008 / 0025980 and International Publication No. 2009 / 101611, the disclosures of which are incorporated herein by reference in their entirety. In exemplary embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof for use in the methods described herein comprises a light chain variable region having an amino acid sequence selected from SEQ ID NOs: 15-18 of U.S. Patent Application Publication No. 2008 / 0025980, namely, SEQ ID NO: 15 (SEQ ID NO: 11) of U.S. Patent Application Publication No. 2008 / 0025980, SEQ ID NO: 16 (SEQ ID NO: 12) of U.S. Patent Application Publication No. 2008 / 0025980, SEQ ID NO: 17 (SEQ ID NO: 13) of U.S. Patent Application Publication No. 2008 / 0025980, and SEQ ID NO: 18 (SEQ ID NO: 14) of U.S. Patent Application Publication No. 2008 / 0025980, and / or a heavy chain having an amino acid sequence selected from SEQ ID NOs: 20-24 of U.S. Patent Application Publication No. 2008 / 0025980, namely, SEQ ID NO: 20 (SEQ ID NO: 15) of U.S. Patent Application Publication No. 2008 / 0025980, SEQ ID NO: 21 (SEQ ID NO: 16) of U.S. Patent Application Publication No. 2008 / 0025980, SEQ ID NO: 22 (SEQ ID NO: 17) of U.S. Patent Application Publication No. 2008 / 0025980, SEQ ID NO: 23 (SEQ ID NO: 18) of U.S. Patent Application Publication No. 2008 / 0025980, and SEQ ID NO: 24 (SEQ ID NO: 19) of U.S. Patent Application Publication No. 2008 / 0025980.

[0089] In certain embodiments, the targeting moiety comprises a light chain (SEQ ID NO: 14) comprising SEQ ID NO: 18 of U.S. Patent Application Publication No. 2008 / 0025980 and a heavy chain (SEQ ID NO: 17) comprising SEQ ID NO: 22 of U.S. Patent Application Publication No. 2008 / 0025980.

[0090] In certain embodiments, the targeting moiety comprises AMP-514 (also known as MEDI-0680).

[0091] In certain embodiments, the targeting moiety comprises AMP-224, a PD-L2-Fc fusion protein as disclosed in WO 2010 / 027827 and WO 2011 / 066342, the entire disclosures of which are incorporated herein by reference. In such embodiments, the targeting moiety may comprise a targeting domain (SEQ ID NO: 20) comprising SEQ ID NO: 4 of WO 2010 / 027827, and / or a B7-DC fusion protein (SEQ ID NO: 21) comprising SEQ ID NO: 83 of WO 2010 / 027827.

[0092] In certain embodiments, the targeting moiety comprises either the AUNP12 peptide or other peptides as disclosed in US Patent Application Publication No. 2011 / 0318373 or US Patent No. 8,907,053. For example, the targeting moiety may comprise AUNP12 having the following sequence (SEQ ID NO: 22) (i.e., Compound 8 of US Patent Application Publication No. 2011 / 0318373 or SEQ ID NO: 49).

Chemical formula

[0093] In certain embodiments, the targeting moiety comprises 1E3, an anti-PD-1 antibody or a fragment thereof as disclosed in US Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 1E3 or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 23), and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 24).

[0094] In one embodiment, the targeting moiety comprises 1E8, an anti-PD-1 antibody as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, or a fragment thereof, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 1E8 or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25, and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 26.

[0095] In one embodiment, the targeting moiety comprises 1H3, an anti-PD-1 antibody as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, or a fragment thereof, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 1H3 or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 27), and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 28).

[0096] In one embodiment, the targeting moiety comprises, for example, an anti-PD-1 nanobody as disclosed in U.S. Patent No. 8,907,065 and International Publication No. 2008 / 071447, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, the anti-PD-1 nanobody comprises SEQ ID NOs: 347-351 of U.S. Patent No. 8,907,065 (SEQ ID NO: 347 (SEQ ID NO: 29) of U.S. Patent No. 8,907,065, SEQ ID NO: 348 (SEQ ID NO: 30) of U.S. Patent No. 8,907,065, SEQ ID NO: 349 (SEQ ID NO: 31) of U.S. Patent No. 8,907,065, SEQ ID NO: 350 (SEQ ID NO: 32) of U.S. Patent No. 8,907,065, and SEQ ID NO: 351 (SEQ ID NO: 33) of U.S. Patent No. 8,907,065).

[0097] In certain embodiments, it includes any one of the anti-PD-1 antibodies or a fragment thereof as disclosed in 2010 / 036959, and the entire content of those documents is incorporated herein by reference. In an exemplary embodiment, the antibody or antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 25-29 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 25 (SEQ ID NO: 34) of US Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 26 (SEQ ID NO: 35) of US Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 27 (SEQ ID NO: 36) of US Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 28 (SEQ ID NO: 37) of US Patent Application Publication No. 2011 / 0271358, and SEQ ID NO: 29 (SEQ ID NO: 38) of US Patent Application Publication No. 2011 / 0271358), and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 30-33 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 30 (SEQ ID NO: 39) of US Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 31 (SEQ ID NO: 40) of US Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 32 (SEQ ID NO: 41) of US Patent Application Publication No. 2011 / 0271358, and SEQ ID NO: 33 (SEQ ID NO: 42) of US Patent Application Publication No. 2011 / 0271358).

[0098] In various embodiments, the chimeric protein of the present disclosure comprises one or more anti-PD-1 antibodies or antibody fragments thereof selected from TSR-042 (Tesaro), REGN2810 (Regeneron Pharmaceuticals), PDR001 (Novartis Pharmaceuticals), and BGB-A317 (BeiGene).

[0099] In various embodiments, the chimeric proteins of the present disclosure have one or more targeting moieties that target PD-L1. In some embodiments, the chimeric protein has one or more targeting moieties that selectively bind to the PD-L1 polypeptide. In some embodiments, the chimeric protein comprises one or more antibodies, antibody derivatives or antibody formats, peptides or polypeptides, or fusion proteins that selectively bind to the PD-L1 polypeptide.

[0100] In certain embodiments, the targeting moiety comprises MEDI4736 (also known as durvalumab), an anti-PD-L1 antibody, or a fragment thereof. MEDI4736 is selective for PD-L1 and inhibits the binding of PD-L1 to the PD-1 receptor and the CD80 receptor. MEDI4736 and antigen-binding fragments thereof for use in the methods described herein comprise a heavy chain and a light chain, or a heavy chain variable region and a light chain variable region. The sequence of MEDI4736 is disclosed in International Publication No. WO 2016 / 06272, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, MEDI4736 or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 43, and / or a light chain comprising the amino acid sequence of SEQ ID NO: 44.

[0101] In an exemplary embodiment, MEDI4736 or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region (SEQ ID NO: 45) comprising the amino acid sequence of SEQ ID NO: 4 of International Publication No. WO 2016 / 06272, and / or a light chain variable region (SEQ ID NO: 46) comprising the amino acid sequence of SEQ ID NO: 3 of International Publication No. WO 2016 / 06272.

[0102] In certain embodiments, the targeting moiety comprises atezolizumab (also known as MPDL3280A, RG7446), an anti-PD-L1 antibody, or a fragment thereof. In an exemplary embodiment, atezolizumab or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 47, and / or a light chain comprising the amino acid sequence of SEQ ID NO: 48.

[0103] In certain embodiments, the targeting moiety comprises the anti-PD-L1 antibody Avelumab (also known as MSB0010718C) or a fragment thereof. In an exemplary embodiment, the Avelumab or antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain comprising the amino acid sequence of (SEQ ID NO: 49), and / or a light chain comprising the amino acid sequence of (SEQ ID NO: 50).

[0104] In certain embodiments, the targeting moiety comprises the anti-PD-L1 antibody BMS-936559 (also known as 12A4, MDX-1105) or a fragment thereof as disclosed in US Patent Application Publication No. 2013 / 0309250 and International Publication No. WO 2007 / 005874, the disclosures of which are hereby incorporated by reference in their entirety. In an exemplary embodiment, the BMS-936559 or antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 51), and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 52).

[0105] In certain embodiments, the targeting moiety comprises the anti-PD-L1 antibody 3G10 or a fragment thereof as disclosed in US Patent Application Publication No. 2013 / 0309250 and International Publication No. WO 2007 / 005874, the disclosures of which are hereby incorporated by reference in their entirety. In an exemplary embodiment, the 3G10 or antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 53), and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 54).

[0106] In one embodiment, the targeting moiety comprises 10A5, an anti-PD-L1 antibody as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and International Publication No. 2007 / 005874, or a fragment thereof, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 10A5 or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 55), and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 56).

[0107] In one embodiment, the targeting moiety comprises 5F8, an anti-PD-L1 antibody as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and International Publication No. 2007 / 005874, or a fragment thereof, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 5F8 or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 57), and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 58).

[0108] In one embodiment, the targeting moiety comprises 10H10, an anti-PD-L1 antibody as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and International Publication No. 2007 / 005874, or a fragment thereof, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 10H10, or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 59), and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 60).

[0109] In one embodiment, the targeting moiety comprises 1B12, an anti-PD-L1 antibody as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and International Publication No. 2007 / 005874, or a fragment thereof, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 1B12 or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 61), and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 62).

[0110] In one embodiment, the targeting moiety comprises 7H1, an anti-PD-L1 antibody as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and International Publication No. 2007 / 005874, or a fragment thereof, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 7H1 or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 63), and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 64).

[0111] In one embodiment, the targeting moiety comprises 11E6, an anti-PD-L1 antibody as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and International Publication No. 2007 / 005874, or a fragment thereof, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 11E6 or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 65), and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 66).

[0112] In one embodiment, the targeting moiety comprises 12B7 or a fragment thereof, which is an anti-PD-L1 antibody as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and International Publication No. 2007 / 005874, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 12B7 or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 67), and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 68).

[0113] In one embodiment, the targeting moiety comprises 13G4 or a fragment thereof, which is an anti-PD-L1 antibody as disclosed in U.S. Patent Application Publication No. 2013 / 0309250 and International Publication No. 2007 / 005874, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 13G4 or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 69), and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 70).

[0114] In one embodiment, the targeting moiety comprises 1E12 or a fragment thereof, which is an anti-PD-L1 antibody as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 1E12 or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 71), and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 72).

[0115] In one embodiment, the targeting moiety comprises 1F4 or a fragment thereof, which is an anti-PD-L1 antibody as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 1F4 or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 73), and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 74).

[0116] In one embodiment, the targeting moiety comprises 2G11, an anti-PD-L1 antibody as disclosed in US Patent Application Publication No. 2014 / 0044738, or a fragment thereof, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 2G11 or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 75), and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 76).

[0117] In one embodiment, the targeting moiety comprises 3B6, an anti-PD-L1 antibody as disclosed in US Patent Application Publication No. 2014 / 0044738, or a fragment thereof, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 3B6 or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 77), and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 78).

[0118] In one embodiment, the targeting moiety comprises 3D10, an anti-PD-L1 antibody as disclosed in US Patent Application Publication No. 2014 / 0044738 and International Publication No. 2012 / 145493, or a fragment thereof, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, 3D10 or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 79), and / or a light chain variable region comprising the amino acid sequence of (SEQ ID NO: 80).

[0119] In certain embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in U.S. Patent Application Publication No. 2011 / 0271358 and International Publication No. 2010 / 036959, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, the antibody or antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 34-38 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 34 (SEQ ID NO: 81) of U.S. Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 35 (SEQ ID NO: 82) of U.S. Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 36 (SEQ ID NO: 83) of U.S. Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 37 (SEQ ID NO: 84) of U.S. Patent Application Publication No. 2011 / 0271358, and SEQ ID NO: 38 (SEQ ID NO: 85) of U.S. Patent Application Publication No. 2011 / 0271358), and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 39-42 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 39 (SEQ ID NO: 86) of U.S. Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 40 (SEQ ID NO: 87) of U.S. Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 41 (SEQ ID NO: 88) of U.S. Patent Application Publication No. 2011 / 0271358, and SEQ ID NO: 42 (SEQ ID NO: 89) of U.S. Patent Application Publication No. 2011 / 0271358).

[0120] In certain embodiments, the targeting moiety comprises 2.7A4 or a fragment thereof, an anti-PD-L1 antibody as disclosed in International Publication No. 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 2.7A4 or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 of International Publication No. 2011 / 066389 (SEQ ID NO: 90), and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7 of International Publication No. 2011 / 066389 (SEQ ID NO: 91).

[0121] In one embodiment, the targeting moiety comprises 2.9D10, an anti-PD-L1 antibody as disclosed in WO 2011 / 066389, US Patent No. 8,779,108, and US Patent Application Publication No. 2014 / 0356353, or a fragment thereof, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 2.9D10 or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12 of WO 2011 / 066389 (SEQ ID NO: 92), and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17 of WO 2011 / 066389 (SEQ ID NO: 93).

[0122] In one embodiment, the targeting moiety comprises 2.14H9, an anti-PD-L1 antibody as disclosed in WO 2011 / 066389, US Patent No. 8,779,108, and US Patent Application Publication No. 2014 / 0356353, or a fragment thereof, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 2.14H9 or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 22 of WO 2011 / 066389 (SEQ ID NO: 94), and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 27 of WO 2011 / 066389 (SEQ ID NO: 95).

[0123] In one embodiment, the targeting moiety comprises 2.20A8, an anti-PD-L1 antibody as disclosed in WO 2011 / 066389, US Patent No. 8,779,108, and US Patent Application Publication No. 2014 / 0356353, or a fragment thereof, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 2.20A8 or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 32 of WO 2011 / 066389 (SEQ ID NO: 96), and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 37 of WO 2011 / 066389 (SEQ ID NO: 97).

[0124] In one embodiment, the targeting moiety comprises 3.15G8, an anti-PD-L1 antibody as disclosed in WO 2011 / 066389, US Patent No. 8,779,108, and US Patent Application Publication No. 2014 / 0356353, or a fragment thereof, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 3.15G8 or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 42 of WO 2011 / 066389 (SEQ ID NO: 98), and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 47 of WO 2011 / 066389 (SEQ ID NO: 99).

[0125] In one embodiment, the targeting moiety comprises 3.18G1, an anti-PD-L1 antibody as disclosed in WO 2011 / 066389, US Patent No. 8,779,108, and US Patent Application Publication No. 2014 / 0356353, or a fragment thereof, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 3.18G1, or an antigen-binding fragment thereof for use in the methods described herein, comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 52 of WO 2011 / 066389 (SEQ ID NO: 100), and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 57 of WO 2011 / 066389 (SEQ ID NO: 101).

[0126] In one embodiment, the targeting moiety comprises 2.7A4OPT, an anti-PD-L1 antibody as disclosed in WO 2011 / 066389, US Patent No. 8,779,108, US Patent Application Publication No. 2014 / 0356353, and US Patent Application Publication No. 2014 / 0356353, or a fragment thereof, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, 2.7A4OPT or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 62 of WO 2011 / 066389 (SEQ ID NO: 102), and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 67 of WO 2011 / 066389 (SEQ ID NO: 103).

[0127] In one embodiment, the targeting moiety comprises 2.14H9OPT or a fragment thereof, which is an anti-PD-L1 antibody as disclosed in International Publication No. WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353, the disclosures of which are hereby incorporated by reference in their entireties. In an exemplary embodiment, the 2.14H9OPT or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain variable region (SEQ ID NO: 104) comprising the amino acid sequence of SEQ ID NO: 72 of International Publication No. WO 2011 / 066389, and / or a light chain variable region (SEQ ID NO: 105) comprising the amino acid sequence of SEQ ID NO: 77 of International Publication No. WO 2011 / 066389.

[0128] In one embodiment, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO 2016 / 061142, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, the antibody or antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 18, 30, 38, 46, 50, 54, 62, 70, and 78 of WO 2016 / 061142 (SEQ ID NO: 18 (SEQ ID NO: 106) of WO 2016 / 061142, SEQ ID NO: 30 (SEQ ID NO: 107) of WO 2016 / 061142, SEQ ID NO: 38 (SEQ ID NO: 108) of WO 2016 / 061142, SEQ ID NO: 46 (SEQ ID NO: 109) of WO 2016 / 061142, SEQ ID NO: 50 (SEQ ID NO: 110) of WO 2016 / 061142, SEQ ID NO: 54 (SEQ ID NO: 111) of WO 2016 / 061142, SEQ ID NO: 62 (SEQ ID NO: 112) of WO 2016 / 061142, SEQ ID NO: 70 (SEQ ID NO: 113) of WO 2016 / 061142, and SEQ ID NO: 78 (SEQ ID NO: 114) of WO 2016 / 061142), and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 22, 26, 34, 42, 58, 66, 74, 82, and 86 of WO 2016 / 061142 (SEQ ID NO: 22 (SEQ ID NO: 115) of WO 2016 / 061142, SEQ ID NO: 26 (SEQ ID NO: 116) of WO 2016 / 061142, SEQ ID NO: 34 (SEQ ID NO: 117) of WO 2016 / 061142, SEQ ID NO: 42 (SEQ ID NO: 118) of WO 2016 / 061142, SEQ ID NO: 58 (SEQ ID NO: 119) of WO 2016 / 061142, SEQ ID NO: 66 (SEQ ID NO: 120) of WO 2016 / 061142, SEQ ID NO: 74 (SEQ ID NO: 121) of WO 2016 / 061142, SEQ ID NO: 82 (SEQ ID NO: 122) of WO 2016 / 061142, and SEQ ID NO: 86 (SEQ ID NO: 123) of WO 2016 / 061142).

[0129] In one embodiment, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO 2016 / 022630, the entire content of which is incorporated herein by reference. In an exemplary embodiment, the antibody or antigen-binding fragment thereof for use in the methods described herein has a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, and 46 of WO 2016 / 022630 (SEQ ID NO: 2 (SEQ ID NO: 124) of WO 2016 / 022630, SEQ ID NO: 6 (SEQ ID NO: 125) of WO 2016 / 022630, SEQ ID NO: 10 (SEQ ID NO: 126) of WO 2016 / 022630, SEQ ID NO: 14 (SEQ ID NO: 127) of WO 2016 / 022630, SEQ ID NO: 18 (SEQ ID NO: 128) of WO 2016 / 022630, SEQ ID NO: 22 (SEQ ID NO: 129) of WO 2016 / 022630, SEQ ID NO: 26 (SEQ ID NO: 130) of WO 2016 / 022630, SEQ ID NO: 30 (SEQ ID NO: 131) of WO 2016 / 022630, SEQ ID NO: 34 (SEQ ID NO: 132) of WO 2016 / 022630, SEQ ID NO: 38 (SEQ ID NO: 133) of WO 2016 / 022630, SEQ ID NO: 42 (SEQ ID NO: 134) of WO 2016 / 022630, and SEQ ID NO: 46 (SEQ ID NO: 135) of WO 2016 / 022630), and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 of WO 2016 / 022630 (SEQ ID NO: 4 (SEQ ID NO: 136) of WO 2016 / 022630, SEQ ID NO: 8 (SEQ ID NO: 137) of WO 2016 / 022630, SEQ ID NO: 12 (SEQ ID NO: 138) of WO 2016 / 022630, SEQ ID NO: 16 (SEQ ID NO: 139) of WO 2016 / 022630, SEQ ID NO: 20 (SEQ ID NO: 140) of WO 2016 / 022630, SEQ ID NO: 24 (SEQ ID NO: 141) of WO 2016 / 022630, SEQ ID NO: 28 (SEQ ID NO: 142) of WO 2016 / 022630, SEQ ID NO: 32 (SEQ ID NO: 143) of WO 2016 / 022630, SEQ ID NO: 36 (SEQ ID NO: 144) of WO 2016 / 022630,It includes a light chain comprising an amino acid sequence selected from SEQ ID NO: 40 (SEQ ID NO: 145), SEQ ID NO: 44 (SEQ ID NO: 146), and SEQ ID NO: 48 (SEQ ID NO: 147) of WO 2016 / 022630.

[0130] In certain embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO 2015 / 112900, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, the antibody or antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 38, 50, 82, and 86 of WO 2015 / 112900 (SEQ ID NO: 38 (SEQ ID NO: 148), SEQ ID NO: 50 (SEQ ID NO: 149), SEQ ID NO: 82 (SEQ ID NO: 150), and SEQ ID NO: 86 (SEQ ID NO: 151) of WO 2015 / 112900), and / or a light chain comprising an amino acid sequence selected from SEQ ID NO: 42, 46, 54, 58, 62, 66, 70, 74, and 78 of WO 2015 / 112900 (SEQ ID NO: 42 (SEQ ID NO: 152), SEQ ID NO: 46 (SEQ ID NO: 153), SEQ ID NO: 54 (SEQ ID NO: 154), SEQ ID NO: 58 (SEQ ID NO: 155), SEQ ID NO: 62 (SEQ ID NO: 156), SEQ ID NO: 66 (SEQ ID NO: 157), SEQ ID NO: 70 (SEQ ID NO: 158), SEQ ID NO: 74 (SEQ ID NO: 159), and SEQ ID NO: 78 (SEQ ID NO: 160) of WO 2015 / 112900).

[0131] In certain embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies disclosed in WO 2010 / 077634 and US Patent No. 8,217,149, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain region (SEQ ID NO: 161) comprising the amino acid sequence of SEQ ID NO: 20 of WO 2010 / 077634, and / or a light chain variable region (SEQ ID NO: 162) comprising the amino acid sequence of SEQ ID NO: 21 of WO 2010 / 077634.

[0132] In certain embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies obtainable from hybridomas available under the CNCM accession numbers CNCM I-4122, CNCM I-4080, and CNCM I-4081, as disclosed in US Patent Application Publication No. 2012 / 0039906, the entire disclosure of which is incorporated herein by reference.

[0133] In certain embodiments, the targeting moiety comprises an anti-PD-L1 nanobody as disclosed, for example, in US Patent No. 8,907,065 and WO 2008 / 071447, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, the anti-PD-L1 nanobody comprises SEQ ID NOs: 394-399 of US Patent No. 8,907,065 (SEQ ID NO: 394 (SEQ ID NO: 163), SEQ ID NO: 395 (SEQ ID NO: 164), SEQ ID NO: 396 (SEQ ID NO: 165), SEQ ID NO: 397 (SEQ ID NO: 166), SEQ ID NO: 398 (SEQ ID NO: 167), and SEQ ID NO: 399 (SEQ ID NO: 168) of US Patent No. 8,907,065).

[0134] In various embodiments, the chimeric proteins of the present disclosure have one or more targeting moieties that target PD-L2. In some embodiments, the chimeric protein has one or more targeting moieties that selectively bind to the PD-L2 polypeptide. In some embodiments, the chimeric protein comprises one or more antibodies, antibody derivatives or antibody formats, peptides or polypeptides, or fusion proteins that selectively bind to the PD-L2 polypeptide.

[0135] In certain embodiments, the targeting moiety comprises an anti-PD-L2 nanobody, such as those disclosed in, for example, U.S. Patent No. 8,907,065 and International Publication No. 2008 / 071447, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, those anti-PD-L2 nanobodies include SEQ ID NOs: 449-455 of U.S. Patent No. 8,907,065 (SEQ ID NO: 449 (SEQ ID NO: 169) of U.S. Patent No. 8,907,065, SEQ ID NO: 450 (SEQ ID NO: 170) of U.S. Patent No. 8,907,065, SEQ ID NO: 451 (SEQ ID NO: 171) of U.S. Patent No. 8,907,065, SEQ ID NO: 452 (SEQ ID NO: 172) of U.S. Patent No. 8,907,065, SEQ ID NO: 453 (SEQ ID NO: 173) of U.S. Patent No. 8,907,065, SEQ ID NO: 454 (SEQ ID NO: 174) of U.S. Patent No. 8,907,065, and SEQ ID NO: 455 (SEQ ID NO: 175) of U.S. Patent No. 8,907,065).

[0136] In one embodiment, the targeting moiety comprises any one of the anti-PD-L2 antibodies disclosed in U.S. Patent Application Publication No. 2011 / 0271358 and International Publication No. 2010 / 036959, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, the antibody or antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 43-47 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 43 (SEQ ID NO: 176) of U.S. Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 44 (SEQ ID NO: 177) of U.S. Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 45 (SEQ ID NO: 178) of U.S. Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 46 (SEQ ID NO: 179) of U.S. Patent Application Publication No. 2011 / 0271358, and SEQ ID NO: 47 (SEQ ID NO: 180) of U.S. Patent Application Publication No. 2011 / 0271358), and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 48-51 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 48 (SEQ ID NO: 181) of U.S. Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 49 (SEQ ID NO: 182) of U.S. Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 50 (SEQ ID NO: 183) of U.S. Patent Application Publication No. 2011 / 0271358, and SEQ ID NO: 51 (SEQ ID NO: 184) of U.S. Patent Application Publication No. 2011 / 0271358).

[0137] In various embodiments, the targeting moiety of the invention can include a sequence that targets PD-1, PD-L1, and / or PD-L2 and that is at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to any of the sequences disclosed herein (e.g., a sequence that is about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to any of the sequences disclosed herein).

[0138] In various embodiments, the targeting moiety of the invention can include any combination of a heavy chain sequence, a light chain sequence, a heavy chain variable region sequence, a light chain variable region sequence, a complementarity determining region (CDR) sequence, and a framework region sequence that targets PD-1, PD-L1, and / or PD-L2 as disclosed herein.

[0139] Other antibodies, antibody derivatives or antibody formats, peptides or polypeptides, or fusion proteins that selectively bind to or target PD-1, PD-L1, and / or PD-L2 are described in International Publication No. WO 2011 / 066389, US Patent Application Publication No. US 2008 / 0025980, US Patent Application Publication No. US 2013 / 0034559, US Patent No. 8,779,108, US Patent Application Publication No. US 2014 / 0356353, US Patent No. 8,609,089, US Patent Application Publication No. US 2010 / 028330, US Patent Application Publication No. US 2012 / 0114649, International Publication No. WO 2010 / 027827, International Publication No. WO 2011 / 066342, US Patent No. 8,907,065, International Publication No. WO 2016 / 062722, International Publication No. WO 2009 / 101611, International Publication No. WO 2010 / 027827, International Publication No. WO 2011 / 066342, International Publication No. WO 2007 / 005874, International Publication No. WO 2001 / 014556, US Patent Application Publication No. US 2011 / 0271358, International Publication No. WO 2010 / 036959, International Publication No. WO 2010 / 077634, US Patent No. 8,217,149, US Patent Application Publication No. US 2012 / 0039906, International Publication No. WO 2012 / 145493, US Patent Application Publication No. US 2011 / 0318373, US Patent No. 8,779,108, US Patent Application Publication No. US 2014 / 0044738, International Publication No. WO 2009 / 089149, International Publication No. WO 2007 / 00587, International Publication No. WO 2016 / 061142, International Publication No. WO 2016 / 02263, International Publication No. WO 2010 / 077634, and International Publication No. WO 2015 / 112900, the disclosures of which are hereby incorporated by reference in their entirety.

[0140] In certain embodiments, the chimeric proteins of the disclosure have (i) a targeting moiety that directs T cells, for example, mediated by targeting to CD8, and (ii) a targeting moiety that directs tumor cells together with any of the signaling agents described herein (e.g., consensus interferon or variants thereof). In certain embodiments, the chimeric proteins of the disclosure have a targeting moiety that directs CD8 on T cells and a second targeting moiety that directs PD-L1 or PD-L2 on tumor cells.

[0141] In certain embodiments, the chimeric protein of the present disclosure has (i) a targeting moiety that targets T cells, which is mediated, for example, by targeting to CD4, and (ii) a targeting moiety that targets tumor cells together with any of the signaling substances described herein (e.g., consensus interferon or a variant thereof). In certain embodiments, the chimeric protein of the present disclosure has a targeting moiety that targets CD4 on T cells and a second targeting moiety that targets PD-L1 or PD-L2 on tumor cells.

[0142] In certain embodiments, the chimeric protein of the present disclosure has (i) a targeting moiety that targets T cells, which is mediated, for example, by targeting to CD3, CXCR3, CCR4, CCR9, CD70, CD103, or one or more immune checkpoint markers, and (ii) a targeting moiety that targets tumor cells together with any of the signaling substances described herein (e.g., consensus interferon or a variant thereof). In certain embodiments, the chimeric protein of the present disclosure has a targeting moiety that targets CD3 on T cells and a second targeting moiety that targets PD-L1 or PD-L2 on tumor cells.

[0143] In some embodiments, the chimeric protein of the present disclosure has one or more targeting moieties that target CD3 expressed on T cells. In some embodiments, the chimeric protein has one or more targeting moieties that selectively bind to the CD3 polypeptide. In some embodiments, the chimeric protein comprises one or more antibodies, antibody derivatives or antibody formats, peptides or polypeptides, or fusion proteins that selectively bind to the CD3 polypeptide.

[0144] In certain embodiments, the targeting moiety comprises muromonab-CD3, an anti-CD3 antibody (also known as orthoclone OKT3), or a fragment thereof. Muromonab-CD3 is disclosed in U.S. Patent No. 4,361,549 and Wilde, et al. (1996) 51: pp. 865-894, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, muromonab-CD3 or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain comprising the amino acid sequence of (SEQ ID NO: 185), and / or a light chain comprising the amino acid sequence of (SEQ ID NO: 186).

[0145] In certain embodiments, the targeting moiety comprises otelixizumab, an anti-CD3 antibody, or a fragment thereof. Otelixizumab is disclosed in U.S. Patent Application Publication No. 2016 / 0000916 and Chatenoud, et al. (2012) 9: pp. 372-381, the entire disclosures of which are incorporated herein by reference. In an exemplary embodiment, otelixizumab or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 187, and / or a light chain comprising the amino acid sequence of SEQ ID NO: 188.

[0146] In certain embodiments, the targeting moiety comprises teprilizumab (also known as MGA031 and hOKT3γ1(Ala-Ala)), an anti-CD3 antibody, or a fragment thereof. Teprilizumab is disclosed in Chatenoud, et al. (2012) 9: pp. 372-381, the entire disclosure of which is incorporated herein by reference. In an exemplary embodiment, teprilizumab or an antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 189, and / or a light chain comprising the amino acid sequence of SEQ ID NO: 190.

[0147] In certain embodiments, the targeting moiety comprises bispecificity monoclonal antibody against CD3 antibody visilizumab (also known as Nuvion®, HuM291) or a fragment thereof. Visilizumab is disclosed in U.S. Patent No. 5,834,597, International Publication No. 2004 / 052397, and Cole, et al. Transplantation, (1999) 68: pp. 563-571, the disclosures of which are incorporated herein by reference in their entirety. In an exemplary embodiment, the visilizumab or its antigen-binding fragment for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 191, and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 192.

[0148] In certain embodiments, the targeting moiety comprises foralumab (also known as NI-0401), an anti-CD3 antibody, or a fragment thereof. In various embodiments, the targeting moiety comprises any one of the anti-CD3 antibodies disclosed in U.S. Patent Application Publication No. 2014 / 0193399, U.S. Patent No. 7,728,114, U.S. Patent Application Publication No. 2010 / 0183554, and U.S. Patent No. 8,551,478, the disclosures of which are incorporated herein by reference in their entirety.

[0149] In an exemplary embodiment, the anti-CD3 antibody or its antigen-binding fragment for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequences of SEQ ID NOs: 2 and 6 of U.S. Patent No. 7,728,114 (SEQ ID NO: 2 (SEQ ID NO: 193) of U.S. Patent No. 7,728,114 and SEQ ID NO: 6 (SEQ ID NO: 194) of U.S. Patent No. 7,728,114), and / or a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 4 and 8 of U.S. Patent No. 7,728,114 (SEQ ID NO: 4 (SEQ ID NO: 195) of U.S. Patent No. 7,728,114 and SEQ ID NO: 8 (SEQ ID NO: 196) of U.S. Patent No. 7,728,114).

[0150] In certain embodiments, the targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 of U.S. Patent No. 7,728,114 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4 of U.S. Patent No. 7,728,114. In certain embodiments, the targeting moiety comprises any one of the anti-CD3 antibodies disclosed in U.S. Patent Application Publication No. 2016 / 0168247, the entire content of which is incorporated herein by reference. In an exemplary embodiment, the antibody or antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 6-9 of U.S. Patent Application Publication No. 2016 / 0168247 (SEQ ID NO: 6 (SEQ ID NO: 197) of U.S. Patent Application Publication No. 2016 / 0168247, SEQ ID NO: 7 (SEQ ID NO: 198) of U.S. Patent Application Publication No. 2016 / 0168247, SEQ ID NO: 8 (SEQ ID NO: 199) of U.S. Patent Application Publication No. 2016 / 0168247, and SEQ ID NO: 9 (SEQ ID NO: 200) of U.S. Patent Application Publication No. 2016 / 0168247), and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 10-12 of U.S. Patent Application Publication No. 2016 / 0168247 (SEQ ID NO: 10 (SEQ ID NO: 201) of U.S. Patent Application Publication No. 2016 / 0168247, SEQ ID NO: 11 (SEQ ID NO: 202) of U.S. Patent Application Publication No. 2016 / 0168247, and SEQ ID NO: 12 (SEQ ID NO: 203) of U.S. Patent Application Publication No. 2016 / 0168247).

[0151] In certain embodiments, the targeting moiety comprises any one of the anti-CD3 antibodies disclosed in U.S. Patent Application Publication No. 2015 / 0175699, the entire content of which is incorporated herein by reference. In an exemplary embodiment, the antibody or antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 9 (SEQ ID NO: 204) of U.S. Patent Application Publication No. 2015 / 0175699, and / or a light chain comprising an amino acid sequence selected from SEQ ID NO: 10 (SEQ ID NO: 205) of U.S. Patent Application Publication No. 2015 / 0175699.

[0152] In one embodiment, the targeting moiety comprises any one of the anti-CD3 antibodies disclosed in U.S. Patent No. 8,784,821, the entire content of which is incorporated herein by reference. In an exemplary embodiment, the antibody or antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, and 114 of U.S. Patent No. 8,784,821 (SEQ ID NO: 2 (SEQ ID NO: 206) of U.S. Patent No. 8,784,821, SEQ ID NO: 18 (SEQ ID NO: 207) of U.S. Patent No. 8,784,821, SEQ ID NO: 34 (SEQ ID NO: 208) of U.S. Patent No. 8,784,821, SEQ ID NO: 50 (SEQ ID NO: 209) of U.S. Patent No. 8,784,821, SEQ ID NO: 66 (SEQ ID NO: 210) of U.S. Patent No. 8,784,821, SEQ ID NO: 82 (SEQ ID NO: 211) of U.S. Patent No. 8,784,821, SEQ ID NO: 98 (SEQ ID NO: 212) of U.S. Patent No. 8,784,821, and SEQ ID NO: 114 (SEQ ID NO: 213) of U.S. Patent No. 8,784,821), and / or a light chain comprising an amino acid sequence selected from SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, and 122 of U.S. Patent No. 8,784,821 (SEQ ID NO: 10 (SEQ ID NO: 214) of U.S. Patent No. 8,784,821, SEQ ID NO: 26 (SEQ ID NO: 215) of U.S. Patent No. 8,784,821, SEQ ID NO: 42 (SEQ ID NO: 216) of U.S. Patent No. 8,784,821, SEQ ID NO: 58 (SEQ ID NO: 217) of U.S. Patent No. 8,784,821, SEQ ID NO: 74 (SEQ ID NO: 218) of U.S. Patent No. 8,784,821, SEQ ID NO: 90 (SEQ ID NO: 219) of U.S. Patent No. 8,784,821, SEQ ID NO: 106 (SEQ ID NO: 220) of U.S. Patent No. 8,784,821, and SEQ ID NO: 122 (SEQ ID NO: 221) of U.S. Patent No. 8,784,821).

[0153] In certain embodiments, the targeting moiety comprises any one of the anti-CD3 binding constructs disclosed in U.S. Patent Application Publication No. 2015 / 0118252, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, the antibody or antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 6 and 86 of U.S. Patent Application Publication No. 2015 / 0118252 (SEQ ID NO: 6 (SEQ ID NO: 222) of U.S. Patent Application Publication No. 2015 / 0118252 and SEQ ID NO: 86 (SEQ ID NO: 223) of U.S. Patent Application Publication No. 2015 / 0118252), and / or a light chain comprising an amino acid sequence selected from SEQ ID NO: 3 of U.S. Patent Application Publication No. 2015 / 0175699 (SEQ ID NO: 3 (SEQ ID NO: 224) of U.S. Patent Application Publication No. 2015 / 0118252).

[0154] In certain embodiments, the targeting moiety comprises any one of the anti-CD3 binding proteins disclosed in U.S. Patent Application Publication No. 2016 / 0039934, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, the antibody or antigen-binding fragment thereof for use in the methods described herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 6-9 of U.S. Patent Application Publication No. 2016 / 0039934 (SEQ ID NO: 6 (SEQ ID NO: 225) of U.S. Patent Application Publication No. 2016 / 0039934, SEQ ID NO: 7 (SEQ ID NO: 226) of U.S. Patent Application Publication No. 2016 / 0039934, SEQ ID NO: 8 (SEQ ID NO: 227) of U.S. Patent Application Publication No. 2016 / 0039934, and SEQ ID NO: 9 (SEQ ID NO: 228) of U.S. Patent Application Publication No. 2016 / 0039934), and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 1-4 of U.S. Patent Application Publication No. 2016 / 0039934 (SEQ ID NO: 1 (SEQ ID NO: 229) of U.S. Patent Application Publication No. 2016 / 0039934, SEQ ID NO: 2 (SEQ ID NO: 230) of U.S. Patent Application Publication No. 2016 / 0039934, SEQ ID NO: 3 (SEQ ID NO: 231) of U.S. Patent Application Publication No. 2016 / 0039934, and SEQ ID NO: 4 (SEQ ID NO: 232) of U.S. Patent Application Publication No. 2016 / 0039934).

[0155] In various embodiments, the targeting moiety of the invention can comprise a sequence that targets CD3 and is at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% identical to any of the sequences disclosed herein (e.g., a sequence that is about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to any of the sequences disclosed herein).

[0156] In various embodiments, the targeting moiety of the present invention can include any combination of a heavy chain sequence, a light chain sequence, a heavy chain variable region sequence, a light chain variable region sequence, a complementarity determining region (CDR) sequence, and a framework region sequence that targets CD3 as disclosed herein. In various embodiments, the targeting moiety of the present invention can include the heavy chain sequence, the light chain sequence, the heavy chain variable region sequence, the light chain variable region sequence, the complementarity determining region (CDR) sequence, and the framework region sequence of any one of the CD3-specific antibodies including, but not limited to, X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, WT31, and F101.01. These CD3-specific antibodies are well known in the art and are specifically described in Tunnacliffe, (1989) Int. Immunol. 1, pp. 546-550, the entire disclosure of which is incorporated herein by reference.

[0157] Other antibodies, antibody derivatives or antibody formats, peptides or polypeptides, or fusion proteins that selectively bind to or target CD3 are disclosed in US Patent Application Publication No. 2016 / 0000916, US Patent Nos. 4,361,549, 5,834,597, 6,491,916, 6,406,696, 6,143,297, 6,750,325, and International Publication No. 2004 / 052397, the entire disclosures of which are incorporated herein by reference.

[0158] In certain embodiments, the chimeric protein of the present disclosure has (i) a targeting moiety that is directed to T cells, for example, mediated by targeting to PD-1, and (ii) a targeting moiety that is directed to tumor cells together with any one of the signaling agents (e.g., consensus interferon or variants thereof) described herein.

[0159] As a non-limiting example, in various embodiments, the chimeric proteins of the present disclosure have a B-cell targeting moiety that mediates targeting to, for example, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD70, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a / b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138, or CDw150, and (ii) a tumor cell targeting moiety together with any of the signaling substances described herein (e.g., consensus interferon or a variant thereof). In certain embodiments, the chimeric proteins of the present disclosure have a targeting moiety that targets CD20.

[0160] In certain embodiments, the chimeric proteins of the present disclosure have a B-cell targeting moiety that mediates targeting to, for example, CD19, CD20, or CD70, and (ii) a tumor cell targeting moiety together with any of the signaling substances described herein (e.g., consensus interferon or a variant thereof).

[0161] In certain embodiments, the chimeric proteins of the present disclosure have a B-cell targeting moiety that mediates targeting to, for example, CD20, and (ii) a tumor cell targeting moiety together with any of the signaling substances described herein (e.g., consensus interferon or a variant thereof). In certain embodiments, the chimeric proteins of the present disclosure have a targeting moiety that targets CD20 on B cells and a second targeting moiety that targets PD-L1 or PD-L2 on tumor cells. As an example, in some embodiments, the CD20 targeting moiety is a recombinant heavy-chain-only antibody (VHH) having the following sequence.

Chemical formula

[0162] As a non-limiting example, in various embodiments, the chimeric proteins of the present disclosure have a targeting moiety that targets NK cells and is mediated, for example, to 2B4 / SLAMF4, KIR2DS4, CD155 / PVR, KIR3DL1, CD94, LMIR1 / CD300A, CD69, LMIR2 / CD300c, CRACC / SLAMF7, LMIR3 / CD300LF, DNAM-1, LMIR5 / CD300LB, Fc-εRII, LMIR6 / CD300LE, Fc-γR1 / CD64, MICA, Fc-γRIIB / CD32b, MICB, Fc-γRIIC / CD32c, MULT-1, Fc-γRIIA / CD32a, nectin-2 / CD112, Fc-γRIII / CD16, NKG2A, FcRH1 / IRTA5, NKG2C, FcRH2 / IRTA4, NKG2D, FcRH4 / IRTA1, NKp30, FcRH5 / IRTA2, NKp44, Fc receptor-like molecule 3 / CD16-2, NKp46 / NCR1, NKp80 / KLRF1, NTB-A / SLAMF6, Rae-1, Rae-1α, Rae-1β, Rae-1δ, H60, Rae-1ε, ILT2 / CD85j, Rae-1γ, ILT3 / CD85k, TREM-1, ILT4 / CD85d, TREM-2, ILT5 / CD85a, TREM-3, KIR / CD158, TREML1 / TLT-1, KIR2DL1, ULBP-1, KIR2DL3, ULBP-2, KIR2DL4 / CD158d, or ULBP-3, and (ii) a targeting moiety that targets tumor cells together with any of the signaling substances described herein (e.g., consensus interferon or a variant thereof).

[0163] In certain embodiments, the chimeric proteins of the present disclosure have a targeting moiety that targets NK cells and is mediated, for example, to Kir1α, DNAM-1, or CD64, and (ii) a targeting moiety that targets tumor cells together with any of the signaling substances described herein (e.g., consensus interferon or a variant thereof).

[0164] In certain embodiments, the chimeric proteins of the present disclosure have a targeting moiety that targets NK cells, which is mediated, for example, to target KIR1, and (ii) a targeting moiety that targets tumor cells together with any of the signaling substances described herein (e.g., consensus interferon or a variant thereof). In certain embodiments, the chimeric proteins of the present disclosure have a targeting moiety that targets KIR1 on NK cells and a second targeting moiety that targets PD-L1 or PD-L2 on tumor cells.

[0165] In certain embodiments, the chimeric proteins of the present disclosure have a targeting moiety that targets NK cells, which is mediated, for example, to target TIGIT or KIR1, and (ii) a targeting moiety that targets tumor cells together with any of the signaling substances described herein (e.g., consensus interferon or a variant thereof). In certain embodiments, the chimeric proteins of the present disclosure have a targeting moiety that targets TIGIT on NK cells and a second targeting moiety that targets PD-L1 or PD-L2 on tumor cells.

[0166] As a non-limiting example, in various embodiments the chimeric proteins of the present disclosure include (i) for example, CLEC-9A, XCR1, RANK, CD36 / SRB3, LOX-1 / SR-E1, CD68, MARCO, CD163, SR-A1 / MSR, CD5L, SREC-1, CL-P1 / COLEC12, SREC-II, LIMPIIISRB2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBB ligand / TNFSF9, IL-12 / IL-23 p40, 4-amino-1,8-naphthalimide, ILT2 / CD85j, CCL21 / 6Ckine, ILT3 / CD85k, 8-oxo-dG, ILT4 / CD85d, 8D6A, ILT5 / CD85a, A2B5, integrin α4 / CD49d, Aag, integrin β2 / CD18, AMICA, langerin, B7-2 / CD86, leukotriene B4R1, B7-H3, LMIR1 / CD300A, BLAME / SLAMF8, LMIR2 / CD300c, C1qR1 / CD93, LMIR3 / CD300LF, CCR6, LMIR5 / CD300LBTargeting moieties directed to dendritic cells that mediate targeting to, for example, CCR7, LMIR6 / CD300LE, CD40 / TNFRSF5, MAG / Siglec-4-a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1 / CLEC5A, CD84 / SLAMF5, MMR, CD97, NCAML1, CD2F-10 / SLAMF9, Osteoactivin GPNMB, Chern23, PD-L2, CLEC-1, RP105, CLEC-2, Siglec-2 / CD22, CRACC / SLAMF7, Siglec-3 / CD33, DC-SIGN, Siglec-5, DC-SIGNR / CD299, Siglec-6, DCAR, Siglec-7, DCIR / CLEC4A, Siglec-9, DEC-205, Siglec-10, Dectin-1 / CLEC7A, Siglec-F, Dectin-2 / CLEC6A, SIGNR1 / CD209, DEP-1 / CD148, SIGNR4, DLEC, SLAM, EMMPRIN / CD147, TCCR / WSX-1, Fc-γR1 / CD64, TLR3, Fc-γRIIB / CD32b, TREM-1, Fc-γRIIC / CD32c, TREM-2, Fc-γRIIA / CD32a, TREM-3, Fc-γRIII / CD16, TREML1 / TLT-1, ICAM-2 / CD102, or vanilloid R1, and (ii) targeting moieties directed to tumor cells in combination with any of the signaling agents described herein (e.g., consensus interferon or variants thereof).

[0167] In certain embodiments, the chimeric proteins of the disclosure have (i) targeting moieties directed to dendritic cells that mediate targeting to, for example, CLEC-9A, DC-SIGN, CD64, CLEC4A, or DEC205, and (ii) targeting moieties directed to tumor cells in combination with any of the signaling agents described herein (e.g., consensus interferon or variants thereof). In certain embodiments, the chimeric proteins of the disclosure have a targeting moiety directed to CLEC9A on dendritic cells and a second targeting moiety directed to PD-L1 or PD-L2 on tumor cells.

[0168] In certain embodiments, the chimeric proteins of the present disclosure have (i) a targeting moiety that targets dendritic cells, which is mediated, for example, to target CLEC9A, and (ii) a targeting moiety that targets tumor cells together with any one of the signaling agents (e.g., consensus interferon or a variant thereof) described herein. In certain embodiments, the chimeric proteins of the present disclosure have a targeting moiety that targets CLEC9A on dendritic cells and a second targeting moiety that targets PD-L1 or PD-L2 on tumor cells.

[0169] In certain embodiments, the chimeric proteins of the present disclosure have (i) a targeting moiety that targets dendritic cells, which is mediated, for example, to target XCR1, and (ii) a targeting moiety that targets tumor cells together with any one of the signaling agents (e.g., consensus interferon or a variant thereof) described herein. In certain embodiments, the chimeric proteins of the present disclosure have a targeting moiety that targets XCR1 on dendritic cells and a second targeting moiety that targets PD-L1 or PD-L2 on tumor cells.

[0170] In certain embodiments, the chimeric proteins of the present disclosure have (i) a targeting moiety that targets dendritic cells, which is mediated, for example, to target RANK, and (ii) a targeting moiety for tumor cells together with any one of the signaling agents (e.g., consensus interferon or a variant thereof) described herein. In certain embodiments, the chimeric proteins of the present disclosure have a targeting moiety that targets RANK on dendritic cells and a second targeting moiety that targets PD-L1 or PD-L2 on tumor cells.

[0171] As a non-limiting example, in various embodiments, the chimeric proteins of the present disclosure include (i) for example, SIRP1a, B7-1 / CD80, ILT4 / CD85d, B7-H1, ILT5 / CD85a, common beta chain, integrin alpha4 / CD49d, BLAME / SLAMF8, integrin alphaX / CD11c, CCL6 / C10, integrin beta2 / CD18, CD155 / PVR, integrin beta3 / CD61, CD31 / PECAM-1, Latexin, CD36 / SR-B3, leukotriene B4R1, CD40 / TNFRSF5, LIMPIIISR-B2, CD43, LMIR1 / CD300A, CD45, LMIR2 / CD300c, CD68, LMIR3 / CD300LF, CD84 / SLAMF5, LMIR5 / CD300LB, CD97, LMIR6 / CD300LE, CD163, LRP-1, CD2F-10 / SLAMF9, MARCO, CRACC / SLAMF7, MD-1, ECF-L, MD-2, EMMPRIN / CD147, MGL2, endoglin / CD105, osteoactivin / GPNMB, Fc-gammaRI / CD64, osteopontin, Fc-gammaRIIB / CD32b, PD-L2, Fc-gammaRIIC / CD32c, siglec-3 / CD33, Fc-gammaRIIA / CD32a, SIGNR1 / CD209, Fc-gammaRIII / CD16, SLAM, GM-CSFRα, TCCR / WSX-1, ICAM-2 / CD102, TLR3, IFN-γR1, TLR4, IFN-γR2, TREM-1, IL-1RII, TREM-2, ILT2 / CD85j, TREM-3, ILT3 / CD85k, TREML1 / TLT-1, 2B4 / SLAMF4, IL-10Rα, ALCAM, IL-10Rβ, aminopeptidase N / ANPEP, ILT2 / CD85j, common β chain, ILT3 / CD85k, C1qR1 / CD93, ILT4 / CD85d, CCR1, ILT5 / CD85a, CCR2, CD206, integrin α4 / CD49d, CCR5, integrin αM / CD11b, CCR8, integrin αX / CD11c, CD155 / PVR, integrin β2 / CD18, CD14, integrin β3 / CD61, CD36 / SR-B3, LAIR1, CD43, LAIR2, CD45, leukotriene B4-R1, CD68, LIMPIIISR-B2, CD84 / SLAMF5, LMIR1 / CD300A, CD97, LMIR2 / CD300c, CD163, LMIR3 / CD300LF, coagulation factor III / tissue factor, LMIR5 / CD300LB, CX3CR1, CX3CL1, LMIR6 / CD300LE, CXCR4, LRP-1, CXCR6, M-CSF R, DEP-1 / CD148, MD-1, DNAM-1, MD-2, EMMPRIN / CD147, MMR, endoglin / CD105, NCAM-L1, Fc-γRI / CD64, PSGL-1, Fc-γRIIIICD16, RP105, G-CSF R, L-selectin, GM-CSF Rα, siglec-3 / CD33, HVEM / TNFRSF14, SLAM, ICAM-1 / CD54, TCCR / WSX-1, ICAM-2 / CD102, TREM-1, IL-6R, TREM-2, CXCR1 / IL-8RA, TREM-3, or TREML1 / TLT-1, and (ii) a targeting moiety that targets tumor cells in combination with any of the signaling agents (e.g., consensus interferon or a variant thereof) described herein.

[0172] In certain embodiments, the chimeric proteins of the present disclosure have a targeting moiety that targets monocytes / macrophages, which is mediated, for example, to the targeting of B7-H1, CD31 / PECAM-1, CD163, CCR2, or macrophage mannose receptor CD206, and (ii) a targeting moiety that targets tumor cells together with any of the signaling agents described herein (e.g., consensus interferon or a variant thereof).

[0173] In certain embodiments, the chimeric proteins of the present disclosure have a targeting moiety that targets monocytes / macrophages, which is mediated, for example, to the targeting of SIRP1a, and (ii) a targeting moiety that targets tumor cells together with any of the signaling agents described herein (e.g., consensus interferon or a variant thereof). In certain embodiments, the chimeric proteins of the present disclosure have a targeting moiety that targets SIRP1a on macrophage cells and a second targeting moiety that targets PD-L1 or PD-L2 on tumor cells.

[0174] In various embodiments, the chimeric proteins of the present disclosure have one or more targeting moieties that target one or more checkpoint markers, such as PD-1 / PD-L1 or PD-L2, CD28 / CD80 or CD86, CTLA4 / CD80 or CD86, ICOS / ICOSL or B7RP1, BTLA / HVEM, KIR, LAG3, CD137 / CD137L, OX40 / OX40L, CD27, CD40L, TIM3 / Gal9, and A2aR. In certain embodiments, the chimeric proteins of the present disclosure have (i) a targeting moiety that targets a checkpoint marker on a T cell, such as PD-1, and (ii) a targeting moiety that targets a tumor cell, such as PD-L1 or PD-L2, together with any of the signaling agents (e.g., consensus interferon or a variant thereof) described herein. In certain embodiments, the chimeric proteins of the present disclosure have a targeting moiety that targets PD-1 on a T cell and a second targeting moiety that targets PD-L1 on a tumor cell. In another embodiment, the chimeric proteins of the present disclosure have a targeting moiety that targets PD-1 on a T cell and a second targeting moiety that targets PD-L2 on a tumor cell.

[0175] In some embodiments, the chimeric proteins of the present disclosure include two or more targeting moieties for the same or different immune cells. In some embodiments, the chimeric proteins of the present disclosure have (i) one or more targeting moieties that target immune cells selected from T cells, B cells, dendritic cells, macrophages, NK cells, or subsets thereof, and (ii) one or more targeting moieties that target the same or different immune cells selected from T cells, B cells, dendritic cells, macrophages, NK cells, or subsets thereof, together with any of the signaling agents (e.g., consensus interferon or a variant thereof) described herein.

[0176] In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target T cells and one or more targeting moieties that target the same or different T cells. In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target T cells and one or more targeting moieties that target B cells. In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target T cells and one or more targeting moieties that target dendritic cells. In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target T cells and one or more targeting moieties that target macrophages. In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target T cells and one or more targeting moieties that target NK cells. For example, in an exemplary embodiment, the chimeric protein of the present disclosure may include a targeting moiety against CD8 and a targeting moiety against Clec9A. In another exemplary embodiment, the chimeric protein of the present disclosure may include a targeting moiety against CD8 and a targeting moiety against CD3. In another exemplary embodiment, the chimeric protein of the present disclosure may include a targeting moiety against CD8 and a targeting moiety against PD-1.

[0177] In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target B cells and one or more targeting moieties that target the same or different B cells. In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target B cells and one or more targeting moieties that target T cells. In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target B cells and one or more targeting moieties that target dendritic cells. In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target B cells and one or more targeting moieties that target macrophages. In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target B cells and one or more targeting moieties that target NK cells.

[0178] In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target dendritic cells and one or more targeting moieties that target the same or different dendritic cells. In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target dendritic cells and one or more targeting moieties that target T cells. In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target dendritic cells and one or more targeting moieties that target B cells. In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target dendritic cells and one or more targeting moieties that target macrophages. In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target dendritic cells and one or more targeting moieties that target NK cells.

[0179] In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target macrophages and one or more targeting moieties that target the same or different macrophages. In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target macrophages and one or more targeting moieties that target T cells. In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target macrophages and one or more targeting moieties that target B cells. In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target macrophages and one or more targeting moieties that target dendritic cells. In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target macrophages and one or more targeting moieties that target NK cells.

[0180] In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target NK cells and one or more targeting moieties that target the same or different NK cells. In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target NK cells and one or more targeting moieties that target T cells. In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target NK cells and one or more targeting moieties that target B cells. In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target NK cells and one or more targeting moieties that target macrophages. In certain embodiments, the chimeric proteins of the present disclosure include one or more targeting moieties that target NK cells and one or more targeting moieties that target dendritic cells.

[0181] In certain embodiments, the chimeric proteins of the present disclosure include a targeting moiety that targets tumor cells and a second targeting moiety that targets the same or different tumor cells. In such embodiments, the targeting moiety can bind to any of the tumor antigens described herein.

[0182] p39 In some embodiments, the chimeric protein of the present invention comprises one or more targeting moieties having a recognition domain that binds to a target of interest (e.g., an antigen, a receptor) comprising a target found in one or more types of cells selected from adipocytes (e.g., white adipocytes, brown adipocytes), hepatic lipid cells, hepatocytes, renal cells (e.g., juxtarenal cells, renal salivary glands, mammary glands, etc.), duct cells (duct cells of seminal vesicles, prostate, etc.), intestinal brush border cells (including microvilli), exocrine gland striated cells, gallbladder epithelial cells, non-ciliated cells of the vas deferens, principal cells of the epididymis, basal cells of the epididymis, endothelial cells, ameloblast epithelial cells (tooth enamel secretion), semicircular canal epithelial cells of the ear's vestibular system (proteoglycan secretion), interdental epithelial cells of the organ of Corti (secretory tectorial membrane covering hair cells), fibroblast cells of loose connective tissue, corneal fibroblast cells (corneal stromal cells), tendon fibroblast cells, fibroblast cells of bone marrow reticular tissue, non-epithelial fibroblast cells, pericytes, nucleus pulposus cells of the intervertebral disc, cementoblast / cementocyte (secretion of periodontal bone-like ewan cells), odontoblast / odontocyte (tooth dentin secretion), hyaline cartilage cells, fibrocartilage cells, elastic cartilage cells, osteoblast / osteocyte, osteoprogenitor cells (stem cells of osteoblasts), vitreous cells of the eye's vitreous body, stellate cells of the ear's perilymphatic space, hepatic stellate cells (Ito cells), pancreatic stellate cells, skeletal muscle cells, satellite cells, cardiomyocytes, smooth muscle cells, myoepithelial cells of the iris, myoepithelial cells of exocrine glands, exocrine epithelial cells (e.g., salivary gland cells, mammary gland cells, lacrimal gland cells, sweat gland cells, sebaceous gland cells, prostate gland cells, gastric gland cells, pancreatic acinar cells, lung cells), hormone-secreting cells (e.g., pituitary cells, neurosecretory cells, intestinal and respiratory tract cells, thyroid cells, parathyroid cells, adrenal cells, testicular Leydig cells, pancreatic islet cells), keratinized epithelial cells, wet stratified barrier epithelial cells, nerve cells (e.g., interneurons, principal cells, stellate cells, oligodendrocytes, and sensory transduction cells such as Schwann cells, autonomic nerve cells, sensory organ and peripheral nerve support cells, and central nervous system nerve cells, and glial cells).

[0183] Form of the targeting moiety In various embodiments, the targeting moiety of the chimeric protein of the present disclosure is a proteinaceous substance capable of specific binding, such as an antibody or a derivative thereof. In certain embodiments, the targeting moiety comprises an antibody. In various embodiments, the antibody is a full-length multimeric protein comprising two heavy chains and two light chains. Each heavy chain comprises one variable region (e.g., V H ) and at least three constant regions (e.g., CH1, CH2, and CH3), and each light chain comprises one variable region (V L ) and one constant region (C L ). The variable regions determine the specificity of the antibody. Each variable region comprises three hypervariable regions, also known as complementarity-determining regions (CDRs), flanked by four relatively conserved framework regions (FRs). These three CDRs are called CDR1, CDR2, and CDR3 and contribute to the binding specificity of the antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody.

[0184] In some embodiments, the targeting moiety comprises an antibody derivative or antibody format. In some embodiments, the targeting moiety of the chimeric proteins of the present disclosure is a single domain antibody, recombinant heavy chain antibody (VHH), single chain antibody (scFv), shark heavy chain antibody (VNAR), microprotein (cysteine knot protein, knottin), DARPin, tetranectin, affibody, transbody, anticalin, adnectin, affilin, microbody, peptide aptamer, alterase, plastic antibody, phylomer, stradbody, maxibody, evibody, fynomer, armadillo repeat protein, Kunitz domain, avimer, atrimer, probody, immunobody, triomab, tribody, pepbody, vaccibody, unibody, affimer, duoibody, Fv, Fab, Fab’, F(ab’)2, peptidomimetic molecule, or synthetic molecule as described in U.S. Patent Specifications or U.S. Patent Application Specifications such as U.S. Patent No. 7,417,130, U.S. Patent Application Publication No. 2004 / 132094, U.S. Patent No. 5,831,012, U.S. Patent Application Publication No. 2004 / 023334, U.S. Patent No. 7,250,297, U.S. Patent No. 6,818,418, U.S. Patent Application Publication No. 2004 / 209243, U.S. Patent No. 7,838,629, U.S. Patent No. 7,186,524, U.S. Patent No. 6,004,746, U.S. Patent No. 5,475,096, U.S. Patent Application Publication No. 2004 / 146938, U.S. Patent Application Publication No. 2004 / 157209, U.S. Patent No. 6,994,982, U.S. Patent No. 6,794,144, U.S. Patent Application Publication No. 2010 / 239633, U.S. Patent No. 7,803,907, U.S. Patent Application Publication No. 2010 / 119446, and / or U.S. Patent No. 7,166,697, the entire contents of which are incorporated herein by reference. See also Storz, MAbs., May - Jun. 2011, 3(3): pp. 310 - 317.

[0185] In certain embodiments, the targeting moiety includes single domain antibodies such as VHHs derived from organisms that produce VHH antibodies such as camelid VHHs, shark VHHs, or designed VHHs. A VHH is an antibody-derived therapeutic protein that includes structural and functional properties unique to natural heavy chain antibodies. VHH technology is based on fully functional antibodies derived from camel antibodies that lack a light chain. These heavy chain antibodies include a single variable domain (VHH) and two constant domains (CH2 and CH3). VHHs are commercially available under the trademarks NANOBODY or NANOBODIES.

[0186] In certain embodiments, the targeting moiety includes a VHH. In some embodiments, the VHH is a humanized VHH or a camelized VHH.

[0187] In some embodiments, the VHH includes a fully human VH domain, such as a HUMABODY (Crescendo Biologics, Cambridge, UK). In some embodiments, the fully human VH domain, such as a HUMABODY, is monovalent, bivalent, or trivalent. In some embodiments, the fully human VH domain, such as a HUMABODY, is monospecific or multispecific, such as monospecific, bispecific, or trispecific. Exemplary fully human VH domains, such as a HUMABODY, are described, for example, in International Publication Nos. 2016 / 113555 and 2016 / 113557, the entire disclosures of which are incorporated herein by reference.

[0188] In various embodiments, the targeting moiety of the chimeric protein of the present disclosure is a proteinaceous substance capable of specific binding to a cell receptor, such as a natural ligand to a cell receptor. In various embodiments, the cell receptor is found on one or more types of immune cells, including but not limited to T cells, cytotoxic T lymphocytes, helper T cells, natural killer (NK) cells, natural killer T (NKT) cells, antitumor macrophages (e.g., M1 macrophages), B cells, dendritic cells, or subsets thereof. In some embodiments, the cell receptor is found on megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, or subsets thereof.

[0189] In some embodiments, the targeting moiety is a natural ligand such as a chemokine. Exemplary chemokines that can be included in the chimeric proteins of the present invention include, but are not limited to, CCL1, CCL2, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CLL25, CCL26, CCL27, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, HCC-4, and LDGF-PBP. In an exemplary embodiment, the targeting moiety may be XCL1, a chemokine that recognizes and binds to the dendritic cell receptor XCR1. In another exemplary embodiment, the targeting moiety is CCL1, a chemokine that recognizes and binds to CCR8. In another exemplary embodiment, the targeting moiety is CCL2, a chemokine that recognizes and binds to CCR2 or CCR9. In another exemplary embodiment, the targeting moiety is CCL3, a chemokine that recognizes and binds to CCR1, CCR5, or CCR9. In another exemplary embodiment, the targeting moiety is CCL4, a chemokine that recognizes and binds to CCR1, CCR5, or CCR9. In another exemplary embodiment, the targeting moiety is CCL5, a chemokine that recognizes and binds to CCR1, CCR3, CCR4, or CCR5. In another exemplary embodiment, the targeting moiety is CCL6, a chemokine that recognizes and binds to CCR1. In another exemplary embodiment, the targeting moiety is CCL7, a chemokine that recognizes and binds to CCR2 or CCR9. In another exemplary embodiment, the targeting moiety is CCL8, a chemokine that recognizes and binds to CCR1, CCR2, CCR2B, CCR5, or CCR9. In another exemplary embodiment, the targeting moiety is CCL9, a chemokine that recognizes and binds to CCR1. In another exemplary embodiment, the targeting moiety is CCL10, a chemokine that recognizes and binds to CCR1.In another exemplary embodiment, the targeting moiety is CCL11, a chemokine that recognizes and binds to CCR2, CCR3, CCR5, or CCR9. In another exemplary embodiment, the targeting moiety is CCL13, a chemokine that recognizes and binds to CCR2, CCR3, CCR5, or CCR9. In another exemplary embodiment, the targeting moiety is CCL14, a chemokine that recognizes and binds to CCR1 or CCR9. In another exemplary embodiment, the targeting moiety is CCL15, a chemokine that recognizes and binds to CCR1 or CCR3. In another exemplary embodiment, the targeting moiety is CCL16, a chemokine that recognizes and binds to CCR1, CCR2, CCR5, or CCR8. In another exemplary embodiment, the targeting moiety is CCL17, a chemokine that recognizes and binds to CCR4. In another exemplary embodiment, the targeting moiety is CCL19, a chemokine that recognizes and binds to CCR7. In another exemplary embodiment, the targeting moiety is CCL20, a chemokine that recognizes and binds to CCR6. In another exemplary embodiment, the targeting moiety is CCL21, a chemokine that recognizes and binds to CCR7. In another exemplary embodiment, the targeting moiety is CCL22, a chemokine that recognizes and binds to CCR4. In another exemplary embodiment, the targeting moiety is CCL23, a chemokine that recognizes and binds to CCR1. In another exemplary embodiment, the targeting moiety is CCL24, a chemokine that recognizes and binds to CCR3. In another exemplary embodiment, the targeting moiety is CL25, a chemokine that recognizes and binds to CCR9. In another exemplary embodiment, the targeting moiety is CCL26, a chemokine that recognizes and binds to CCR3. In another exemplary embodiment, the targeting moiety is CCL27, a chemokine that recognizes and binds to CCR10. In another exemplary embodiment, the targeting moiety is CCL28, a chemokine that recognizes and binds to CCR3 or CCR10. In another exemplary embodiment, the targeting moiety is CXCL1, a chemokine that recognizes and binds to CXCR1 or CXCR2. In another exemplary embodiment, the targeting moiety is CXCL2, a chemokine that recognizes and binds to CXCR2.In another exemplary embodiment, the targeting moiety is CXCL3, a chemokine that recognizes and binds to CXCR2. In another exemplary embodiment, the targeting moiety is CXCL4, a chemokine that recognizes and binds to CXCR3B. In another exemplary embodiment, the targeting moiety is CXCL5, a chemokine that recognizes and binds to CXCR2. In another exemplary embodiment, the targeting moiety is CXCL6, a chemokine that recognizes and binds to CXCR1 or CXCR2. In another exemplary embodiment, the targeting moiety is CXCL8, a chemokine that recognizes and binds to CXCR1 or CXCR2. In another exemplary embodiment, the targeting moiety is CXCL9, a chemokine that recognizes and binds to CXCR3. In another exemplary embodiment, the targeting moiety is CXCL10, a chemokine that recognizes and binds to CXCR3. In another exemplary embodiment, the targeting moiety is CXCL11, a chemokine that recognizes and binds to CXCR3 or CXCR7. In another exemplary embodiment, the targeting moiety is CXCL12, a chemokine that recognizes and binds to CXCR4 or CXCR7. In another exemplary embodiment, the targeting moiety is CXCL13, a chemokine that recognizes and binds to CXCR5. In another exemplary embodiment, the targeting moiety is CXCL16, a chemokine that recognizes and binds to CXCR6. In another exemplary embodiment, the targeting moiety is LDGF-PBP, a chemokine that recognizes and binds to CXCR2. In another exemplary embodiment, the targeting moiety is XCL2, a chemokine that recognizes and binds to XCR1. In another exemplary embodiment, the targeting moiety is CX3CL1, a chemokine that recognizes and binds to CX3CR1.

[0190] In various embodiments, the chimeric proteins of the present disclosure include various combinations of targeting moieties. In an exemplary embodiment, the chimeric protein of the present disclosure may include two targeting moieties, both of which are antibodies or antibody derivatives. In another exemplary embodiment, the chimeric protein of the present disclosure may include two targeting moieties, both of which are natural ligands of cell receptors. In other exemplary embodiments, the chimeric protein of the present disclosure may include two targeting moieties, one of which is an antibody or antibody derivative and the other of which is a natural ligand of a cell receptor.

[0191] In various embodiments, the recognition domain of the chimeric protein of the present disclosure modulates (e.g., partially or completely neutralizes) the function of a target of interest (e.g., an antigen, a receptor), e.g., substantially inhibits, suppresses, or neutralizes a biological effect of the antigen. For example, various recognition domains may target one or more tumor antigens that actively suppress or have the ability to suppress the immune system, such as in a patient having a tumor. For example, in some embodiments, the chimeric protein of the present disclosure modulates the function of one or more of immunosuppressive signals (e.g., checkpoint inhibitors), such as TIM-3, BTLA, PD-1, CTLA-4, B7-H4, GITR, galectin-9, HVEM, PD-L1, PD-L2, B7-H3, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, and TMIGD2. For example, in some embodiments, the chimeric protein of the present disclosure is modified to interfere with, prevent, suppress, and / or inhibit the transmission of immunosuppressive signals, e.g., by binding of PD-1 to PD-L1 or PD-L2 and / or binding of CTLA-4 to one or more of AP2M1, CD80, CD86, SHP-2, and PPP2R5A.

[0192] In various embodiments, the recognition domain of the chimeric protein of the present disclosure binds to a target of interest (e.g., an antigen, a receptor) without modulating its function. For example, the recognition domain is a binding antibody or is similar to a binding antibody. For example, in various embodiments, the recognition domain simply targets an antigen or a receptor without substantially inhibiting, suppressing, or modulating the biological action of the antigen or receptor. For example, among the small antibody formats described above (e.g., as compared to a full-length antibody, etc.), there are those that have the ability to target hard-to-access epitopes and result in specific binding at a wider range of positions. In various embodiments, the recognition domain binds to an epitope that is physically distant from a site of the antigen or receptor that is important for biological activity (e.g., the active site of the antigen).

[0193] Such non-neutralizing binding is used in various embodiments of the invention, including methods of using the chimeric proteins of the disclosure to directly or indirectly recruit activated immune cells to a site of need via an effector antigen, such as any effector antigen described herein. For example, in various embodiments, the chimeric proteins of the disclosure can be used to directly or indirectly recruit cytotoxic T cells to tumor cells via CD8 in methods of reducing or eliminating a tumor (e.g., the chimeric proteins of the disclosure may comprise an anti-CD8 recognition domain and an anti-tumor antigen recognition domain). In such embodiments, CD8-expressing cytotoxic T cells are directly or indirectly recruited, but it is desirable not to functionally modulate CD8 activity. In contrast, in these embodiments, CD8 signaling is important for tumor-reducing or tumor-eliminating effects. As another example, the chimeric proteins of the disclosure are used to directly or indirectly recruit dendritic cells (DCs) via CLEC9A in various methods of reducing or eliminating a tumor (e.g., the chimeric proteins of the disclosure may comprise an anti-CLEC9A recognition domain and an anti-tumor antigen recognition domain). In such embodiments, CLEC9A-expressing DCs are directly or indirectly recruited, but it is desirable not to functionally modulate CLEC9A activity. In contrast, in these embodiments, CLEC9A signaling is important for tumor-reducing or tumor-eliminating effects.

[0194] In various embodiments, the recognition domain of the chimeric proteins of the disclosure binds, for example, to XCR1 on dendritic cells. For example, in some embodiments, the recognition domain comprises all or a portion of XCL1, or a non-neutralizing anti-XCR1 agent.

[0195] In various embodiments, the recognition domain of the chimeric protein of the present disclosure binds to an immunomodulatory (e.g., immunostimulatory or immunosuppressive) antigen. In various embodiments, the immunomodulatory antigen is one or more of 4-1BB, OX-40, HVEM, GITR, CD27, CD28, CD30, CD40, ICOS ligand, OX-40 ligand, LIGHT (CD258), GITR ligand, CD70, B7-1, B7-2, CD30 ligand, CD40 ligand, ICOS, ICOS ligand, CD137 ligand, and TL1A. In various embodiments, such immunostimulatory antigens are expressed on tumor cells. In various embodiments, the recognition domain of the chimeric protein of the present disclosure binds to such immunostimulatory antigens without modulating their function, and thus can mobilize these cells without reducing or eliminating their potential tumor-reducing or tumor-eliminating ability.

[0196] In various embodiments, the recognition domain of the chimeric protein of the present disclosure may be in the form of a chimeric protein that includes two recognition domains having neutralizing activity, two recognition domains having non-neutralizing (e.g., binding) activity, or one recognition domain having neutralizing activity and one recognition domain having non-neutralizing (e.g., binding) activity.

[0197] Other signaling substances In one aspect, the present invention provides a chimeric protein comprising one or more signaling agents (e.g., immunomodulatory factors) in addition to consensus interferon or a variant described herein. In an exemplary embodiment, the chimeric protein may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more types of signaling agents in addition to consensus interferon or a variant described herein. In various embodiments, the additional signaling agent is modified such that its affinity or activity for one or more of its receptors is reduced, thereby attenuating its activity (including agonism or antagonism) and / or preventing non-specific signaling or unwanted sequestration of the chimeric protein.

[0198] In various embodiments, the additional signaling agent has one or more mutations that attenuate its antagonist activity, which is antagonist in the wild type. In various embodiments, the additional signaling agent is antagonist due to one or more mutations, e.g., an agonist signaling agent is converted to an antagonist signaling agent, and such a converted signaling agent may also have one or more mutations that attenuate its antagonist activity, if desired (e.g., as described in WO 2015 / 007520, which is hereby incorporated by reference in its entirety).

[0199] In various embodiments, the additional signaling agent is selected from modified cytokines, growth factors, and hormones. Representative examples of such cytokines, growth factors, and hormones include lymphokines, monokines; conventional polypeptide hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine, insulin; proinsulin; relaxin; prolactin; placental lactogen; tumor necrosis factor-α and tumor necrosis factor-β; Müllerian inhibiting substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-α; platelet-derived growth factor; transforming growth factors (TGF) such as TGF-α and TGF-β; insulin-like growth factor-I and insulin-like growth factor-II; osteogenic factors; interferons such as interferon-α, interferon-β, and interferon-γ (and type I, type II, and type III interferons); colony-stimulating factors (CSF) such as macrophage-CSF (M-CSF), granulocyte macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF); interleukins (IL) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, and IL-18; tumor necrosis factors such as TNF-α or TNF-β, and other polypeptide factors including, for example, LIF and kit ligand (KL), but are not limited thereto. As used herein, cytokines, growth factors, and hormones include proteins of natural origin, or proteins produced from recombinant bacterial cell culture systems, recombinant eukaryotic cell culture systems, or recombinant mammalian cell culture systems, and biological activity equivalents of native sequence cytokines.

[0200] In some embodiments, the additional signaling molecule is a modified form of a growth factor selected from, but not limited to, transforming growth factors (TGFs) such as TGF-α and TGF-β, epidermal growth factor (EGF), insulin-like growth factors such as insulin-like growth factor-I and insulin-like growth factor-II, fibroblast growth factor (FGF), heregulin, platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF).

[0201] In one embodiment, the growth factor is a modified form of fibroblast growth factor (FGF). Examples of FGF include, but are not limited to, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, mouse FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23.

[0202] In one embodiment, the growth factor is a modified form of vascular endothelial growth factor (VEGF). Examples of VEGF include VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PGF, and various isoforms of VEGF-A including, but not limited to, VEGF 121 , VEGF 121 b, VEGF 145 , VEGF 165 , VEGF 165 b, VEGF 189 , and VEGF 206 and their isoforms including various isoforms of VEGF-A such as these.

[0203] In one embodiment, the growth factor is a modified form of transforming growth factor (TGF). Examples of TGF include TGF-α and TGF-β, and their subtypes including various subtypes of TGF-β including TGFβ1, TGFβ2, and TGFβ3, but are not limited to these.

[0204] In some embodiments, the additional signaling molecule is a modified form of a hormone selected from, but not limited to, human chorionic gonadotropin, gonadotropin-releasing hormone, androgen, estrogen, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, prolactin, growth hormone, adrenocorticotropic hormone, antidiuretic hormone, oxytocin, thyroid-stimulating hormone-releasing hormone, growth hormone-releasing hormone, adrenocorticotropic hormone-releasing hormone, somatostatin, dopamine, melatonin, thyroxine, calcitonin, parathyroid hormone, glucocorticoid, mineralocorticoid, adrenaline, noradrenaline, progesterone, insulin, glucagon, amylin, calcitriol, calciferol, atrial natriuretic peptide, gastrin, secretin, cholecystokinin, neuropeptide Y, ghrelin, PYY3-36, insulin-like growth factor (IGF), leptin, thrombopoietin, erythropoietin (EPO), and angiotensinogen.

[0205] In some embodiments, the additional signaling molecule is one or more of immunomodulatory factors such as interleukin, interferon, and tumor necrosis factor.

[0206] In some embodiments, the additional signaling molecule is an interleukin, which includes, for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36, or a fragment, variant, analog, or family member thereof. Interleukins are a group of multifunctional cytokines synthesized by lymphocytes, monocytes, and macrophages. Known functions include stimulating the proliferation of immune cells (e.g., helper T cells, B cells, eosinophils, and lymphocytes), chemotaxis of neutrophils and T lymphocytes, and / or inhibition of interferons. Interleukin activity can be determined using assay methods known in the art, namely, the assay methods of Matthew et al., Lymphokines and Interferens: A Practical Approach, edited by Clemens et al., IRL Press, Washington D.C., 1987, pp. 221-225, and Orencole and Dinarello, (1989) Cytokine, 1, pp. 14-20.

[0207] In some embodiments, the signaling molecule is a modified interferon such as type I, type II, and type III interferons. Examples of interferons include, for example, interferon-α-1, 2, 4, 5, 6, 7, 8, 10, 13, 14, 16, 17, and 21, interferon-β and interferon-γ, interferon κ, interferon ε, interferon τ, and interferon ω.

[0208] In some embodiments, the additional signaling agent is a modified form of a tumor necrosis factor (TNF) or a protein of the TNF family, including but not limited to TNF-α, TNF-β, LT-β, CD40L, CD27L, CD30L, FASL, 4-1BBL, OX40L, and TRAIL.

[0209] In various embodiments, the additional signaling agent is a modified form (e.g., a variant) of a signaling agent having one or more mutations. In various embodiments, due to the mutation, the modified signaling agent has a decrease in one or more of its activities, such as a decrease in binding affinity, a decrease in intrinsic activity, and a decrease in a specific biological activity, compared to the unmodified or unmutated, i.e., wild-type, signaling agent (e.g., when comparing the same signaling agent between the wild-type and the modified form (e.g., the variant form)). In various embodiments, due to the mutation, the modified signaling agent has a decrease in one or more of its activities, such as a decrease in binding affinity, a decrease in intrinsic activity, and a decrease in a specific biological activity, compared to the unmodified or unmutated, i.e., unmutated consensus sequence, interferon. In some embodiments, mutations that attenuate or decrease binding or affinity include mutations that substantially decrease or eliminate binding or affinity. In some embodiments, the mutations that attenuate or decrease binding or affinity are different from the mutations that substantially decrease or eliminate binding or affinity. As a result, in various embodiments, due to the mutation, the signaling agent is safer compared to the unmutated, i.e., wild-type, signaling agent (e.g., when comparing the same signaling agent between the wild-type and the modified form (e.g., the variant form)), for example, systemic toxicity is reduced, side effects are reduced, and off-target effects are reduced. In various embodiments, due to those mutations, the signaling agent is safer compared to the unmutated interferon, e.g., the interferon of the unmutated consensus sequence, for example, systemic toxicity is reduced, side effects are reduced, and off-target effects are reduced.

[0210] In various embodiments, the additional signaling agent is modified to have one or more mutations that reduce the binding affinity or activity for one or more of its receptors. In some embodiments, the signaling agent is modified to have one or more mutations that substantially reduce or eliminate the binding affinity or activity for the receptor. In some embodiments, the activity by the wild-type signaling agent is agonism in the receptor (e.g., activation of cellular action at the treatment site). For example, the wild-type signaling agent may activate its receptor. In such embodiments, due to the mutation, the modified signaling agent will have reduced or eliminated activation activity in the receptor. For example, due to the mutation, the modified signaling agent may deliver a reduced activation signal to the target cell, or the activation signal may be eliminated. In some embodiments, the activity by the wild-type signaling agent is antagonism in the receptor (e.g., inhibition or attenuation of cellular action at the treatment site). For example, the wild-type signaling agent may attenuate or inhibit the receptor. In these embodiments, due to the mutation, the modified signaling agent will have reduced or eliminated antagonizing activity in the receptor. For example, due to the mutation, the modified signaling agent may deliver a reduced inhibitory signal to the target cell, or the inhibitory signal may be eliminated. In various embodiments, the signaling agent is antagonistic due to one or more mutations, for example, an agonistic signaling agent is converted to an antagonistic signaling agent (as described, for example, in WO 2015 / 007520, which is hereby incorporated by reference in its entirety). Optionally, the converted signaling agent may have one or more mutations that reduce its binding affinity or activity for one or more of its receptors, or that substantially reduce or eliminate its binding affinity or activity for one or more of its receptors.

[0211] In some embodiments, a decrease in affinity or activity at a receptor can be restored by binding to one or more of the targeting moieties. In another embodiment, a decrease in affinity or activity at a receptor is not substantially restored by the activity of one or more of the targeting moieties.

[0212] In various embodiments, because the targeting moiety compensates for lost / inadequate binding (and / or, without limitation, binding activity, etc.) required for substantial activation, the additional signaling agent has activity on the target cell. In various embodiments, the modified signaling agent is substantially inactive en route to the therapeutic active site, acts substantially on specifically targeted cell types, and greatly reduces unwanted side effects.

[0213] In some embodiments, the additional signaling agent can include one or more mutations that attenuate or reduce binding or affinity for a receptor (i.e., the therapeutic receptor), and one or more mutations that substantially reduce or eliminate binding or affinity at a second receptor. In such embodiments, these mutations may be at the same position or at different positions (i.e., the same mutation or multiple mutations). In some embodiments, the mutations that decrease binding and / or activity at one receptor are different from the mutations that substantially reduce or eliminate binding at another receptor. In some embodiments, the mutations that decrease binding and / or activity at one receptor are the same as the mutations that substantially reduce or eliminate binding at another receptor. In some embodiments, the chimeric protein of the present disclosure has mutations that attenuate binding and / or activity at the therapeutic receptor, enabling a more controlled on-target therapeutic effect (e.g., compared to a wild-type signaling agent), and mutations that substantially reduce or eliminate binding and / or activity at another receptor, reducing side effects (e.g., compared to a wild-type signaling agent), and has a modified signaling agent with both mutations.

[0214] In some embodiments, a substantial decrease or elimination of binding or activity is not substantially restored by the targeting moiety. In some embodiments, a substantial decrease or elimination of binding or activity is recoverable by the targeting moiety. In various embodiments, a substantial decrease or elimination of binding or activity at the second receptor can also prevent deleterious effects mediated by the other receptor. Alternatively, or additionally, a substantial decrease or elimination of binding or activity at the other receptor reduces or eliminates sequestration of the therapeutic chimeric protein away from the site of therapeutic action, thereby improving the therapeutic effect. For example, in some embodiments, this obviates the need for high doses of the chimeric proteins of the present disclosure to compensate for loss at the other receptor. Such an ability to reduce the dose further reduces the likelihood of side effects.

[0215] In various embodiments, an additional modified signaling agent decreases, substantially decreases, or eliminates, for example, binding (e.g., K D ), and / or activation (e.g., when the modified signaling agent is an agonist of the receptor, measurable as, for example, K A and / or EC 50 ), and / or inhibition (e.g., when the modified signaling agent is an antagonist of the receptor, measurable as, for example, K I and / or IC 50when it is an antagonist of a measurable receptor), and includes one or more mutations. In various embodiments, the activity (including agonism or antagonism) is attenuated by a decrease in affinity at the receptor of the signaling substance. In said embodiments, the modified signaling substance has an affinity for the receptor that is about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10% - 20%, about 20% - 40%, about 50%, about 40% - 60%, about 60% - 80%, about 80% - 100% lower than that of the wild-type signaling substance. In some embodiments, the binding affinity is at least about 2-fold lower, about 3-fold lower, about 4-fold lower, about 5-fold lower, about 6-fold lower, about 7-fold lower, about 8-fold lower, about 9-fold lower, at least about 10-fold lower, at least about 15-fold lower, at least about 20-fold lower, at least about 25-fold lower, at least about 30-fold lower, at least about 35-fold lower, at least about 40-fold lower, at least about 45-fold lower, at least about 50-fold lower, at least about 100-fold lower, at least about 150-fold lower, or about 10 - 50-fold lower, about 50 - 100-fold lower, about 100 - 150-fold lower, about 150 - 200-fold lower, or 200-fold lower compared to the wild-type signaling substance (including, as non-limiting examples, comparison with an unmutated consensus interferon).

[0216] In embodiments having a mutation that reduces binding in a receptor for a chimeric protein and a mutation that substantially reduces or eliminates binding in a second receptor, the attenuation or reduction of the binding affinity of a modified signaling substance for one receptor is less than a substantial reduction or elimination of the affinity for the other receptor. In some embodiments, the attenuation or reduction of the binding affinity of a modified signaling substance for one receptor is about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% less than a substantial reduction or elimination of the affinity for the other receptor. In various embodiments, a substantial reduction or elimination means a greater degree of reduction in binding affinity and / or activity than an attenuation or reduction.

[0217] In various embodiments, an additional modified signaling substance comprises one or more mutations that reduce the intrinsic activity of the signaling substance to about 75%, about 70%, about 60%, about 50%, about 40%, about 30%, about 25%, about 20%, about 10%, about 5%, about 3%, or about 1% compared to, for example, the wild-type signaling substance (including comparison with an unmutated consensus interferon as a non-limiting example).

[0218] In various embodiments, an additional modified signaling substance comprises one or more mutations that will reduce the affinity and / or activity of the signaling substance for a receptor for any one of the cytokines, growth factors, and hormones as described herein.

[0219] In some embodiments, the additional modified signaling agent comprises one or more mutations that will result in a reduced affinity for the receptor of the signaling agent, which affinity is lower than the binding affinity of the targeting moiety for the receptor. In some embodiments, this difference in binding affinity is the difference between the binding affinity of the signaling agent / receptor and the binding affinity of the targeting moiety / receptor in the same cell. In some embodiments, this difference in binding affinity allows the signaling agent, e.g., the mutant signaling agent, to localize its on-target action and minimize the off-target action underlying the side effects seen with the wild-type signaling agent. In some embodiments, this binding affinity is at least about 2-fold lower, at least about 5-fold lower, at least about 10-fold lower, at least about 15-fold lower, at least about 25-fold lower, at least about 50-fold lower, at least about 100-fold lower, or at least about 150-fold lower.

[0220] Receptor binding activity can be measured using methods known in the art. For example, affinity and / or binding activity can be evaluated by Scatchard plot analysis of binding data and computer fitting (e.g., Scatchard, 1949), or by reflectometric interference spectroscopy under flow-through conditions as described by Brecht et al. (1993), the entire contents of which are incorporated herein by reference.

[0221] The amino acid sequences of the wild-type signaling substances described in this specification are well known in the art. Thus, in various embodiments, additional modified signaling substances have at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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%, or at least about 99% sequence identity (e.g., about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity) with the known wild-type amino acid sequences of the signaling substances described herein.

[0222] In various embodiments, an additional modified signaling agent comprises an amino acid sequence having at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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%, or at least about 99% sequence identity (e.g., about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity) with any of the sequences disclosed herein.

[0223] In various embodiments, an additional modified signaling agent comprises an amino acid sequence having one or more amino acid mutations. In some embodiments, the one or more amino acid mutations can independently be selected from substitutions, insertions, deletions, and truncations.

[0224] In some embodiments, the amino acid mutation is an amino acid substitution, and the amino acid mutation can include conservative substitutions and / or non-conservative substitutions as described herein.

[0225] As described herein, additional modified signaling agents have mutations that affect the affinity and / or activity in one or more receptors. In various embodiments, the affinity and / or activity is reduced in therapeutic receptors, e.g., receptors that mediate a desired therapeutic effect (e.g., agonism or antagonism). In various embodiments, the modified signaling agent has a mutation that substantially reduces or eliminates the affinity and / or activity in a receptor that does not mediate a desired therapeutic effect (e.g., as a result of promiscuous binding). One receptor of any of the modified signaling agents, e.g., cytokines, growth factors, and hormones as described herein, is known in the art.

[0226] Examples of mutations that cause a reduction in affinity and / or activity (e.g., agonist activity) in a receptor are found in International Publication No. WO 2013 / 107791 (e.g., regarding interferon), International Publication No. WO 2015 / 007542 (e.g., regarding interleukin), and International Publication No. WO 2015 / 007903 (e.g., regarding TNF), the entire contents of each of which are incorporated herein by reference. Examples of mutations that result in a reduction in affinity and / or activity (e.g., antagonist activity) in a therapeutic receptor are found in International Publication No. WO 2015 / 007520, the entire contents of which are incorporated herein by reference.

[0227] In some embodiments, the additional modified signaling agent comprises one or more mutations that reduce the affinity and / or activity of the signaling agent for a type I cytokine receptor, a type II cytokine receptor, a chemokine receptor, a receptor within the tumor necrosis factor receptor (TNFR) superfamily, a TGF-β receptor, a receptor within the immunoglobulin (Ig) superfamily, and / or a receptor within the tyrosine kinase superfamily.

[0228] In various embodiments, the receptor for the additional signaling molecule is a type I cytokine receptor. Type I cytokine receptors are known in the art and include, but are not limited to, receptors for IL2 (β-subunit), IL3, IL4, IL5, IL6, IL7, IL9, IL11, IL12, GM-CSF, G-CSF, LIF, and CNTF, and also include receptors for thrombopoietin (TPO), prolactin, and growth hormone. Examples of type I cytokine receptors include, but are not limited to, GM-CSF receptor, G-CSF receptor, LIF receptor, CNTF receptor, TPO receptor, and type I IL receptor.

[0229] In various embodiments, the receptor for the additional signaling molecule is a type II cytokine receptor. Type II cytokine receptors are multimeric receptors composed of heterologous subunits and are mainly receptors for interferons. Receptors of this family include, but are not limited to, receptors for interferon-α, interferon-β, and interferon-γ, IL10, IL22, and tissue factor. Examples of type II cytokine receptors include, but are not limited to, IFN-α receptor (e.g., IFNAR1 and IFNAR2), IFN-β receptor, IFN-γ receptor (e.g., IFNGR1 and IFNGR2), and type II IL receptor.

[0230] In various embodiments, the receptor for the additional signaling substance is a G protein-coupled receptor. Chemokine receptors are G protein-coupled receptors having a seven-transmembrane structure and are coupled to G proteins for signal transduction. Chemokine receptors include, but are not limited to, CC chemokine receptors, CXC chemokine receptors, CX3C chemokine receptors, and XC chemokine receptors (XCR1). Exemplary chemokine receptors include, but are not limited to, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR3B, CXCR4, CXCR5, CSCR6, CXCR7, XCR1, and CX3CR1.

[0231] In various embodiments, the receptor for the additional signaling substance is a TNFR family member. Tumor necrosis factor receptor (TNFR) family members commonly have a cysteine-rich domain (CRD) formed from three disulfide bonds around a CXXCXXC core motif that forms an extended molecule. Exemplary tumor necrosis factor receptor family members include CD120a (TNFRSF1A), CD120b (TNFRSF1B), lymphotoxin β receptor (LTBR, TNFRSF3), CD134 (TNFRSF4), CD40 (CD40, TNFRSF5), FAS (FAS, TNFRSF6), TNFRSF6B (TNFRSF6B), CD27 (CD27, TNFRSF7), CD30 (TNFRSF8), CD137 (TNFRSF9), TNFRSF10A (TNFRSF10A), TNFRSF10B, (TNFRSF10B), TNFRSF10C (TNFRSF10C), TNFRSF10D (TNFRSF10D), RANK (TNFRSF11A), osteoprotegerin (TNFRSF11B), TNFRSF12A (TNFRSF12A), TNFRSF13B (TNFRSF13B), TNFRSF13C (TNFRSF13C), TNFRSF14 (TNFRSF14), nerve growth factor receptor (NGFR, TNFRSF16), TNFRSF17 (TNFRSF17), TNFRSF18 (TNFRSF18), TNFRSF19 (TNFRSF19), TNFRSF21 (TNFRSF21), and TNFRSF25 (TNFRSF25).

[0232] In various embodiments, the receptor for the additional signaling substance is a TGF-β receptor. The TGF-β receptor is a single-pass serine / threonine kinase receptor. TGF-β receptors include, but are not limited to, TGFBR1, TGFBR2, and TGFBR3.

[0233] In various embodiments, the receptor for the additional signaling substance is an Ig superfamily receptor. Receptors of the immunoglobulin (Ig) superfamily share structural homology with immunoglobulins. Examples of receptors of the Ig superfamily include, but are not limited to, interleukin-1 receptor, CSF-1R, PDGFR (such as PDGFRA and PDGFRB), and SCFR.

[0234] In various embodiments, the receptor for the additional signaling substance is a tyrosine kinase superfamily receptor. Receptors of the tyrosine kinase superfamily are well known in the art. There are approximately 58 known receptor tyrosine kinases (RTKs), which are grouped into 20 subfamilies. Examples of receptors of the tyrosine kinase superfamily include, but are not limited to, FGF receptors and their various isoforms, such as FGFR1, FGFR2, FGFR3, FGFR4, and FGFR5.

[0235] In one embodiment, the additional modified signaling substance is interferon α. In said embodiment, the modified IFN-α substance has a reduced affinity and / or activity for the IFN-α / β receptor (IFNAR), i.e., the IFNAR1 chain and / or the IFNAR2 chain. In some embodiments, the modified IFN-α substance has a substantially reduced or eliminated affinity and / or activity for the IFN-α / β receptor (IFNAR), i.e., the IFNAR1 chain and / or the IFNAR2 chain.

[0236] Mutant forms of interferon α are known to those skilled in the art. In an exemplary embodiment, the modified signaling substance is an IFN-α2a allele having the amino acid sequence of SEQ ID NO: 233.

[0237] In an exemplary embodiment, the modified signaling substance is an IFN-α2b allele having the amino acid sequence of SEQ ID NO: 234 (which differs from IFN-α2a at the amino acid position 23).

[0238] In some embodiments, in the IFN-α2 variants (IFN-α2a or IFN-α2b), mutations are introduced into one or more amino acids at positions 144-154, for example, at positions 148, 149, and / or 153 of the amino acids. In some embodiments, the IFN-α2 variant comprises one or more mutations selected from L153A, R149A, and M148A. Such variants are described, for example, in International Publication No. WO 2013 / 107791 and Piehler, et al., (2000) J. Biol. Chem., 275: pp. 40425-33, the entire contents of which are incorporated herein by reference.

[0239] In some embodiments, the IFN-α2 variant has a reduced affinity and / or activity for IFNAR1. In some embodiments, the IFN-α2 variant comprises one or more mutations selected from F64A, N65A, T69A, L80A, Y85A, and Y89A as described in International Publication No. WO 2010 / 030671, the entire contents of which are incorporated herein by reference.

[0240] In some embodiments, the IFN-α2 variant comprises one or more mutations selected from K133A, R144A, R149A, and L153A as described in International Publication No. WO 2008 / 124086, the entire contents of which are incorporated herein by reference.

[0241] In some embodiments, the IFN-α2 variant comprises one or more mutations selected from R120E and R120E / K121E as described in International Publication No. WO 2015 / 007520 and International Publication No. WO 2010 / 030671, the entire contents of which are incorporated herein by reference. In such embodiments, the IFN-α2 variant antagonizes wild-type IFN-α2 activity. In such embodiments, the mutant IFN-α2 has a reduced affinity and / or activity for IFNAR1 while maintaining the affinity and / or activity for IFNR2.

[0242] In some embodiments, the human IFN-α2 variant comprises (1) one or more mutations selected from R120E and R120E / K121E, which, although not wishing to be bound by theory, cause an antagonistic effect, and (2) one or more mutations selected from K133A, R144A, R149A, and L153A, which, although not wishing to be bound by theory, attenuate the effect, for example, in IFNAR2. In one embodiment, the human IFN-α2 variant comprises R120E and L153A.

[0243] In some embodiments, the human IFN-α2 variant comprises one or more mutations selected from L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, D114R, L117A, R120A, R125A, K134A, R144A, A145G, A145M, M148A, R149A, S152A, L153A, and N156A, as disclosed in International Publication No. WO 2013 / 059885, the entire disclosure of which is incorporated herein by reference. In some embodiments, the human IFN-α2 variant comprises the mutations H57Y, E58N, Q61S, and / or L30A, as disclosed in International Publication No. WO 2013 / 059885. In some embodiments, the human IFN-α2 variant comprises the mutations H57Y, E58N, Q61S, and / or R33A, as disclosed in International Publication No. WO 2013 / 059885. In some embodiments, the human IFN-α2 variant comprises the mutations H57Y, E58N, Q61S, and / or M148A, as disclosed in International Publication No. WO 2013 / 059885. In some embodiments, the human IFN-α2 variant comprises the mutations H57Y, E58N, Q61S, and / or L153A, as disclosed in International Publication No. WO 2013 / 059885. In some embodiments, the human IFN-α2 variant comprises the mutations N65A, L80A, Y85A, and / or Y89A, as disclosed in International Publication No. WO 2013 / 059885. In some embodiments, the human IFN-α2 variant comprises the mutations N65A, L80A, Y85A, Y89A, and / or D114A, as disclosed in International Publication No. WO 2013 / 059885.

[0244] In one embodiment, the additional modified signaling substance is interferon β. In said embodiment, the modified interferon β substance also has a reduced affinity and / or activity for the IFN-α / β receptor (IFNAR), i.e., the IFNAR1 chain and / or the IFNAR2 chain. In some embodiments, the modified interferon β substance has a substantially reduced or eliminated affinity and / or activity for the IFN-α / β receptor (IFNAR), i.e., the IFNAR1 chain and / or the IFNAR2 chain.

[0245] In one embodiment, the additional modified signaling substance is interferon γ. In said embodiment, the modified interferon γ substance has a reduced affinity and / or activity for the interferon-γ receptor (IFNGR), i.e., the IFNGR1 chain and the IFNGR2 chain. In some embodiments, the modified interferon γ substance has a substantially reduced or eliminated affinity and / or activity for the interferon-γ receptor (IFNGR), i.e., the IFNGR1 chain and the IFNGR2 chain.

[0246] In some embodiments, an additional modified signaling substance is vascular endothelial growth factor (VEGF). VEGF plays a major role not only in physiological angiogenesis but also in pathological angiogenesis, controls vascular permeability, and is a potent growth factor that can act as a growth factor on VEGF receptor-expressing cells. In particular, other functions include stimulation of cell migration in macrophage lineage cells and endothelial cells. There are several members of the VEGF family of growth factors, and at least three types of receptors (VEGFR-1, VEGFR-2, and VEGFR-3). Members of the VEGF family can bind to and activate two or more types of VEGFRs. For example, VEGF-A binds to VEGFR-1 and VEGFR-2, while VEGF-C can bind to VEGFR-2 and VEGFR-3. Activation of VEGFR-1 and VEGFR-2 controls angiogenesis, while activation of VEGFR-3 is related to lymphangiogenesis. The main angiogenesis-promoting signal results from the activation of VEGFR-2. Activation of VEGFR-1 has been reported to potentially play a negative role in angiogenesis. Transmission of the VEGFR-1 signal is important for in vivo tumor growth via bone marrow-derived VEGFR-1-positive cells (which contribute to the formation of pre-metastatic niches in bone). Several therapies based on VEGF-A-directed / neutralizing therapeutic antibodies have been developed for use in the treatment of various human tumors that mainly depend on angiogenesis. However, these therapies do not have no side effects. This may not be surprising considering that these antibodies act as general non-cell / tissue-specific VEGF / VEGFR interaction inhibitors. Therefore, it is preferable to limit VEGF (e.g., VEGF-A) / VEGFR-2 inhibition to specific target cells (e.g., tumor vascular endothelial cells).

[0247] In some embodiments, VEGF is VEGF-A, VEGF-B, VEFG-C, VEGF-D, or VEGF-E, as well as VEGF 121 , VEGF 121 b, VEGF 145 , VEGF 165 , VEGF165 b, VEGF 189 and VEGF 206 and the like, and their isoforms including various isoforms of VEGF-A. In some embodiments, the modified signaling substance has a reduced affinity and / or activity for VEGFR-1 (Flt-1) and / or VEGFR-2 (KDR / Flk-1). In some embodiments, the modified signaling substance has a substantially reduced or eliminated affinity and / or activity for VEGFR-1 (Flt-1) and / or VEGFR-2 (KDR / Flk-1). In certain embodiments, the modified signaling substance has a reduced affinity and / or activity for VEGFR-2 (KDR / Flk-1), and / or a substantially reduced or eliminated affinity and / or activity for VEGFR-1 (Flt-1). The said embodiments are used, for example, in a method of wound healing or the treatment of ischemia-related diseases (although not wishing to be bound by theory, through the effect of VEGFR-2 on endothelial cell function and angiogenesis). In various embodiments, binding to VEGFR-1 (Flt-1) associated with cancer and inflammation-promoting activity is avoided. In various embodiments, VEGFR-1 (Flt-1) acts as a decoy receptor, and thus the affinity at this receptor is substantially reduced or eliminated, avoiding sequestration of the therapeutic agent. In certain embodiments, the modified signaling substance has a substantially reduced or eliminated affinity and / or activity for VEGFR-1 (Flt-1), and / or a substantially reduced or eliminated affinity and / or activity for VEGFR-2 (KDR / Flk-1). In some embodiments, VEGF is VEGF-C or VEGF-D. In the said embodiments, the modified signaling substance has a reduced affinity and / or activity for VEGFR-3. Alternatively, the modified signaling substance has a substantially reduced or eliminated affinity and / or activity for VEGFR-3.

[0248] Therapies for promoting angiogenesis are also important in various diseases (such as ischemic heart disease, bleeding, etc.), and these therapies include VEGF-based therapies. Activation of VEGFR-2 is angiogenic (acting on endothelial cells). Activation of VEFGR-1 causes stimulation of the migration of inflammatory cells (including macrophages, for example), which may lead to inflammation-related high vascular permeability. Activation of VEFGR-1 can also promote bone marrow-related tumor niche formation. Therefore, in this case, a VEGF-based therapy selective for VEGFR-2 activation would be desirable. Furthermore, cell-specific targeting, for example, cell-specific targeting to endothelial cells, would be desirable.

[0249] In some embodiments, the additional modified signaling substance has a reduced affinity and / or activity (such as antagonist activity) for VEGFR-2, and / or the affinity and / or activity for VEGFR-1 is substantially reduced or eliminated. When targeted to tumor vascular endothelial cells via a targeting moiety that binds to a tumor endothelial cell marker (such as PSMA and others), such a construct inhibits VEGFR-2 activation, particularly on cells positive for such markers, while not activating VEGFR-1 in transit to and (when inactive) on the target cells, thus precluding induction of an inflammatory response, etc. This results in an angiogenesis inhibition therapy for a greater variety of tumors that is more selective and safer compared to VEGF-A neutralization therapy.

[0250] In some embodiments, the additional modified signaling agent has a reduced affinity and / or activity for VEGFR-2 (e.g., agonist activity), and / or the affinity and / or activity for VEGFR-1 is substantially reduced or eliminated. Through targeting to the vascular endothelium, in some embodiments, such constructs promote angiogenesis without inducing VEGFR-1 related inflammatory responses. Thus, such constructs will have a targeted angiogenesis inducing effect with a substantially reduced risk of side effects caused by systemic activation of VEGFR-2 and VEGR-1.

[0251] In an exemplary embodiment, the modified signaling agent is VEGF 165 and has the amino acid sequence of SEQ ID NO: 235.

[0252] In another exemplary embodiment, the additional modified signaling agent is VEGF 165b and has the amino acid sequence of SEQ ID NO: 236.

[0253] In these embodiments, the modified signaling agent has a mutation at the 83rd amino acid (e.g., a substitution mutation at the 83rd amino acid, e.g., I83K, I83R, or I83H). Without wishing to be bound by theory, it is believed that such mutations may cause a decrease in receptor binding affinity. See, for example, U.S. Patent No. 9,078,860, which is hereby incorporated by reference in its entirety.

[0254] In one embodiment, the additional modified signaling substance is TNF-α. TNF is a pleiotropic cytokine with a wide variety of functions including cell growth control, differentiation, apoptosis, tumorigenesis, viral replication, autoimmunity, immune cell function and trafficking, inflammation, and septic shock, and binds to two distinct membrane receptors on target cells, namely TNFR1 (p55) and TNFR2 (p75). While TNFR1 exhibits a very broad expression pattern, TNFR2 is preferentially expressed in certain populations of lymphocytes, Tregs, endothelial cells, certain types of neurons, microglia, cardiomyocytes, and mesenchymal stem cells. Although there is some overlap, very different biological pathways are activated in response to receptor activation. In principle, without wishing to be bound by theory, TNFR1 signaling is associated with the induction of apoptosis (cell death), and TNFR2 signaling is associated with the activation of cell survival signals (e.g., activation of the NFkB pathway). Administration of TNF is toxic systemically, which is mainly due to the involvement of TNFR1. However, activation of TNFR2 is also associated with a wide range of activities, and it should be noted that TNF targeting and control of TNF activity are important from the perspective of developing TNF-based therapies, as in the case of TNFR1.

[0255] In some embodiments, the additional modified signaling substance has a reduced affinity and / or activity for TNFR1 and / or TNFR2. In some embodiments, the modified signaling substance has a substantially reduced or eliminated affinity and / or activity for TNFR1 and / or TNFR2. While TNFR1 is expressed in most tissues and is involved in cell death signaling, in contrast, TNFR2 is involved in cell survival signaling. Thus, in embodiments related to methods of treating cancer, the modified signaling substance has a reduced affinity and / or activity for TNFR1 and / or a substantially reduced or eliminated affinity and / or activity for TNFR2. In these embodiments, the chimeric protein can be targeted to cells in which apoptosis is desired, such as tumor cells or tumor vascular endothelial cells. For example, in embodiments related to methods of promoting cell survival in neurodegeneration for the treatment of neurodegenerative disorders, the modified signaling substance has a reduced affinity and / or activity for TNFR2 and / or a substantially reduced or eliminated affinity and / or activity for TNFR1. In other words, the chimeric proteins of the present disclosure include, in some embodiments, modified TNF-α substances that enable preference for either a cell death signal or a survival signal.

[0256] In some embodiments, the chimeric protein has a modified TNF with a reduced affinity and / or activity for TNFR1 and / or a substantially reduced or eliminated affinity and / or activity for TNFR2. Such chimeras are, in some embodiments, more potent apoptosis inducers compared to chimeras having only mutations that cause a reduction in the affinity and / or activity for wild-type TNF and / or TNFR1. Such chimeras are, in some embodiments, used to induce tumor cell death or tumor vascular endothelial cell death (e.g., in the treatment of cancer). Also, in some embodiments, these chimeras further assist in TNFR1-mediated antitumor activity in vivo by avoiding or suppressing the activation of T cells, such as via TNFR2. reg Avoiding or suppressing the activation of cells further aids in TNFR1-mediated antitumor activity in vivo.

[0257] In some embodiments, the chimeric protein has a modified TNF with reduced affinity and / or activity for TNFR2 and / or reduced or eliminated affinity and / or activity for TNFR1. In some embodiments, such chimeras are more potent activators of cell survival in certain types of cells, which can be specific therapeutic targets in various disease contexts including, but not limited to, stimulation of neurogenesis. Additionally, such TNFR2-preferring chimeras are also useful for the treatment of autoimmune diseases (such as Crohn's disease, diabetes, MS, colitis, etc., and many other diseases described herein). In some embodiments, the chimeras target autoreactive T cells. In some embodiments, the chimeras promote the activation of T reg cells and the indirect suppression of cytotoxic T cells.

[0258] In some embodiments, the chimeras (e.g., modified TNF with reduced affinity and / or activity for TNFR2 and / or reduced or eliminated affinity and / or activity for TNFR1) cause cell death of autoreactive T cells, for example, by activation of TNFR2 and / or avoidance of TNFR1. Without wishing to be bound by theory, these autoreactive T cells alter their apoptosis / cell survival signals, for example, by changes in the activity / signal transduction of the NFkB pathway.

[0259] In some embodiments, TNFR2-based chimeras have other therapeutic uses, particularly in diseases including but not limited to various autoimmune diseases, heart diseases, demyelinating neurodegenerative disorders, and infectious diseases.

[0260] In one embodiment, wild-type TNF-α has the amino acid sequence of SEQ ID NO: 237.

[0261] In the foregoing embodiment, the modified TNF-α substance has a mutation that produces a modified TNF-α with reduced receptor-binding affinity at one or more amino acid positions among the 29th, 31st, 32nd, 84th, 85th, 86th, 87th, 88th, 89th, 145th, 146th, and 147th positions. See, for example, U.S. Patent No. 7,993,636, the entire contents of which are incorporated herein by reference.

[0262] In some embodiments, the modified human TNF-α moiety has a mutation at one or more amino acid positions among R32, N34, Q67, H73, L75, T77, S86, Y87, V91, I97, T105, P106, A109, P113, Y115, E127, N137, D143, and A145, as described in International Publication No. WO 2015 / 007903, the entire contents of which are incorporated herein by reference (numbering based on the human TNF sequence of Genbank accession number BAG70306, update number BAG70306.1, GI:197692685). In some embodiments, the modified human TNF-α moiety has a substitution mutation selected from R32G, N34G, Q67G, H73G, L75G, L75A, L75S, T77A, S86G, Y87Q, Y87L, Y87A, Y87F, V91G, V91A, I97A, I97Q, I97S, T105G, P106G, A109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, A145G, and A145T. In one embodiment, the human TNF-α moiety has a mutation selected from Y87Q, Y87L, Y87A, and Y87F. In another embodiment, the human TNF-α moiety has a mutation selected from I97A, I97Q, and I97S. In other embodiments, the human TNF-α moiety has a mutation selected from Y115A and Y115G.

[0263] In some embodiments, the modified TNF-α substance has one or more mutations selected from N39Y, S147Y, and Y87H, as described in International Publication No. WO 2008 / 124086, the entire contents of which are incorporated herein by reference.

[0264] In certain embodiments, the additional modified signaling substance is TNF-β. TNF-β can form homodimers or heterodimers with LT-β (LT-α1β2). In some embodiments, the modified signaling substance has substantially reduced or eliminated affinity and / or activity for TNFR1 and / or TNFR2 and / or herpesvirus entry mediator (HEVM) and / or LT-βR.

[0265] In certain embodiments, wild-type TNF-β has the amino acid sequence of SEQ ID NO: 238.

[0266] In such embodiments, the modified TNF-β substance may include mutations that produce a modified TNF-β with reduced receptor binding affinity for TNFR2 in one or more amino acids at positions 106-113. In certain embodiments, the modified signaling substance has one or more substitution mutations at amino acid positions 106-113. In an exemplary embodiment, those substitution mutations are selected from Q107E, Q107D, S106E, S106D, Q107R, Q107N, Q107E / S106E, Q107E / S106D, Q107D / S106E, and Q107D / S106D. In another embodiment, the modified signaling substance has an insertion of about 1 to about 3 amino acids at positions 106-113.

[0267] In some embodiments, the additional modified agent can be a TNF family member (e.g., TNF-α, TNF-β) that is a single-chain trimeric form as described in International Publication No. WO 2015 / 007903, the entire contents of which are incorporated herein by reference.

[0268] In some embodiments, the modified agent is a TNF family member (e.g., TNF-α, TNF-β) that has reduced affinity and / or activity for TNFR1, i.e., antagonist activity (e.g., natural antagonist activity, or antagonist activity as a result of one or more mutations, see, e.g., International Publication No. WO 2015 / 007520, the entire content of which is incorporated herein by reference). In these embodiments, the modified agent may be a TNF family member (e.g., TNF-α, TNF-β) that has substantially reduced or eliminated affinity and / or activity for TNFR2. In some embodiments, the modified agent is a TNF family member (e.g., TNF-α, TNF-β) that has reduced affinity and / or activity for TNFR2, i.e., antagonist activity (e.g., natural antagonist activity, or antagonist activity as a result of one or more mutations, see, e.g., International Publication No. WO 2015 / 007520, the entire content of which is incorporated herein by reference). In these embodiments, the modified agent may be a TNF family member (e.g., TNF-α, TNF-β) that has substantially reduced or eliminated affinity and / or activity for TNFR1. The constructs of the embodiments are used, for example, in methods for attenuating the TNF response in a cell-specific manner. In some embodiments, the antagonist TNF family member (e.g., TNF-α, TNF-β) is single-chain trimeric as described in International Publication No. WO 2015 / 007903.

[0269] In certain embodiments, the additional modified signaling substance is TRAIL. In some embodiments, the modified TRAIL substance has reduced affinity and / or activity for DR4 (TRAIL-RI), and / or DR5 (TRAIL-RII), and / or DcR1, and / or DcR2. In some embodiments, the modified TRAIL substance has substantially reduced or eliminated affinity and / or activity for DR4 (TRAIL-RI), and / or DR5 (TRAIL-RII), and / or DcR1, and / or DcR2.

[0270] In certain embodiments, wild-type TRAIL has the amino acid sequence of SEQ ID NO: 239.

[0271] In the above embodiments, the modified TRAIL substance may have mutations at the amino acid positions of T127 - R132, E144 - R149, E155 - H161, Y189 - Y209, T214 - I220, K224 - A226, W231, E236 - L239, E249 - K251, T261 - H264, and H270 - E271 (numbering based on the human sequence of Genbank accession number NP_003801, update number NP_003801.1, GI: 4507593. See the above reference).

[0272] In certain embodiments, the additional modified signaling substance is TGFα. In the above embodiments, the modified TGFα substance has reduced affinity and / or activity for the epidermal growth factor receptor (EGFR). In some embodiments, the modified TGFα substance has substantially reduced or eliminated affinity and / or activity for the epidermal growth factor receptor (EGFR).

[0273] In certain embodiments, an additional modified signaling substance is TGFβ. In such embodiments, the modified signaling substance has a reduced affinity and / or activity for TGFBR1 and / or TGFBR2. In some embodiments, the modified signaling substance has a substantially reduced or eliminated affinity and / or activity for TGFBR1 and / or TGFBR2. In some embodiments, the modified signaling substance may have a reduced, substantially reduced, or eliminated affinity and / or activity for TGFBR3, which may, although not wishing to be bound by theory, have a role in storing ligands for the TGF-β receptor. In some embodiments, TGFβ may prefer TGFBR1 over TGFBR2 or TGFBR2 over TGFBR1. Similarly, although not wishing to be bound by theory, LAP may have a role in storing ligands for the TGF-β receptor. In some embodiments, the modified signaling substance has a reduced affinity and / or activity for TGFBR1 and / or TGFBR2 and / or a substantially reduced or eliminated affinity and / or activity for latent associated peptide (LAP). In some embodiments, such chimeras are used in camurati-engelmann disease or other diseases associated with inappropriate TGFβ signaling.

[0274] In some embodiments, an additional modified agent is a TGF family member (such as TGFα, TGFβ) having a reduced affinity and / or activity in one or more of TGFBR1, TGFBR2, TGFBR3, i.e., antagonist activity (e.g., natural antagonist activity or antagonist activity as a result of one or more mutations, see, e.g., WO 2015 / 007520, which is incorporated herein by reference in its entirety). In these embodiments, the modified agent is a TGF family member (such as TGFα, TGFβ) that may have a substantially reduced or eliminated affinity and / or activity in one or more of TGFBR1, TGFBR2, TGFBR3.

[0275] In some embodiments, the additional modified agent is a TGF family member (e.g., TGFα, TGFβ) that has reduced affinity and / or activity for TGFBR1 and / or TGFBR2, i.e., antagonist activity (e.g., natural antagonist activity or antagonist activity as a result of one or more mutations; see, e.g., WO 2015 / 007520, which is hereby incorporated by reference in its entirety). In these embodiments, the modified agent may be a TGF family member (e.g., TGFα, TGFβ) that has substantially reduced or eliminated affinity and / or activity for TGFBR3.

[0276] In one embodiment, the additional modified signaling molecule is IL-1. In one embodiment, the modified signaling molecule is IL-1α or IL-1β. In some embodiments, the modified signaling molecule has reduced affinity and / or activity for IL-1R1 and / or IL-1RAcP. In some embodiments, the modified signaling molecule has substantially reduced or eliminated affinity and / or activity for IL-1R1 and / or IL-1RAcP. In some embodiments, the modified signaling molecule has reduced affinity and / or activity for IL-1R2. In some embodiments, the modified signaling molecule has substantially reduced or eliminated affinity and / or activity for IL-1R2. For example, in some embodiments, the modified IL-1 substances of the present disclosure avoid interaction at IL-1R2, thus substantially reducing its function as a decoy and / or sink for therapeutic agents.

[0277] In one embodiment, wild-type IL-1β has the amino acid sequence of SEQ ID NO: 240.

[0278] IL1 is a pro-inflammatory cytokine and an important immune system regulator. IL1 is a potent activator of CD4 T cell responses, increasing the proportion of Th17 cells and enhancing the proliferation of IFNγ-producing cells and IL-4-producing cells. IL-1 is also a potent regulator of CD8 + T cells, enhancing the proliferation, differentiation, migration to the periphery, and memory of antigen-specific CD8 + T cells. The IL-1 receptor includes IL-1R1 and IL-1R2. Binding to IL-1R1 and signal transduction from IL-1R1 constitute the mechanism by which IL-1 mediates much of its biological (and pathological) activity. IL1-R2 can function as a decoy receptor, thereby reducing the availability of IL-1 for interaction with IL-1R1 and signal transduction from IL-1R1.

[0279] In some embodiments, the modified IL-1 has a reduced affinity and / or activity for IL-1R1 (e.g., agonist activity). In some embodiments, the modified IL-1 has a substantially reduced or eliminated affinity and / or activity for IL-1R2. In such embodiments, recoverable IL-1 / IL-1R1 signal transduction, prevention of the disappearance of the therapeutic chimera in IL-R2, and as a result, a decrease in the required IL-1 dosage (e.g., compared to wild type or a chimera having only an attenuated mutation for IL-R1) are seen. Such constructs are used, for example, in methods of treating cancer that include stimulating the immune system to initiate an anti-cancer response.

[0280] In some embodiments, the modified IL-1 has a reduced affinity and / or activity for IL-1R1 (e.g., antagonist activity, e.g., natural antagonist activity, or antagonist activity as a result of one or more mutations, see, e.g., WO 2015 / 007520, which is hereby incorporated by reference in its entirety). In some embodiments, the modified IL-1 has a substantially reduced or eliminated affinity and / or activity for IL-1R2. In such embodiments, irreversible IL-1 / IL-1R1 signaling, and prevention of the loss of the therapeutic chimeric in IL-R2, and as a result a decrease in the required IL-1 dose (e.g., compared to wild-type or a chimeric having only a attenuating mutation for IL-R1) is seen. Such constructs are used, for example, in methods of treating autoimmune diseases including suppressing the immune system.

[0281] In the above-described embodiment, the modified signaling substance produces a modified human IL-1β having a deletion of amino acids 52 to 54, which has a reduced binding affinity for type I IL-1R and a reduced biological activity. For example, see International Publication No. WO 1994 / 000491, the entire content of which is incorporated herein by reference. In some embodiments, the modified human IL-1β exhibits a reduced binding to IL-1R as described, for example, in International Publication No. WO 2015 / 007542 and International Publication No. WO 2015 / 007536, the entire contents of which are incorporated herein by reference, and has one or more substitution mutations selected from A117G / P118G, R120X, L122A, T125G / L126G, R127G, Q130X, Q131G, K132A, S137G / Q138Y, L145G, H146X, L145A / L147A, Q148X, Q148G / Q150G, Q150G / D151A, M152G, F162A, F162A / Q164E, F166A, Q164E / E167K, N169G / D170G, I172A, V174A, K208E, K209X, K209A / K210A, K219X, E221X, E221S / N224A, N224S / K225S, E244K, N245Q (where X can be any amino acid change, for example, a non-conservative change) (numbering based on the human IL-1β sequence of Genbank accession number NP_000567, update number NP-000567.1, GI:10835145). In some embodiments, the modified human IL-1β may have one or more mutations selected from R120A, R120G, Q130A, Q130W, H146A, H146G, H146E, H146N, H146R, Q148E, Q148G, Q148L, K209A, K209D, K219S, K219Q, E221S, and E221K. In one embodiment, the modified human IL-1β includes the mutations Q131G and Q148G. In one embodiment, the modified human IL-1β includes the mutations Q148G and K208E. In one embodiment, the modified human IL-1β includes the mutations R120G and Q131G. In one embodiment, the modified human IL-1β includes the mutations R120G and H146G. In one embodiment, the modified human IL-1β includes the mutations R120G and K208E.In certain embodiments, the modified human IL-1β comprises the mutations R120G, F162A, and Q164E.

[0282] In certain embodiments, the additional modified signaling molecule is IL-2. In said embodiments, the modified signaling molecule has a reduced affinity and / or activity for IL-2Rα and / or IL-2Rβ and / or IL-2Rγ. In some embodiments, the modified signaling molecule has a reduced affinity and / or activity for IL-2Rβ and / or IL-2Rγ. In some embodiments, the modified signaling molecule has a substantially reduced or eliminated affinity and / or activity for IL-2Rα. Said embodiments may be suitable for the treatment of cancer, for example when the modified IL-2 is agonistic at IL-2Rβ and / or IL-2Rγ. For example, the constructs of the present disclosure are advantageous for attenuating the activity of CD8 + T cells that have IL2 receptor β and γ (and can provide an anti-tumor effect), and are disadvantageous for T reg that have IL2 receptor α, β, and γ (and can provide an immunosuppressive tumor-promoting effect). Further, in some embodiments, by preferring IL-2Rβ and / or IL-2Rγ over IL-2Rα, side effects of IL-2 such as pulmonary edema are avoided. Also, an IL-2-based chimera may be useful for the treatment of autoimmune diseases, for example when the modified IL-2 is antagonistic at IL-2Rβ and / or IL-2Rγ (e.g., natural antagonist activity, or antagonist activity as a result of one or more mutations, for which see, e.g., WO 2015 / 007520, which is incorporated herein by reference in its entirety). For example, the constructs of the present disclosure are advantageous for attenuating the activity of CD8 + T cells (and thus attenuating the immune response) that have IL2 receptor β and γ, and are disadvantageous for T reg that have IL2 receptor α, β, and γ. Alternatively, in some embodiments, the chimera having IL-2 is advantageous for the activation of T reg and thus immunosuppression, and CD8 +It is disadvantageous for T cell activation. For example, these constructs are used for the treatment of diseases, or diseases that can benefit from immunosuppression, such as autoimmune diseases.

[0283] In some embodiments, the chimeric protein is CD8 + A targeting moiety as described herein that targets T cells, and a modified IL-2 substance with reduced affinity and / or activity for IL-2Rβ and / or IL-2Rγ, and / or substantially reduced or removed affinity and / or activity for IL-2Rα. In some embodiments, these constructs are targeted CD8 + Provide T cell activity and are generally inactive (or have substantially reduced activity) against T reg cells. In some embodiments, such constructs have enhanced immunostimulatory effects compared to wild-type IL-2 (for example, without wishing to be bound by theory, by not stimulating Tregs), while removing or reducing the systemic toxicity associated with IL-2.

[0284] In one embodiment, wild-type IL-2 has the amino acid sequence of SEQ ID NO: 241.

[0285] In the above embodiment, the modified IL-2 substance has one or more mutations in amino acid L72 (L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, or L72K), amino acid F42 (F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, or F42K), and amino acid Y45 (Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R or Y45K). Without wishing to be bound by theory, it is believed that these modified IL-2 substances have reduced affinity for the high-affinity IL-2 receptor and maintained affinity for the intermediate-affinity IL-2 receptor compared to wild-type IL-2. See, for example, US Patent Application Publication No. 2012 / 0244112, which is hereby incorporated by reference in its entirety.

[0286] In certain embodiments, an additional modified signaling agent is IL-3. In some embodiments, the modified signaling agent has reduced affinity and / or activity for an IL-3 receptor, which is a heterodimer that includes a unique α chain paired with a common β (βc or CD131) subunit. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for an IL-3 receptor, which is a heterodimer that includes a unique α chain paired with a common β (βc or CD131) subunit.

[0287] In certain embodiments, an additional modified signaling agent is IL-4. In such embodiments, the modified signaling agent has reduced affinity and / or activity for type 1 and / or type 2 IL-4 receptors. In such embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for type 1 and / or type 2 IL-4 receptors. The type 1 IL-4 receptor is composed of an IL-4Rα subunit and a common γ chain and specifically binds IL-4. The type 2 IL-4 receptor includes an IL-4Rα subunit bound to a different subunit known as IL-13Rα1. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for the type 2 IL-4 receptor.

[0288] In certain embodiments, wild-type IL-4 has the amino acid sequence of SEQ ID NO: 242.

[0289] In the above-described embodiment, the modified IL-4 substance has one or more mutations in amino acid R121 (R121A, R121D, R121E, R121F, R121H, R121I, R121K, R121N, R121P, R121T, R121W), amino acid E122 (E122F), Y124 (Y124A, Y124Q, Y124R, Y124S, Y124T), and amino acid S125 (S125A). Without wishing to be bound by theory, it is believed that these modified IL-4 substances maintain the activity mediated by the type I receptor but significantly reduce the biological activities mediated by other receptors. See, for example, U.S. Patent No. 6,433,157, the entire contents of which are incorporated herein by reference.

[0290] In one embodiment, the additional modified signaling substance is IL-6. IL-6 signals through a cell surface type I cytokine receptor complex that includes a ligand-binding IL-6R chain (CD126) and a signaling element gp130. IL-6 can also bind to soluble IL-6R (sIL-6R), which is the extracellular portion of IL-6R. The sIL-6R / IL-6 complex may be involved in neurite outgrowth and neuronal survival and may thus be important for nerve regeneration via remyelination. Thus, in some embodiments, the modified signaling substance has a reduced affinity and / or activity for IL-6R / gp130 and / or sIL-6R. In some embodiments, the modified signaling substance has a substantially reduced or eliminated affinity and / or activity for IL-6R / gp130 and / or sIL-6R.

[0291] In one embodiment, wild-type IL-6 has the amino acid sequence of SEQ ID NO: 243.

[0292] In the above embodiment, the modified signal transduction substance has one or more mutations in the 58th, 160th, 163rd, 171st, or 177th amino acid. Although not wishing to be bound by theory, these modified IL-6 substances are thought to exhibit a decrease in binding affinity for IL-6Rα and a decrease in biological activity. See, for example, International Publication No. 97 / 10338, the entire contents of which are incorporated herein by reference.

[0293] In one embodiment, an additional modified signal transduction substance is IL-10. In the above embodiment, the modified signal transduction substance has a decreased affinity and / or activity for IL-10 receptor-1 and IL-10 receptor-2. In some embodiments, the modified signal transduction substance has a substantially decreased or eliminated affinity and / or activity for IL-10 receptor-1 and IL-10 receptor-2.

[0294] In one embodiment, an additional modified signal transduction substance is IL-11. In the above embodiment, the modified signal transduction substance has a decreased affinity and / or activity for IL-11Rα and / or IL-11Rβ and / or gp130. In the above embodiment, the modified signal transduction substance has a substantially decreased or eliminated affinity and / or activity for IL-11Rα and / or IL-11Rβ and / or gp130.

[0295] In one embodiment, an additional modified signal transduction substance is IL-12. In the above embodiment, the modified signal transduction substance has a decreased affinity and / or activity for IL-12Rβ1 and / or IL-12Rβ2. In the above embodiment, the modified signal transduction substance has a substantially decreased or eliminated affinity and / or activity for IL-12Rβ1 and / or IL-12Rβ2.

[0296] In certain embodiments, the additional modified signaling substance is IL-13. In said embodiments, the modified signaling substance has a reduced affinity and / or activity for the IL-4 receptor (IL-4Rα) and IL-13Rα1. In some embodiments, the modified signaling substance has a substantially reduced or eliminated affinity and / or activity for the IL-4 receptor (IL-4Rα) or IL-13Rα1.

[0297] In certain embodiments, wild-type IL-13 has the amino acid sequence of SEQ ID NO: 244.

[0298] In said embodiments, the modified IL-13 substance has one or more mutations in the 13th, 16th, 17th, 66th, 69th, 99th, 102nd, 104th, 105th, 106th, 107th, 108th, 109th, 112th, 113th, and 114th amino acids. Without wishing to be bound by theory, these modified IL-13 substances are thought to exhibit reduced biological activity. See, for example, WO 2002 / 018422, which is incorporated herein by reference in its entirety.

[0299] In certain embodiments, the additional modified signaling substance is IL-18. In some embodiments, the modified signaling substance has a reduced affinity and / or activity for IL-18Rα and / or IL-18Rβ. In some embodiments, the modified signaling substance has a substantially reduced or eliminated affinity and / or activity for IL-18Rα and / or IL-18Rβ. In some embodiments, the modified signaling substance has a substantially reduced or eliminated affinity and / or activity for type II IL-18Rα, an isoform of IL-18Rα that lacks the TIR domain required for signaling.

[0300] In certain embodiments, wild-type IL-18 has the amino acid sequence of SEQ ID NO: 245.

[0301] In the above-described embodiment, the modified IL-18 substance may contain one or more mutations in an amino acid or amino acid region selected from Y37-K44, R49-Q54, D59-R63, E67-C74, R80, M87-A97, N127-K129, Q139-M149, K165-K171, R183, and Q190-N191, as described in International Publication No. 2015 / 007542, the entire contents of which are incorporated herein by reference (numbering based on the human IL-18 sequence of Genbank accession number AAV38697, update number AAV38697.1, GI:54696650).

[0302] In one embodiment, the additional modified signaling substance is IL-33. In the above-described embodiment, the modified signaling substance has a reduced affinity and / or activity for the ST-2 receptor and IL-1RAcP. In some embodiments, the modified signaling substance has a substantially reduced or eliminated affinity and / or activity for the ST-2 receptor and IL-1RAcP.

[0303] In one embodiment, wild-type IL-33 has the amino acid sequence of SEQ ID NO: 246.

[0304] In the above-described embodiment, the modified IL-33 substance may contain one or more mutations in an amino acid or amino acid region selected from I113-Y122, S127-E139, E144-D157, Y163-M183, E200, Q215, L220-C227, and T260-E269, as described in International Publication No. 2015 / 007542, the entire contents of which are incorporated herein by reference (numbering based on the human sequence of Genbank accession number NP_254274, update number NP_254274.1, GI:15559209).

[0305] In one embodiment, the modified signaling substance is epidermal growth factor (EGF). EGF is a member of the family of potent growth factors. The members include EGF, HB-EGF, and others such as TGFα, amphiregulin, neuregulin, epiregulin, betacellulin. The EGF family receptors include EGFR (ErbB1), ErbB2, ErbB3, and ErbB4. These receptors may function as homodimeric receptor subtypes and / or heterodimeric receptor subtypes. Different EGF family members exhibit different selectivities for different receptor subtypes. For example, EGF binds to ErbB1 / ErbB1, ErbB1 / ErbB2, ErbB4 / ErbB2, and several other heterodimeric subtypes. HB-EGF has a similar pattern but also binds to ErbB4 / 4. The regulation of EGF (EGF-like) growth factor signaling, whether positive or negative, is of high therapeutic interest. For example, in the treatment of various cancers in which EGFR signaling constitutes a major growth-promoting signal, the inhibition of EGFR signaling is of interest. Alternatively, the stimulation of EGFR signaling is of therapeutic interest, for example, in promoting wound healing (acute and chronic), in the treatment of oral mucositis (a major side effect of various cancer therapies including but not limited to radiotherapy).

[0306] In some embodiments, the additional modified signaling agent has reduced affinity and / or activity for ErbB1, ErbB2, ErbB3, and / or ErbB4. Such embodiments are used, for example, in methods of treating wounds. In some embodiments, the modified signaling agent binds to one or more of ErbB1, ErbB2, ErbB3, and ErbB4 and antagonizes the activity of the receptor. In such embodiments, the modified signaling agent has reduced affinity and / or activity for ErbB1, ErbB2, ErbB3, and / or ErbB4, thereby being antagonized such that the activity of the receptor is attenuated. Such embodiments are used, for example, in the treatment of cancer. In one embodiment, the modified signaling agent has reduced affinity and / or activity for ErbB1. ErbB1 is a therapeutic target of kinase inhibitors (e.g., gefitinib, erlotinib, afatinib, brigatinib, and icotinib) that have mostly side effects due to their lack of selectivity. In some embodiments, attenuation of ErbB1 signaling by the antagonist is more targeted and has fewer side effects than other agents targeting the EGF receptor.

[0307] In some embodiments, the additional modified signaling agent has a reduced affinity and / or activity for ErbB1 (e.g., antagonist activity, e.g., natural antagonist activity, or antagonist activity as a result of one or more mutations, see, e.g., WO 2015 / 007520, which is incorporated herein by reference in its entirety), and / or a substantially reduced or eliminated affinity and / or activity for ErbB4 or other subtypes that can interact. Specific targeting via the targeting moiety results in cell-selective inhibition of ErbB1 / ErbB1 receptor activation (antagonism, e.g., natural antagonist activity, or antagonist activity as a result of one or more mutations, for which see, e.g., WO 2015 / 007520, which is incorporated herein by reference in its entirety), but is not related to other receptor subtypes that may be associated with inhibition-related side effects. Thus, in contrast to EGFR kinase inhibitors that inhibit EGFR activity in all cell types in the body, such constructs provide a cell-selective anti-EGFR (ErbB1) drug effect with reduced side effects (e.g., tumor cells in which EGFR signaling is activated due to receptor amplification, overexpression, etc.).

[0308] In some embodiments, the additional modified signaling agent has a reduced affinity and / or activity for ErbB4 and / or other subtypes that can interact (e.g., agonist activity). Specific targeting to specific target cells via the targeting moiety results in selective activation of ErbB1 signaling (e.g., in epithelial cells). In some embodiments, such constructs are used for the treatment of wounds with reduced side effects (promoting wound healing), particularly chronic conditions, and applications other than topical application of therapeutic agents (e.g., systemic wound healing).

[0309] In certain embodiments, the modified signaling substance is insulin or an insulin analog. In some embodiments, the modified insulin or insulin analog has a reduced affinity and / or activity for the insulin receptor and / or the IGF1 receptor or the IGF2 receptor. In some embodiments, the modified insulin or insulin analog has a substantially reduced or eliminated affinity and / or activity for the insulin receptor and / or the IGF1 receptor or the IGF2 receptor. Attenuation of the response in the insulin receptor enables the management of diabetes, obesity, metabolic disorders, etc., while separating from the IGF1 receptor or the IGF2 receptor avoids the cancer promoting effect.

[0310] In certain embodiments, the modified signaling substance is insulin-like growth factor-I or insulin-like growth factor-II (IGF-1 or IGF-2). In certain embodiments, the modified signaling substance is IGF-1. In such embodiments, the modified signaling substance has a reduced affinity and / or activity for the insulin receptor and / or the IGF1 receptor. In certain embodiments, the modified signaling substance IGF1 receptor binds and may antagonize the activity of the receptor. In such embodiments, the modified signaling substance has a reduced affinity and / or activity for the IGF1 receptor, thereby being antagonized such that the activity of the receptor is attenuated. In some embodiments, the modified signaling substance has a substantially reduced or eliminated affinity and / or activity for the insulin receptor and / or the IGF1 receptor. In some embodiments, the modified signaling substance antagonizes the IGF2 receptor such that the activity of the receptor is attenuated by a decrease in its affinity and / or activity for the IGF2 receptor. In certain embodiments, the modified signaling substance has a substantially reduced or eliminated affinity and / or activity for the insulin receptor and thus does not interfere with insulin signaling. This is applied to the treatment of cancer in various embodiments. In various embodiments, the agent of the present disclosure prevents the IR isoform A from becoming resistant to cancer treatment.

[0311] In certain embodiments, the modified signaling molecule is EPO. In various embodiments, the modified EPO agonist has a reduced affinity and / or activity for the EPO receptor (EPOR) receptor and / or the ephrin receptor (EphR) compared to wild-type EPO or an agonist based on other EPOs described herein. In some embodiments, the modified EPO agonist has a substantially reduced or eliminated affinity and / or activity for the EPO receptor (EPOR) receptor and / or the Eph receptor (EphR). Examples of EPO receptors include, but are not limited to, EPOR homodimers or EPOR / CD131 heterodimers. The beta common receptor (βcR) is also mentioned as an EPO receptor. Examples of Eph receptors include, but are not limited to, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, and EPHB6. In some embodiments, the modified EPO protein contains one or more mutations that cause a reduced affinity of the EPO protein for a receptor that includes one or more EPO receptors or Eph receptors (e.g., heterodimers, heterotrimers including, as non-limiting examples, EPOR-EPHB4, EPOR-βcR-EPOR). The receptors of European Patent Application Publication No. 2492355, the entire content of which is incorporated herein by reference, are also presented, and NEPOR is mentioned as a non-limiting example.

[0312] In certain embodiments, human EPO has the amino acid sequence of SEQ ID NO: 247 (the first 27 amino acids are the signal peptide).

[0313] In certain embodiments, the human EPO protein is a mature (signal peptide excised) EPO that is a glycoprotein consisting of 166 amino acid residues having the sequence of SEQ ID NO: 248.

[0314] The structure of the human EPO protein is expected to include four helix bundles, including helix A, helix B, helix C, and helix D. In various embodiments, the modified EPO protein includes one or more mutations located within four regions of the EPO protein that are important for biological activity, namely, amino acid residues 10 - 20, 44 - 51, 96 - 108, and 142 - 156. In some embodiments, one or more mutations are located at residues 11 - 15, 44 - 51, 100 - 108, and 147 - 151. These residues are located in helix A (Val11, Arg14, and Tyr15), helix C (Ser100, Arg103, Ser104, and Leu108), helix D (Asn147, Arg150, Gly151, and Leu155), and the A / B connecting loop (residues 42 - 51). In some embodiments, the modified EPO protein includes mutations in the residues between amino acids 41 - 52, as well as at amino acids 147, 150, 151, and 155. Without wishing to be bound by theory, mutations in these residues are thought to have a significant effect on both receptor binding and in vitro biological activity. In some embodiments, the modified EPO protein includes mutations at residues 11, 14, 15, 100, 103, 104, and 108. Without wishing to be bound by theory, mutations in these residues are thought to have a moderate effect on receptor binding activity and a greater effect on in vitro biological activity. Examples of substitutions include, but are not limited to, one or more of Val11Ser, Arg14Ala, Arg14Gln, Tyr15Ile, Pro42Asn, Thr44Ile, Lys45Asp, Val46Ala, Tyr51Phe, Ser100Glu, Ser100Thr, Arg103Ala, Ser104Ile, Ser104Ala, Leu108Lys, Asn147Lys, Arg150Ala, Gly151Ala, and Leu155Ala.

[0315] In some embodiments, the modified EPO protein comprises mutations that confer biological activity but not binding, such as those described in Eliot, et al. Mapping of the Active Site of Recombinant Human Erythropoietin, January 15, 1997, Blood:89(2), which is incorporated herein by reference in its entirety.

[0316] In some embodiments, the modified EPO protein comprises one or more mutations related to surface residues of the EPO protein that are involved in contact with the receptor. Without wishing to be bound by theory, mutations of these surface residues are thought to have a low likelihood of affecting protein folding and thus some biological activity is retained. Examples of surface residues where mutations can be introduced include, but are not limited to, residues 147 and 150. In an exemplary embodiment, the mutation is a substitution and includes one or more of N147A, N147K, R150A, and R150E.

[0317] In some embodiments, the modified EPO protein comprises one or more mutations at residues N59, E62, L67, and L70, and one or more mutations that affect disulfide bond formation. Without wishing to be bound by theory, these mutations are thought to affect folding and / or are predicted to be present at buried positions and thus indirectly affect biological activity.

[0318] In one embodiment, the modified EPO protein comprises the K20E substitution that significantly reduces binding to the receptor. See Elliot, et al. (1997) Blood, 89: pp. 493-502, which is incorporated herein by reference in its entirety.

[0319] Other EPO mutations that can be incorporated into the chimeric EPO protein of the present invention are disclosed, for example, in Elliott, et al. (1997) Blood, 89: pp. 493-502, the entire content of which is incorporated herein by reference, and Taylor, et al. (2010) PEDS, 23(4): pp. 251-260, the entire content of which is incorporated herein by reference.

[0320] In various embodiments, the signaling molecule is a toxin or a toxic enzyme. In some embodiments, the toxin or toxic enzyme is derived from plants and bacteria. Examples of toxins or toxic enzymes include, but are not limited to, ribosome-inactivating proteins (RIPs) such as diphtheria toxin, Pseudomonas aeruginosa toxin, anthrax toxin, ricin, and saponin, modeccin, abrin, gelonin, and pokeweed antiviral protein. Other toxins include those disclosed in Mathew, et al. (2009) Cancer Sci, 100(8): pp. 1359-65, the entire disclosure of which is incorporated herein by reference. In such embodiments, the chimeric protein of the present invention can be utilized to induce cell death in a cell type-specific manner. In such embodiments, the toxin can be modified, for example, by mutagenesis, to reduce its affinity and / or activity in order to attenuate its effect, as described for other signaling molecules herein.

[0321] Linker and functional group In some embodiments, the chimeric proteins of the present disclosure may include one or more linkers. In some embodiments, the chimeric proteins of the present disclosure include a linker that connects a targeting moiety and a signaling agent (such as consensus interferon or a variant thereof). In some embodiments, the chimeric proteins of the present disclosure include a linker within a signaling agent (such as consensus interferon or a variant thereof). In some embodiments, linkers can be utilized to link various functional groups, residues, or moieties to the chimeric proteins as described herein. In some embodiments, the linker is a single amino acid or multiple amino acids that do not affect or decrease the stability, orientation properties, binding properties, neutralizing properties, and / or clearance properties of the binding region and the binding protein. In various embodiments, the linker is selected from a peptide, protein, sugar, or nucleic acid.

[0322] In some embodiments, a vector encoding a chimeric protein of the present disclosure linked by any of the linkers described herein as a single nucleotide sequence is provided and can be used to prepare such chimeric proteins.

[0323] In some embodiments, the length of the linker enables efficient binding of the targeting moiety and the signaling agent (such as consensus interferon or a variant thereof) to their receptors. For example, in some embodiments, the length of the linker enables efficient binding of one of the targeting moieties to a receptor on the same cell and the signaling agent.

[0324] In some embodiments, the length of the linker is at least equal to the minimum distance between a binding site of one of the targeting moieties to a receptor on the same cell and the binding site of the signaling agent. In some embodiments, the length of the linker is at least 2-fold, or 3-fold, or 4-fold, or 5-fold, or 10-fold, or 20-fold, or 25-fold, or 50-fold, or 100-fold, or higher than the minimum distance between a binding site of one of the targeting moieties to a receptor on the same cell and the binding site of the signaling agent.

[0325] As described herein, the length of the linker enables efficient binding of one of the targeting moieties and the signaling agent to a receptor on the same cell, and the binding occurs sequentially, for example, the binding of the targeting moiety and the receptor precedes the binding of the signaling agent and the receptor.

[0326] In some embodiments, two linkers are present in a single chimera, each linker connecting the signaling agent to the targeting moiety. In various embodiments, the linker has a length that allows formation of sites on those cells that do not have steric hindrance that would interfere with the regulation of either the diseased cell or the effector cell.

[0327] The present invention contemplates the use of various linker sequences. In various embodiments, the linker may be derived from a native multi-domain protein or a linker based on experiments such as those described in Chichili, et al. (2013) Protein Sci., 22(2): pp. 153-167; Chen, et al. (2013) Adv Drug Deliv Rev., 65(10): pp. 1357-1369, the entire content of which is incorporated herein by reference. In some embodiments, the linker may be designed using linker design databases and computer programs such as those described in Chen, et al. (2013) Adv Drug Deliv Rev., 65(10): pp. 1357-1369 and Crasto, et al. (2000) Protein Eng., 13(5): pp. 309-312, the entire content of which is incorporated herein by reference. In various embodiments, the linker may be functional. For example, but not limited to, the linker may function to improve the folding and / or stability of the chimeric proteins of the present disclosure, improve expression, improve pharmacokinetics, and / or improve biological activity.

[0328] In some embodiments, the linker is a polypeptide. In some embodiments, the length of the linker is less than about 100 amino acids. For example, the length of the linker may be less than about 100 amino acids, less than about 95 amino acids, less than about 90 amino acids, less than about 85 amino acids, less than about 80 amino acids, less than about 75 amino acids, less than about 70 amino acids, less than about 65 amino acids, less than about 60 amino acids, less than about 55 amino acids, less than about 50 amino acids, less than about 45 amino acids, less than about 40 amino acids, less than about 35 amino acids, less than about 30 amino acids, less than about 25 amino acids, less than about 20 amino acids, less than about 19 amino acids, less than about 18 amino acids, less than about 17 amino acids, less than about 16 amino acids, less than about 15 amino acids, less than about 14 amino acids, less than about 13 amino acids, less than about 12 amino acids, less than about 11 amino acids, less than about 10 amino acids, less than about 9 amino acids, less than about 8 amino acids, less than about 7 amino acids, less than about 6 amino acids, less than about 5 amino acids, less than about 4 amino acids, less than about 3 amino acids, or less than about 2 amino acids. In some embodiments, the linker is a polypeptide. In some embodiments, the length of the linker is greater than about 100 amino acids. For example, the length of the linker may be greater than about 100 amino acids, greater than about 95 amino acids, greater than about 90 amino acids, greater than about 85 amino acids, greater than about 80 amino acids, greater than about 75 amino acids, greater than about 70 amino acids, greater than about 65 amino acids, greater than about 60 amino acids, greater than about 55 amino acids, greater than about 50 amino acids, greater than about 45 amino acids, greater than about 40 amino acids, greater than about 35 amino acids, greater than about 30 amino acids, greater than about 25 amino acids, greater than about 20 amino acids, greater than about 19 amino acids, greater than about 18 amino acids, greater than about 17 amino acids, greater than about 16 amino acids, greater than about 15 amino acids, greater than about 14 amino acids, greater than about 13 amino acids, greater than about 12 amino acids, greater than about 11 amino acids, greater than about 10 amino acids, greater than about 9 amino acids, greater than about 8 amino acids, greater than about 7 amino acids, greater than about 6 amino acids, greater than about 5 amino acids, greater than about 4 amino acids, greater than about 3 amino acids, or greater than about 2 amino acids. In some embodiments, the linker is mobile. In another embodiment, the linker lacks mobility.

[0329] In some embodiments of the chimeric protein having two or more targeting moieties, a linker interconnects the two targeting moieties, the length of this linker is short, and a linker connects the targeting moiety and the signaling substance, and this linker is longer than the linker connecting the two targeting moieties. For example, the difference in the amino acid length between the linker connecting the two targeting moieties and the linker connecting the targeting moiety and the signaling substance can be about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 19 amino acids, about 18 amino acids, about 17 amino acids, about 16 amino acids, about 15 amino acids, about 14 amino acids, about 13 amino acids, about 12 amino acids, about 11 amino acids, about 10 amino acids, about 9 amino acids, about 8 amino acids, about 7 amino acids, about 6 amino acids, about 5 amino acids, about 4 amino acids, about 3 amino acids, or about 2 amino acids.

[0330] In various embodiments, the linker is substantially composed of glycine residues and serine residues (e.g., about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 97% glycine and serine). For example, in some embodiments, the linker is (Gly4Ser) n wherein n is from about 1 to about 8, such as 1, 2, 3, 4, 5, 6, 7, or 8 (SEQ ID NOs: 249 to SEQ ID NO: 256 respectively). In one embodiment, the linker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO: 257). Other examples of linkers include LE, GGGGS (SEQ ID NO: 249), (GGGGS) n (n = 1 - 4) (SEQ ID NOs: 249 to SEQ ID NO: 252), (Gly)8 (SEQ ID NO: 258), (Gly)6 (SEQ ID NO: 259), (EAAAK) n (n = 1 - 3) (SEQ ID NOs: 260 to SEQ ID NO: 262), A(EAAAK) nA(n=2 to 5)(SEQ ID NO: 263 to SEQ ID NO: 266), AEAAAKEAAAKA (SEQ ID NO: 263), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 267), the array PAPAP (SEQ ID NO: 268), KESGSVSSEQLAQFRSLD (SEQ ID NO: 269), EGKSSGSGSESKST (SEQ ID NO: 270), GSAGSAAGSGEF (SEQ ID NO: 271), and X represents any amino acid, for example, Ala, Lys, or Glu (XP) n Linkers having the sequences of are included but not limited to these. In various embodiments, the linker is GGS.

[0331] In some embodiments, the linker is one or more of GGGSE (SEQ ID NO: 272), GSESG (SEQ ID NO: 273), GSEGS (SEQ ID NO: 274), GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 275), and linkers consisting of G, S, and E randomly arranged every four amino acids.

[0332] In some embodiments, the linker is the hinge region of an antibody (e.g., IgG, IgA, IgD, and IgE including subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). In various embodiments, the linker is the hinge region of an antibody (e.g., IgG, IgA, IgD, and IgE including subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). The hinge regions found in antibodies of the IgG class, IgA class, IgD class, and IgE class act as flexible spacers, allowing the Fab portions to move freely in space. In contrast to the constant regions, the hinge domain is structurally diverse and differs in both sequence and length among the classes and subclasses of immunoglobulins. For example, the length and flexibility of the hinge region vary among IgG subclasses. The hinge region of IgG1 encompasses amino acids 216 - 231, and because its hinge region is easily bent, the Fab fragment can rotate about its axis of symmetry and move within a sphere centered on the first of the two heavy-chain disulfide bridges. IgG2 has a shorter hinge than IgG1 and contains 12 amino acid residues and 4 disulfide bridges. The hinge region of IgG2 lacks glycine residues, is relatively short, and contains a rigid polyproline double helix stabilized by additional heavy-chain disulfide bonds. These properties limit the mobility of the IgG2 molecule. IgG3 contains 62 amino acids (including 21 prolines and 11 cysteines) and forms a non-flexible polyproline double helix, and differs from other subclasses by its unique long hinge region (about 4 times the length of the IgG1 hinge). In IgG3, the Fab fragment is relatively far from the Fc fragment, resulting in higher mobility of the molecule. The long hinge of IgG3 also contributes to its larger molecular weight compared to other subclasses. The hinge region of IgG4 is shorter than that of IgG1, and its mobility is intermediate between that of the hinge region of IgG1 and that of the hinge region of IgG2. The mobility of the hinge region has been reported to decrease in the order IgG3 > IgG1 > IgG4 > IgG2.

[0333] According to crystallographic studies, the hinge region of an immunoglobulin can be further functionally subdivided into three regions: an upper hinge region, a core region, and a lower hinge region. See Shin, et al. 1992, Immunological Reviews, 130: pp. 87. The upper hinge region includes the amino acids from the carboxy terminus of C H1 to the first residue within the hinge that restricts movement, generally the first cysteine residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the mobility of the antibody segment. The core hinge region has an interchain disulfide bridge, and the lower hinge region is linked to the amino terminus of the C H2 domain and includes the residues within C H2 The core hinge region of wild-type human IgG1 contains the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 276), and upon dimerization by formation of a disulfide bond, a cyclic octapeptide is formed that acts as a pivot axis, thereby conferring mobility. In various embodiments, the linker of the present disclosure includes one, two, or three of the upper hinge region, core region, and lower hinge region of any antibody (e.g., IgG, IgA, IgD, and IgE including subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). The hinge region may contain one or more glycosylation sites, and those sites include a number of structurally different types of sites for carbohydrate attachment. For example, IgA1 contains five glycosylation sites within a 17-amino acid segment of the hinge region, and those sites confer resistance of the hinge region polypeptide to intestinal proteases, which is considered an advantageous property for secretory immunoglobulins. In various embodiments, the linker of the present invention includes one or more glycosylation sites. In various embodiments, the linker is the hinge-CH2-CH3 domain of a human IgG4 antibody.

[0334] Optionally, the chimeric protein of the present disclosure is C H 2 domain and C HIt is capable of being linked to an antibody Fc region that includes one or both of the 3 domains and, optionally, to a hinge region. For example, it is possible to prepare such polypeptides using a vector encoding a chimeric protein of the present disclosure linked to the Fc region as a single nucleotide sequence.

[0335] In some embodiments, the linker is a synthetic linker such as PEG.

[0336] In various embodiments, the linker may be functional. For example, but not limited to, the linker may function to improve the folding and / or stability of the chimeric protein of the present disclosure, improve expression, improve pharmacokinetics, and / or improve biological activity. In another example, the linker may function to target the chimeric protein to a specific cell type or a specific location.

[0337] In various embodiments, the chimeric protein of the present disclosure may include one or more functional groups, residues, or moieties. In various embodiments, the one or more functional groups, residues, or moieties are either bound to or genetically fused to any of the signaling substances or targeting moieties described herein. In some embodiments, the functional group, residue, or moiety confers one or more desired properties or functions to the chimeric protein of the present invention. Examples of such functional groups and examples of methods for introducing those functional groups into the chimeric protein of the present disclosure are known in the art. See, for example, Remington’s Pharmaceutical Sciences, 16th Edition, Mack Publishing Co., Easton, PA (1980).

[0338] In various embodiments, each of the chimeric proteins may be conjugated and / or fused with another agent to extend its half-life or to improve its pharmacodynamic and pharmacokinetic properties. In some embodiments, the chimeric protein may be fused or conjugated with one or more of PEG, XTEN (e.g., rPEG, etc.), polyxen, albumin (e.g., human serum albumin or HAS), elastin-like protein (ELP), PAS, HAP, GLK, CTP, transferrin, etc. In some embodiments, the chimeric protein may be fused or conjugated with an antibody fragment such as an antibody or an Fc fragment. For example, the chimeric protein may be fused to either the N-terminus or the C-terminus of the Fc domain of human immunoglobulin (Ig) G. In various embodiments, each of the individual chimeric proteins is fused with one or more of the agents described in BioDrugs, (2015) 29:215-239, the entire content of which is incorporated herein by reference.

[0339] In some embodiments, the functional group, residue, or moiety includes a suitable pharmaceutically acceptable polymer, such as poly(ethylene glycol) (PEG) or a derivative thereof (e.g., methoxypoly(ethylene glycol), i.e., mPEG, etc.). In some embodiments, the attachment of the PEG moiety increases the half-life of the chimeric protein and / or decreases its immunogenicity. In general, any suitable form of PEGylation can be used, such as those used in the art of antibodies and antibody fragments (including, but not limited to, single domain antibodies such as VHH). See, for example, Chapman, Nat. Biotechnol., 54 pp., 531-545 (2002), Veronese and Harris, Adv. Drug Deliv. Rev., 54, pp. 453-456 (2003), Harris and Chess, Nat. Rev. Drug. Discov., 2 (2003), and International Publication No. 04 / 060965, the entire contents of which are incorporated herein by reference. A variety of reagents for protein PEGylation are commercially available, for example, from Nektar Therapeutics in the United States. In some embodiments, site-specific PEGylation, particularly site-specific PEGylation via cysteine residues, is used (see, for example, Yang, et al. Protein Engineering, 16, 10, pp. 761-770 (2003), the entire contents of which are incorporated herein by reference). In some embodiments, the chimeric protein of the invention is modified to appropriately introduce one or more cysteine residues for PEG attachment using methods known in the art, or an amino acid sequence containing one or more cysteine residues for PEG attachment may be fused to the amino terminus and / or carboxy terminus of the chimeric protein.

[0340] In some embodiments, the functional group, residue, or moiety includes N-linked glycosylation or O-linked glycosylation. In some embodiments, the N-linked glycosylation or O-linked glycosylation is introduced as part of co-translational modification and / or post-translational modification.

[0341] In some embodiments, the functional group, residue, or moiety comprises one or more detectable labels or other signal generating groups or moieties. Suitable labels, as well as methods for attaching those labels, methods for using them, and methods for detecting them are known in the art and include fluorescent labels (such as fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine, as well as fluorescent metals such as Eu or other metals of the lanthanide series), phosphorescent labels, chemiluminescent labels or bioluminescent labels (such as luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate esters, dioxetanes, or GFP and its analogs), radioisotopes, metals, metal chelates or metal cations, or other metals or metal cations particularly suitable for use in in vivo, in vitro, or in situ diagnosis and imaging, as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcal nuclease, δ-V-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triosephosphate isomerase, biotin avidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase, etc.), but are not limited thereto. Other suitable labels include moieties detectable using nuclear magnetic resonance spectroscopy or electron spin resonance spectroscopy. Such labeled VHHs and polypeptides of the present invention can be used, for example, in in vitro assays (including immunoassays known per se such as ELISA, RIA, EIA, and other "sandwich assays"), in vivo assays, or in situ assays, as well as for in vivo diagnostic and imaging purposes depending on the selection of a particular label.

[0342] In some embodiments, a functional group, residue, or moiety comprises a tag attached or genetically fused to the chimeric protein of the present disclosure. In some embodiments, the chimeric protein may comprise a single tag or multiple tags. The tag is, for example, a peptide, sugar, or DNA molecule that does not inhibit or interfere with the binding of the chimeric protein to a target, or to any other antigen of interest, such as a tumor antigen. In various embodiments, the tag is at least about 3-5 amino acids in length, 5-8 amino acids in length, 8-12 amino acids in length, 12-15 amino acids in length, or 15-20 amino acids in length. Examples of tags are described, for example, in U.S. Patent Application Publication No. 2013 / 0058962. In some embodiments, the tag is an affinity tag such as a glutathione-S-transferase (GST) tag and a histidine (His) tag. In one embodiment, the chimeric protein comprises a His tag.

[0343] In some embodiments, a functional group, residue, or moiety comprises a chelating group for chelating one of, for example, a metal or a metal cation. Suitable chelating groups include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0344] In some embodiments, the functional group, residue, or moiety includes a functional group that is part of a specific binding pair, such as a biotin-(strept)avidin binding pair. Such a functional group can be used to link the chimeric protein of the present invention to another protein, polypeptide, chemical compound that is bound to the other of the binding pair, i.e., through the formation of the binding pair. For example, the chimeric protein of the present invention may be bound to biotin and linked to another protein, polypeptide, compound, or carrier that is bound to avidin or streptavidin. For example, such a complexed chimeric protein can be used as a reporter in a diagnostic system where a detectable signal-producing substance is bound to avidin or streptavidin. Such binding pairs can also be used for the binding of the chimeric protein of the present disclosure to a carrier, including a carrier suitable for pharmaceutical purposes. One non-limiting example is the liposome formulation described in Cao and Suresh, Journal of Drug Targeting, 8, 4, pp. 257 (2000). Such binding pairs can also be used to link a therapeutic active substance to the chimeric protein of the present invention.

[0345] Production of chimeric protein Methods for making the chimeric protein of the present invention are described herein. For example, a DNA sequence encoding the chimeric protein of the present invention (e.g., a DNA sequence encoding a signaling substance (e.g., consensus interferon or a variant thereof), a targeting moiety, and a linker) can be chemically synthesized using methods known in the art. A synthetic DNA sequence can be ligated to other appropriate nucleotide sequences, including expression control sequences, to produce a gene expression construct encoding the desired chimeric protein. Thus, in various embodiments, the present invention provides an isolated nucleic acid comprising a nucleotide sequence encoding the chimeric protein of the present invention.

[0346] The nucleic acid encoding the chimeric protein of the present invention can be incorporated (ligated) into an expression vector, and the vector can be introduced into a host cell by a transfection method, a transformation method, or a transduction method. For example, the nucleic acid encoding the chimeric protein of the present invention can be introduced into a host cell by retroviral transduction. Examples of host cells include Escherichia coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells. The transformed host cells can be cultured under conditions that allow the host cells to express the gene encoding the chimeric protein of the present invention. Thus, in various embodiments, the present invention provides an expression vector comprising a nucleic acid encoding the chimeric protein of the present invention. In various embodiments, the present invention further provides a host cell comprising such an expression vector.

[0347] Specific expression and purification conditions will vary depending on the expression system used. For example, when expressing a gene in E. coli, first, the gene is cloned into an expression vector by placing the modified gene downstream of an appropriate bacterial promoter such as Trp or Tac and a prokaryotic signal sequence. In another example, when expressing a modified gene in a eukaryotic host cell such as a CHO cell, first, the modified gene is inserted into an expression vector containing, for example, an appropriate eukaryotic promoter, a secretion signal, an enhancer, and various introns. The gene construct can be introduced into the host cell using a transfection method, a transformation method, or a transduction method.

[0348] The chimeric protein of the present invention can be produced by culturing a host cell transfected with an expression vector encoding the protein under conditions that allow the expression of the chimeric protein. After expression, the protein can be recovered and purified by methods well known in the art, such as using affinity tags such as glutathione-S-transferase (GST) tag and histidine tag, or by chromatography.

[0349] Thus, in various embodiments, the present invention provides a nucleic acid encoding the chimeric protein of the present invention. In various embodiments, the present invention provides a host cell comprising a nucleic acid encoding the chimeric protein of the present invention.

[0350] In various embodiments, the consensus interferon of the present disclosure or a chimeric protein comprising the consensus interferon may be expressed in vivo, for example, within a patient. For example, in various embodiments, the consensus interferon of the present disclosure or a chimeric protein comprising the consensus interferon may be administered in the form of a nucleic acid encoding a consensus interferon chimeric protein of the present disclosure comprising the consensus interferon. In various embodiments, the nucleic acid is DNA or RNA. In some embodiments, the consensus interferon of the present disclosure or a chimeric protein comprising the consensus interferon is encoded by a modified mRNA, i.e., an mRNA comprising one or more modified nucleotides. In some embodiments, the modified mRNA comprises one or more of the modifications found in U.S. Patent No. 8,278,036, the entire contents of which are incorporated herein by reference. In some embodiments, the modified mRNA comprises one or more of m5C, m5U, m6A, s2U, Ψ, and 2'-O-methyl-U. In some embodiments, the present invention relates to the administration of a modified mRNA encoding one or more of the chimeric proteins of the present disclosure. In some embodiments, the present invention relates to a gene therapy vector comprising a modified mRNA. In some embodiments, the present invention relates to a gene therapy method comprising the administration of a modified mRNA. In various embodiments, the nucleic acid is in the form of an oncolytic virus, such as an adenovirus, a reovirus, a measles virus, a herpes simplex virus, a Newcastle disease virus, or a vaccinia virus.

[0351] Pharmaceutically acceptable salts and excipients The chimeric proteins described herein can form pharmaceutically acceptable salts by having a sufficiently basic functional group capable of reacting with an inorganic or organic acid, or a carboxyl group capable of reacting with an inorganic or organic base. As is well known in the art, pharmaceutically acceptable acid addition salts are formed from pharmaceutically acceptable acids. Such salts include, for example, those described in Journal of Pharmaceutical Science, 66, pp. 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use., P.H. Stahl and C.G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are hereby incorporated by reference in their entirety.

[0352] Pharmaceutically acceptable salts include, as non-limiting examples, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, metaphosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate, phenylbutyrate, α-hydroxybutyrate, butyne-1,4-dicarboxylate, hexyne-1,4-dicarboxylate, caprate, caprylate, cinnamate, glycolate, heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, phthalate, terephthalate, propiolate, propionate, phenylpropionate, sebacate, suberate, p-bromobenzenesulfonate, chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate, methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, naphthalene-1,5-sulfonate, xylenesulfonate, and tartrate.

[0353] The term "pharmaceutically acceptable salt" also refers to salts of the compositions of the present invention having acidic functional groups such as carboxylic acid functional groups and bases. Suitable bases include hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals such as aluminum and zinc; ammonia, and organic amines, such as unsubstituted or hydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-lower alkylamine), such as mono-, bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine; N,N-di-lower alkyl-N-(hydroxyl-lower alkyl)-amine, such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine and lysine, but are not limited thereto.

[0354] In some embodiments, the compositions described herein are in the form of pharmaceutically acceptable salts.

[0355] Pharmaceutical Compositions and Formulations In various embodiments, the present invention relates to pharmaceutical compositions comprising the chimeric proteins described herein and a pharmaceutically acceptable carrier or excipient. Any pharmaceutical composition described herein can be administered to a subject as one component of a composition comprising a pharmaceutically acceptable carrier or solvent. Such compositions may contain appropriate amounts of pharmaceutically acceptable excipients to be in a form suitable for proper administration.

[0356] In various embodiments, the pharmaceutical excipient may be a liquid such as water and oil, and the oil includes oils of petroleum origin, animal origin, plant origin, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. These pharmaceutical excipients may be, for example, physiological saline, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. Further, adjuvants, stabilizers, thickeners, lubricants, and coloring agents may be used. In certain embodiments, when administered to a subject, the pharmaceutically acceptable excipient is sterile. Water is a useful excipient when any of the agents described herein are administered intravenously. Aqueous physiological saline solution, as well as aqueous glucose solution and glycerol solution, can also be used as liquid excipients, specifically liquid excipients for injections. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc. Any of the agents described herein may also contain a small amount of wetting agent or emulsifier, or pH buffer. Other examples of suitable pharmaceutical excipients are described in Remington’s Pharmaceutical Sciences, pp. 1447-1676 (edited by Alfonso R. Gennaro, 19th edition, 1995), which is incorporated herein by reference.

[0357] The present invention includes the pharmaceutical compositions (and / or additional therapeutic agents) described herein in various dosage forms. Any of the pharmaceutical compositions (and / or additional therapeutic agents) of the present invention described herein may be in the form of a solution, suspension, emulsion, drops, tablets, pills, pellets, capsules, solution-containing capsules, gelatin capsules, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, lyophilized powders, frozen suspensions, dry powders, or any other suitable form for use. In certain embodiments, the composition is in the form of a capsule. In another embodiment, the composition is in the form of a tablet. In yet another embodiment, the pharmaceutical composition is formulated in the form of a soft gel capsule. In other embodiments, the pharmaceutical composition is formulated in the form of a gelatin capsule. In yet another embodiment, the pharmaceutical composition is formulated as a solution.

[0358] The pharmaceutical compositions (and / or other agents) of the present invention may also contain solubilizing agents if necessary. Also, the active agents can be delivered by suitable solvents or delivery devices known in the art. The combination therapies outlined herein can be co-delivered with a single delivery solvent or delivery device.

[0359] The formulations of the present invention containing the pharmaceutical compositions (and / or other agents) of the present invention may, for convenience, be provided in unit dosage form and may be prepared by any method well known in the art of pharmacy. Such methods generally include the step of bringing into association a carrier which constitutes one or more accessory ingredients with the therapeutic agent. Usually, the formulation is prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired dosage form of the formulation (for example, tableting using conventional methods known in the art after wet or dry granulation, powder blending, etc.).

[0360] In various embodiments, any of the pharmaceutical compositions (and / or other agents) described herein are formulated according to conventional methods as compositions adapted to the methods of administration described herein.

[0361] Examples of administration routes include, for example, oral route, intradermal route, intramuscular route, intraperitoneal route, intravenous route, subcutaneous route, intranasal route, epidural route, sublingual route, intranasal route, intracerebral route, intravaginal route, transdermal route, rectal route, inhalation route, or topical route. Administration may be local or systemic. In some embodiments, administration is achieved orally. In another embodiment, administration is performed by parenteral injection. The method of administration may be left to the discretion of the physician and partly depends on the site of the disease. In most cases, any of the agents described herein will be released into the bloodstream upon administration.

[0362] In certain embodiments, the chimeric proteins described herein are formulated according to conventional methods as compositions adapted for oral administration. Compositions for oral delivery may be in dosage forms such as, for example, tablets, troches, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs. Compositions for oral administration may contain one or more agents, such as sweetening agents, for example, fructose, aspartame, or saccharin; flavoring agents, such as peppermint, wintergreen, or cherry; coloring agents, and preservatives, to provide pharmaceutically palatable formulations. Further, in the case of tablets or pills, the composition can be coated to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over an extended period of time. Selective permeable membranes that cover the osmotic activity driving compound of any chimeric protein described herein are also suitable for oral administration compositions. In these latter platforms, the liquid of the environment around the capsule is absorbed by the driving compound, which swells and extrudes its agent or agent composition through the holes. These delivery platforms can basically generate a zero-order delivery profile, in contrast to the spike-like profile of immediate-release formulations. Time-delay substances such as glyceryl monostearate or glyceryl stearate may also be useful. Oral compositions can contain standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. In certain embodiments, the excipients are of pharmaceutical grade. Suspensions may contain, in addition to the active compound, suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, and mixtures thereof, and the like.

[0363] Suitable dosage forms for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, and intra-articular injections and infusions) include, for example, solutions, suspensions, dispersions, emulsions, etc. These dosage forms may be manufactured in the form of sterile solid compositions (e.g., lyophilized compositions) that can be dissolved or suspended in a sterile injection medium immediately before use. The sterile solid compositions may contain, for example, suspending or dispersing agents known in the art. Suitable formulation components for parenteral administration include sterile diluents such as water for injection, physiological saline solution, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffering agents such as acetate, citrate, or phosphate; and osmotic pressure regulators such as sodium chloride or glucose.

[0364] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (trademark) (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). The carrier must be stable under the manufacturing and storage conditions and must be preserved against microorganisms. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0365] The compositions provided herein can be formulated into aerosol formulations for administration by inhalation (i.e., "nebulized"), either alone or in combination with other suitable components. The aerosol formulations can be admixed with a pressurizable propellant, such as dichlorodifluoromethane, propane, and nitrogen, etc.

[0366] The pharmaceutical compositions (and / or other active agents) of the present invention described herein can all be administered by controlled release means or sustained release means well known to those skilled in the art, or by delivery devices. Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770, 3,916,899, 3,536,809, 3,598,123, 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,556, each of which is incorporated herein by reference in its entirety. Such dosage forms can be useful, for example, in achieving controlled release or sustained release of one or more active ingredients using hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or combinations thereof in various ratios to achieve the desired release profile. Suitable controlled release formulations or sustained release formulations known to those skilled in the art, including the formulations described herein, can be readily selected for use with the active ingredients of the active agents described herein. Accordingly, the present invention provides single unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gel caps, and caplets, that are compatible with controlled release or sustained release.

[0367] Controlled release or sustained release of the active ingredient can be stimulated by, but is not limited to, changes in pH, temperature changes, stimulation by light of an appropriate wavelength, enzyme concentration or availability, water concentration or availability, or various conditions including other physiological conditions or compounds.

[0368] In another embodiment, a controlled release system can be placed near the target area to be treated, and thus only a portion of the systemic dose is required (see, e.g., Goodson, Medical Applications of Controlled Release, supra, 2, pp. 115-138 (1984)). Other controlled release systems discussed in the review by Langer, 1990, Science, 249: pp. 1527-1533) may be used.

[0369] The pharmaceutical formulation is preferably sterile. Sterilization can be achieved, for example, by filtration through a sterile filtration membrane. If the composition is lyophilized, filter sterilization can be carried out before or after lyophilization and reconstitution.

[0370] Administration and Dosage It is understood that the actual dosage of the chimeric protein of the present disclosure administered in accordance with the present invention will vary depending on the particular dosage form and method of administration. One of ordinary skill in the art can take into account a number of factors that can modify the action of the chimeric protein of the present disclosure (e.g., body weight, gender, diet, time of administration, route of administration, rate of excretion, condition of the subject, drug combination, genetic nature, and sensitivity to reaction). Administration can be carried out by continuous administration or one or more divided administrations within the maximum allowable volume. One of ordinary skill in the art can confirm the optimal rate of administration for a given condition using conventional dosage administration tests.

[0371] In some embodiments, an appropriate dosage of the chimeric protein of the present disclosure is in the range of about 0.01 μg to about 100 mg per kg of the subject's body weight, about 0.01 μg to about 10 mg per kg of the subject's body weight, or about 0.01 μg to about 1 mg per kg of the subject's body weight. For example, about 0.01 μg, about 0.02 μg, about 0.03 μg, about 0.04 μg, about 0.05 μg, about 0.06 μg, about 0.07 μg, about 0.08 μg, about 0.09 μg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, about 1.5 mg, about 1.6 mg, about 1.7 mg, about 1.8 mg, 1.9 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, or about 100 mg per kg of body weight, including all values and ranges between the upper and lower values thereof.

[0372] The individual dosages of the chimeric proteins of the present disclosure can be administered, for example, as unit dosage forms (e.g., tablets, capsules, or liquids) containing from about 1 μg to about 100 mg, from about 1 μg to about 90 mg, from about 1 μg to about 80 mg, from about 1 μg to about 70 mg, from about 1 μg to about 60 mg, from about 1 μg to about 50 mg, from about 1 μg to about 40 mg, from about 1 μg to about 30 mg, from about 1 μg to about 20 mg, from about 1 μg to about 10 mg, from about 1 μg to about 5 mg, from about 1 μg to about 3 mg, from about 1 μg to about 1 mg, or from about 1 μg to about 50 μg per unit dosage form. For example, the unit dosage form can be about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, or about 100 mg, including all values and ranges between the upper and lower limit values thereof. In certain embodiments, the chimeric protein is administered as a unit dosage form containing about 9 μg of the chimeric protein of the present disclosure. In another embodiment, the chimeric protein is administered as a unit dosage form containing about 15 μg of the chimeric protein of the present disclosure.

[0373] In certain embodiments, the chimeric protein is administered in a daily dose of from about 1 μg to about 100 mg, from about 1 μg to about 90 mg, from about 1 μg to about 80 mg, from about 1 μg to about 70 mg, from about 1 μg to about 60 mg, from about 1 μg to about 50 mg, from about 1 μg to about 40 mg, from about 1 μg to about 30 mg, from about 1 μg to about 20 mg, from about 01 μg to about 10 mg, from about 1 μg to about 5 mg, from about 1 μg to about 3 mg, or from about 1 μg to about 1 mg. In various embodiments, the chimeric protein is about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, or about 100 mg per day dose, including all values and ranges between the upper and lower values thereof. In certain embodiments, the chimeric protein is administered in a daily dose of about 9 μg. In another embodiment, the chimeric protein is administered in a daily dose of about 15 μg.

[0374] According to certain embodiments of the present invention, the pharmaceutical composition comprising the chimeric protein of the present disclosure may be administered, for example, more than once a day (e.g., about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 times a day), about once a day, about once every two days, about once every three days, about once a week, about once every two weeks, about once a month, about once every two months, about once every three months, about once every six months, or about once a year. In certain embodiments, the pharmaceutical composition comprising the chimeric protein of the present disclosure is administered about three times a week.

[0375] In various embodiments, the chimeric protein of the present disclosure can be administered over a long period of time. For example, the chimeric protein of the present disclosure can be administered for at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, or at least about 12 weeks as described herein. For example, the chimeric protein of the present disclosure can be administered for 12 weeks, 24 weeks, 36 weeks, or 48 weeks. In some embodiments, the chimeric protein of the present disclosure is administered for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months. In some embodiments, the chimeric protein of the present disclosure can be administered for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years.

[0376] Combination therapies and additional therapeutic agents In various embodiments, the pharmaceutical composition of the present invention is co-administered in combination with an additional therapeutic agent. Co-administration may be simultaneous administration or sequential administration.

[0377] In certain embodiments, an additional therapeutic agent and the chimeric protein of the invention are administered to a subject simultaneously. As used herein, the term "simultaneously" means that the additional therapeutic agent and the chimeric protein of the present disclosure are administered within about 60 minutes, such as within about 30 minutes, within about 20 minutes, within about 10 minutes, within about 5 minutes, or within about 1 minute. Administration of the additional therapeutic agent and the chimeric protein of the present disclosure may be by co-administration of a single formulation (e.g., a formulation containing the additional therapeutic agent and the chimeric protein of the present disclosure), or by co-administration of separate formulations (e.g., a first formulation containing the additional therapeutic agent and a second formulation containing the chimeric protein of the present disclosure).

[0378] Co-administration does not require that the therapeutic agents be administered simultaneously, provided that the timing of administration of the therapeutic agents is such that the pharmacological activities of the additional therapeutic agent and the chimeric protein of the present disclosure overlap in time, thereby exerting a combined therapeutic effect. For example, the additional therapeutic agent and the chimeric protein of the present disclosure may be administered sequentially. As used herein, the term "sequentially" means that the additional therapeutic agent and the chimeric protein of the present disclosure are administered at intervals of more than about 60 minutes. For example, the time between sequential administrations of the additional therapeutic agent and the chimeric protein of the present disclosure may be about 60 minutes or more, about 2 hours or more, about 5 hours or more, about 10 hours or more, about 1 day or more, about 2 days or more, about 3 days or more, about 1 week or more, about 2 weeks or more, or about 1 month or more. The optimal dosing time depends on the metabolic rate, excretion rate, and / or pharmacodynamic activity of the additional therapeutic agent and the chimeric protein of the present disclosure being administered. Either the additional therapeutic agent or the chimeric protein of the present disclosure may be administered first.

[0379] Co-administration also does not require that the therapeutic agents be administered to the subject by the same route of administration. Rather, each therapeutic agent may be administered by any suitable route, e.g., parenterally or non-parenterally.

[0380] In some embodiments, the chimeric proteins described herein act synergistically when co-administered with another therapeutic agent. In such embodiments, the chimeric proteins of the disclosure and the additional therapeutic agent may be administered at lower dosages than those used when the agents are used in connection with monotherapy.

[0381] In some embodiments, the present invention relates to chemotherapeutic agents as additional therapeutic agents. For example, without limitation, such combinations of the chimeric proteins of the present disclosure and chemotherapeutic agents are used for the treatment of cancer as described anywhere herein. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN (cyclophosphamide); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocone, meturedopa, and uredopa; ethyleneimines and methylmelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (e.g., bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; calistatin; CC-1065 (including its adozelesin synthetic analogs, carzelesin synthetic analogs, and bizelesin synthetic analogs); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs KW-2189 and CB1-TM1); erythrocin; pancratistatin; sarcodictyin A; spongistatin; nitrogen mustards such as chlorambucil, chloronaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin γII and calicheamicin ωII (see, e.g., Agnew. Chem Intl. Ed. Engl., 33: pp. 183-186 (1994)) and other antibiotics; dynemicin including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein engyin antibiotics chromophore), actinomycins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, cardinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN (doxorubicin) (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin; mitomycins such as mitomycin C; mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomycin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimethoprim; purine analogs such as fludarabine, 6-mercaptopurine, thiampurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, drostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatrexate; dexamethasone; diacontin; elformithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;Pentostatin; Phenamet; Pirarubicin; Losoxantrone; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oregon); Razoxane; Rizoxin; Schizophyllan; Spirogermanium; Tenuazonic acid; Triazicone; 2,2’,2”-Trichlorotriethylamine; ...

Claims

**Claim 1** (a) A variant consensus interferon having only one amino acid mutation which is a mutation from R to A at the 150th amino acid position in consensus interferon of SEQ ID NO: 2 (R150A), wherein the R150A mutation reduces the affinity or activity of the variant consensus interferon for the interferon α / β receptor (IFNAR) as compared to consensus interferon without the mutation, and (b) A targeting moiety consisting of a recombinant heavy chain antibody (VHH) that specifically binds to an antigen or receptor on a tumor cell or an immune cell, comprising wherein the reduction in the affinity or activity of the variant consensus interferon can be restored by the binding of the targeting moiety to the target antigen or receptor, A chimeric protein, wherein the variant consensus interferon and the targeting moiety may be linked by one or more linkers. **Claim 2** The chimeric protein according to claim 1, wherein the variant consensus interferon is PEGylated. **Claim 3** The chimeric protein according to claim 1, wherein the immune cell is selected from T cells, B cells, dendritic cells, macrophages, neutrophils, myeloid-derived suppressor cells, and NK cells. **Claim 4** The chimeric protein according to claim 1, wherein the VHH is a humanized VHH. **Claim 5** The chimeric protein according to claim 1, further comprising an additional targeting moiety. **Claim 6** The chimeric protein according to claim 5, comprising two or more targeting moieties. **Claim 7** The chimeric protein according to claim 1, further comprising one or more additional modified signaling substances. **Claim 8** The chimeric protein according to claim 7, wherein the chimeric protein comprises two signaling substances, two targeting moieties, or both two signaling substances and two targeting moieties. **Claim 9** The chimeric protein according to claim 7, wherein the chimeric protein comprises three signaling substances, three targeting moieties, or both three signaling substances and three targeting moieties. **Claim 10** The chimeric protein according to claim 7, wherein the one or more additional modified signal transduction substances comprise one or more mutations that reduce the affinity or activity of the one or more additional modified signal transduction substances for a receptor as compared to the unmutated signal transduction substance.

11. The chimeric protein according to claim 10, wherein the one or more mutations in the additional modified signal transduction substance result in attenuation of activity.

12. The chimeric protein according to claim 10, wherein the one or more mutations in the one or more additional modified signal transduction substances result in a decrease in affinity or activity that can be restored by the binding of the one or more additional modified signal transduction substances to one or more targeting moieties.

13. A recombinant nucleic acid encoding the chimeric protein according to any one of claims 1 to 12.

14. A host cell comprising the nucleic acid according to claim 13.

15. A medicament for use in the treatment of hepatitis, comprising the chimeric protein according to any one of claims 1 to 12.

16. The medicament according to claim 15, wherein the hepatitis is caused by chronic hepatitis C infection.

17. A medicament for use in the treatment of cancer, comprising the chimeric protein according to any one of claims 1 to 12.

18. The medicament according to claim 17, wherein the cancer is selected from one or more of basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive tract cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastrointestinal cancer, glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, melanoma, multiple myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory tract cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary tract cancer, vulvar cancer, lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloid leukemia, and other carcinomas and sarcomas, and post-transplant lymphoproliferative disorder (PTLD), and abnormal angiogenesis associated with nevus, edema, and MEG syndrome.

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